Agent for preventing or treating steroid osteoporosis

Abstract

The problem addressed is to provide an agent for preventing or treating steroid osteoporosis. The problem is solved by an agent for preventing or treating steroid osteoporosis that contains a Piezo1 and / or an Hes1 enhancer.

Description

Preventive or therapeutic agent for steroid-induced osteoporosis

[0001] The present invention relates to a preventive or therapeutic agent for steroid-induced osteoporosis.

[0002] Glucocorticoid-induced osteoporosis is one of the most important side effects caused by the administration of corticosteroids for reasons such as autoimmune diseases. After a rapid increase in bone resorption, bone formation gradually declines, resulting in an increased risk of fracture. One of the characteristics of fractures in steroid-induced osteoporosis is that fractures occur before a decrease in bone mineral density, suggesting that steroid-induced osteoporosis may affect not only bone mass but also bone quality.

[0003] Previous studies have suggested that osteoclasts play a major role in bone resorption, and osteoblasts in bone formation, but in recent years, osteocytes have been recognized as key cells in regulating bone metabolism through the control of sclerostin, RANKL, OPG, etc. It has become clear that osteocytes form canalicular networks with each other and promote bone formation and bone formation signals in response to mechanical stress.

[0004] In recent years, the function of Piezo1, an ion channel that senses mechanical stress, in bone cells has attracted attention. It has been reported that administration of Yoda1, a Piezo1 activator, to an animal model of fracture shortens the healing time of fractures (Non-Patent Document 1). However, to date, there have been no reports on the Piezo1-mediated mechanical stress response in glucocorticoid-induced osteoporosis.

[0005] International Publication No. 2021 / 067943

[0006] Liu, Y. et al. Mechanosensitive Piezo1 is crucial for periosteal stem cell-mediated fracture healing. Int. J. Biol. Sci. 18, 3961-3980 (2022).

[0007] An object of the present invention is to provide an agent for preventing or treating steroid-induced osteoporosis.

[0008] In view of the above problems, the present inventors have conducted extensive research and found that a preventive or therapeutic agent for steroid-induced osteoporosis containing a Piezo1 and / or Hes1 enhancer can solve the above problems. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects:

[0009] Item 1. A preventive or therapeutic agent for steroid-induced osteoporosis, comprising a Piezo1 and / or Hes1 enhancer.

[0010] Item 2. The preventive or therapeutic agent according to Item 1, wherein the Piezo1 and / or Hes1 enhancer is an enhancer of the expression or function of Piezo1 and / or Hes1.

[0011] Item 3. The preventive or therapeutic agent according to Item 1 or 2, wherein the Piezo1 and / or Hes1 enhancer is at least one selected from the group consisting of a Piezo1 activator, an LSD1 inhibitor, a polynucleotide comprising a Piezo1 coding sequence, a polynucleotide comprising a Hes1 coding sequence, a Piezo1 protein, and a Hes1 protein.

[0012] Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, which comprises a Hes1 enhancer.

[0013] Item 5. The preventive or therapeutic agent according to any one of Items 1 to 4, wherein the Piezo1 and / or Hes1 enhancer is an agent that enhances Piezo1 and / or Hes1 in bone tissue.

[0014] Item 6. The preventive or therapeutic agent according to any one of Items 1 to 5, which is used to inhibit weakening of cortical bone and cancellous bone.

[0015] Item 7. The prophylactic or therapeutic agent according to any one of Items 1 to 6, which is used in combination with a steroid drug or is administered to a subject who has previously been administered a steroid drug.

[0016] Item 8. An agent containing a Piezo1 and / or Hes1 enhancer for use in combination with a steroid or for administration to a subject who has previously received steroids.

[0017] According to the present invention, it is possible to provide an agent for preventing or treating steroid-induced osteoporosis.

[0018] GIOP patients have a reduced canalicular network and Piezo1 expression compared to non-GIOP patients. GIOP: Thin sections derived from femoral cortical bone from GIOP patients (women, 57, 51, and 61 years old). Non-GIOP: Thin sections derived from femoral cortical bone from non-GIOP (osteoarthritis) patients (women, 65, 65, and 63 years old). A, B: Platon silver staining. Scale bar indicates 200 μm. 4x magnification. B: Length of osteocyte dendrites in each specimen. C, D: HE staining. Scale bar indicates 200 μm. 4x magnification. D: Percentage of empty lacunae in each specimen. E, F: TUNEL staining. Arrowheads indicate TUNEL-positive cells. Scale bar indicates 100 μm. 20x magnification. F: Percentage of TUNEL-positive cells. G, H: Piezo1 immunostaining. Arrowheads indicate Piezo1-positive cells. Scale bar indicates 60 μm. 40x magnification. H: Number of Piezo1-positive cells per bone surface area in each specimen. I: Western blotting for Piezo1 and β-Actin. N=3 for each data point. Mean ± SD values ​​are shown. Statistical analysis was performed using Student's t-test with a 95% confidence interval. * p < 0.05, ** p < 0.01, *** p < 0.001. This shows that Yoda1 suppresses the weakening of bone structure in glucocorticoid-induced osteoporosis. A: Administration schedule for DEX and Yoda1. DEX was administered subcutaneously at 1 mg / kg, and Yoda1 was administered intraperitoneally at 5 μmol / kg. Vehicle was administered subcutaneously in water for injection for DEX and intraperitoneally in 5% ethanol for injection for Yoda1. Each condition was administered once daily for five days a week. Vehicle: Vehicle administration group, DEX: DEX administration group, DEX + Yoda1: DEX and Yoda1 combined administration group. B: Overall image of cortical bone, trabecular bone, and bone morphometry analysis area. Scale bars for cortical bone and trabecular bone are 250 μm, and scale bars for overall image of bone morphometry analysis area are 100 μm. C: Micro-CT analysis results. BV / TV: bone volume, Ct. Th: cortical bone width, Tb. N: number of trabeculae, N=9. D: Results of three-point bending bone strength test.Maximum load: Maximum load, Energy absorption: Energy absorption, Stiffness: Stiffness, N=6-10. E: Enlarged view of the osteoid surface for bone morphometry. The osteoid surface is indicated by a white line. The scale bar represents 10 μm and was observed at 80x magnification. F: Ob.S / OS: Osteoblast surface (osteoid surface), G: OV / OS: Osteoid volume (osteoid surface), N=4. H: Enlarged view of the cortical bone periphery for bone morphometry. The cortical bone width is indicated by a black line. The scale bar represents 10 μm and was observed at 80x magnification. I: Ct. Wi: Cortical bone width, J: Ct. Ar.: Cortical bone surface, N=4. K: Enlarged view of alizarin and calcein labeled bone for bone morphometry. Analysis was performed after 4 days of alizarin labeling and 1 day of calcein labeling. The scale bar represents 5 μm and was observed at 100x magnification. L: MAR: mineral apposition rate, M: BFR: bone formation rate, N=4. All graphs are plotted as boxplots. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.001. This figure shows the results of bone histology analysis following one-month administration of DEX and Yoda1. Data refer to the following groups: Vehicle: water for injection, 5% ethanol administration group; DEX: DEX 1 mg / kg administration group; DEX + Yoda1: DEX 1 mg / kg, Yoda1 5 μmol / kg combined administration group. A, B: Piezo1 immunostaining. The scale bar is 200 μm, and the arrowheads indicate Piezo1-positive cells. 20x magnification. B: Number of Piezo1-positive cells per bone surface area. C, D: Sost immunostaining. The scale bar is 200 μm, and the arrowheads indicate Sost-positive cells. 20x magnification. D: Number of Sost-positive cells per bone surface area. E, F: Ploton silver staining. Scale bar indicates 60 μm. 40x magnification. F: Length of osteocyte dendrites. G, H: Alexa488-labeled phalloidin staining. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. 100x magnification. H: Length of osteocyte dendrites. I, J: Osteocalcin immunostaining. Scale bar indicates 200 μm. Arrowheads indicate osteocalcin-positive cells. 20x magnification. J: Number of osteocalcin-positive cells on the trabecular surface.K, L: TRAP staining. The scale bar indicates 200 μm, and the arrowheads indicate TRAP-positive cells. 20x magnification. L: Number of TRAP-positive cells on the trabecular surface. M: Bone resorption surface (ES / BS). H, M: N=4, others: N=5. Box plots. Statistical analysis was performed using one-way ANOVA with Tukey's test. *p<0.05, **p<0.01, ***p<0.001. This shows that Yoda1 prevents the steroid-induced decrease in bone mechanical loading response. A: Administration and bone mechanical loading schedule. Administration conditions: DEX 1 mg / kg subcutaneously, Yoda1 5 μmol / kg intraperitoneally. Vehicle was administered subcutaneously in water for injection for DEX, and 5% ethanol in water for injection was administered intraperitoneally for Yoda1. Each condition was administered once daily, 5 days a week. Bone loading conditions: Mechanical loading was performed using an Electroforce 5500 (TA) with a -13.5N load applied for 0.1 seconds, with 10-second cycles for 40 sets, two to three times per week. Vehicle-Ctrl: Vehicle-administered untreated group; Vehicle-Load: Vehicle-administered loading group; DEX-Load: DEX-administered loading group; DEX + Yoda1-Load: DEX and Yoda1 combined loading group. B: Overall image of cortical and trabecular bone during micro-CT analysis. Scale bars for cortical and trabecular bone are 250 μm. C: Micro-CT analysis results. C: BV / TV: bone volume; Ct. Th: cortical width; Tb. N: trabecular number; Po. (tot): total cortical porosity; N = 6-9. D: Enlarged image of bone morphometry labeled with alizarin and calcein. Analysis was performed after 2 days of tetracycline labeling and 1 day of calcein labeling. The scale bar indicates 5 μm and the image was observed at 100x magnification. E: MAR: mineralization rate, F: BFR bone formation rate. G, H: Osteocalcin immunostaining. The scale bar indicates 200 μm and the black dashed line indicates the periosteal surface. 40x magnification. H: Osteocalcin-positive cell rate on the periosteal surface. I, J: TRAP staining. The scale bar indicates 200 μm and the arrowheads indicate TRAP-positive cells. 20x magnification.J: Number of TRAP-positive cells on the trabecular surface. GM (n=3-4). All graphs are expressed as mean ± SD and plotted as boxplots. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.001). The effects of DEX and Yoda1 on various genes, phosphorylation signals, and cell morphological changes in human bone organ cultures and osteocytes are shown. AD: Human femoral cortical bone organ culture experiment. Ctrl: Control (untreated). DEX: Dexamethasone (DEX) 1 μmol / L. DEX + Yoda1: Dexamethasone 1 μmol / L, Yoda1 10 μmol / L. After femoral removal, cleaned cortical bone was cultured overnight, then treated with each drug for 6 hours. RNA was extracted from the tissue and subjected to qPCR. N=3. Hprt was used as an internal standard and quantification was performed using the ΔΔCt method. N=3. All graphs are expressed as mean ± SD and plotted as boxplots. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.001. E: Western blotting of Piezo1 in MYOY4 cells. Ctrl: Control (untreated). DEX: Dexamethasone 1 μmol / L. DEX + Yoda1 (3), (10): Dexamethasone 1 μmol / L, Yoda1 3, 10 μmol / L. MYOY4 cells were treated with DEX and then various concentrations of Yoda1 were added 24 hours later. Cells were harvested 24 hours later and subjected to Western blotting. β-Actin was used as an internal control. F, G: Western blotting of phosphorylated Akt and ERK. Ctrl: Control (untreated), Yoda1: Yoda1 10 mol / L, DEX: dexamethasone 1 μmol / L, DEX + Yoda1: dexamethasone 1 μmol / L and Yoda1 10 μmol / L. MLOY4 cells were treated with DEX and then various concentrations of Yoda1 were added 24 hours later. Cells were harvested 2 hours later and subjected to Western blotting. β-Actin was used as an internal control.H: Ca into MLOY4 cells. 2+ Ion influx analysis. Ctrl: Control (untreated), DEX: Dexamethasone 1 μmol / L, Piezo1-KD: Piezo1 siRNA transfection treatment. MLOY4 cells were transfected with Piezo1 siRNA by electroporation, and dexamethasone was added 24 hours later. After washing the cells 24 hours later, calcium influx was observed using Fluo8. 2+To stimulate influx, 3 μmol / L Yoda1 was added to the cells using an autosampler. The time course of the fluorescence intensity before addition (F0) and the ratio of the increased fluorescence intensity (ΔF / F0) was plotted. I: Western blotting of CaM kinase II phosphorylation. Ctrl: Control (untreated), Yoda1: 10 μmol / L Yoda1, DEX: 1 μmol / L dexamethasone, DEX + Yoda1: 1 μmol / L dexamethasone and 10 μmol / L Yoda1 treatment. MLOY4 cells were treated with DEX and then various concentrations of Yoda1 were added 24 hours later. Cells were harvested 1 hour later and subjected to Western blotting. J: Effect of CaM kinase II inhibitor addition on Akt phosphorylation. MLOY4 cells were treated with DEX and various concentrations of KN93 added 24 hours later. After 2 hours of preincubation, Yoda1 was added. Cells were harvested 2 hours later and subjected to Western blotting. K, L: Analysis of cell morphology in MLOY4 cells using rhodamine-conjugated phalloidin staining. Ctrl: control (untreated), DEX: 1 μmol / L dexamethasone, DEX + Yoda1 (1): 1 μmol / L dexamethasone and 1 μmol / L Yoda1, DEX + Yoda1 (3): 1 μmol / L dexamethasone and 3 μmol / L Yoda1. MLOY4 cells were treated with various concentrations of DEX and Yoda1. 72 hours later, the cells were stained with rhodamine-conjugated phalloidin and nuclear counterstained with Hoechst. The scale bar indicates 50 μm. Images were observed at 40x magnification using an In Cell Analyzer 6000 (GE). L: The number of actin filament crossing points was automatically calculated using the analysis software (In Cell Developer) included with the In Cell Analyzer. The cell count was calculated by counting only nucleated, live cells stained with Hoechst 33258. N=8. All graphs are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001.This figure shows the results of gene variability analysis and search for candidate Piezo1 transcriptional regulators during bone mechanical loading in mice treated with DEX. A: Administration and bone mechanical loading (loading) schedule. Administration conditions: DEX 1 mg / kg was administered subcutaneously, and vehicle was administered subcutaneously with injectable water. Each condition was administered once daily, five days a week. Bone loading conditions: Using an Electroforce 5500 (TA), a -13.5 N load was applied for 0.1 seconds, with 10 seconds per cycle, for a total of 40 sets, three times a week. Loading was administered only to the left leg, while the right leg served as a control. The tibia was collected 4 hours after the final loading and subjected to RNA sequencing. B: Genes affected during loading in the vehicle-treated group. Horizontal axis: log2 change ratio under loading load. Vertical axis: significant difference in variation analysis due to loading load. DEG: genes with expression changes. DESeq2 was used, and p < 0.05 in the Wald test was used to indicate a significant difference, shown in red. C: Genes with changes when loading was given during DEX administration. D: Comparison of gene variations between the vehicle-administered group and the DEX-administered group under loading load. Piezo1: log2 FC: -0.38, padj: 3.0 e-3, Tnfrsf11b: log2 FC: -0.48, padj: 7.0 e-04, Tnfrsf11a: log2 FC: 0.67, padj: 1.8 e-02. E: GO analysis of genes whose response to loading load was reduced by DEX administration. Gene variation data for the vehicle- and DEX-administered groups was corrected for multiple testing using FDR, and each pathway was plotted in order of decreasing significance. Each count indicates the number of genes in each pathway. F: Venn diagram analysis of candidate molecules that regulate Piezo1 transcription. Piezo1 transcription factor candidates: 145 molecules predicted to bind to the Piezo1 promoter region extracted from ChIP Atlas. Genes decreased expression by DEX: 2,084 genes that showed significant changes between the vehicle-administered group and the DEX1-administered group under non-loading conditions (padj < 0.05).G: Changes in expression of 14 candidate Piezo1 transcription factors when MLOY4 cells are subjected to mechanical stress (LIPUS). Previously reported data on expression changes when MLOY4 cells are stimulated with LIPUS (Shimizu T. et al., Scientific Reports, 11(1), 1-15, 2021). Horizontal axis: expression change ratio when LIPUS is stimulated, vertical axis: significant difference. Red marks: 14 candidate Piezo1 transcription factors. Hes1: hairy and enhancer of split 1, Vdr: vitamin D receptor, Bhlhe41: Basic helix-loop-helix family, member e41. This indicates that Hes1 functions as a transcriptional regulator of Piezo1 and is functionally regulated by DEX and Yoda1. A: Changes in expression of Hes1 and Piezo1 genes when Hes1 is knocked down. MLOY4 cells were electroporated with Hes1 siRNA or negative control siRNA, and RNA was extracted two days later for qPCR. Ctrl: Cells transfected with negative siRNA; Hes1 KD: Cells transfected with Hes1 siRNA. Hprt was used as an internal standard, and quantification was performed using the ΔΔCt method. N=3. All graphs are shown as mean ± SD. Statistical analysis was performed using Student's t test, with confidence intervals at 95%. * p < 0.05, ** p < 0.01, *** p < 0.001. B: Western blotting of Hes1 and Piezo1 following Hes1 knockdown. MLOY4 cells were electroporated with Hes1 siRNA, Piezo1 siRNA, or negative control siRNA, and treated with dexamethasone one day later. Protein was extracted and analyzed by Western blotting one day later. Ctrl: Cells transfected with negative siRNA, DEX: Cells transfected with negative siRNA and treated with 1 μmol / L DEX, Hes1 KD: Cells transfected with Hes1 siRNA, Piezo1 KD: Cells transfected with Piezo1 siRNA.Hprt was used as an internal standard, and quantification was performed using the ΔΔCt method. C: Schematic diagram of the Piezo1 promoter region (1,200 bp) and Hes1 binding region (657 bp) obtained from ChIP Atlas, along with the primers used in the CUT & RUN assay. D: Results of the CUT & RUN assay. MLOY4 cells were electroporated with Hes1 siRNA, Piezo1 siRNA, or negative control siRNA. One day later, they were treated with dexamethasone or Yoda1. Measurements were performed one day later according to the CUT & RUN Assay Kit (Cell Signaling, 86652S) protocol. Ctrl: Untreated; Yoda1: Yoda1 10 μmol / L; DEX: DEX 1 μmol / L; Hes1 KD: Hes1 siRNA. Ctrl was enriched with an IgG antibody, and Yoda1, DEX, and Hes1 KD were enriched with a Hes1 antibody. Percent Input: The same concentration of DNA was added to each condition, and the percent input was calculated from the PCR results. N=3. All graphs show the mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test, with * p < 0.05, ** p < 0.01, and *** p < 0.001. E: Luciferase assay. MLOY4 cells were electroporated with each plasmid vector, treated with DEX 24 hours later, and then treated with Yoda1 24 hours later. Luminescence from NanoLuc and firefly luciferase was measured 4 hours later using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega, N1610) according to the manufacturer's instructions.Empty: Cells transfected with empty vector (pNL3.1). Ctrl: Cells transfected with plasmid DNA (pNL3.1 / Hes1) containing the Hes1 binding region (500 bp) in the NanoLuc expression vector pNL3.1[Nluc / minP] Vector (Promega, N1031). DEX: Cells transfected with pNL3.1 / Hes1 and treated with 1 μmol / L DEX. DEX + Yoda1: Cells transfected with pNL3.1 / Hes1 and treated with 1 μmol / L DEX followed by Yoda1. To correct for transfection efficiency based on firefly luciferase luminescence, pGL4.53[Luc2 / PGK] Vector was transfected at 1 / 10 the amount of each plasmid. N=6. All graphs are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used. F: Effect of DEX on Hes1 gene expression. MLOY4 cells were treated with various concentrations of DEX, and RNA was extracted and qPCR was performed 4 hours later. N = 4. All graphs are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used. Ctrl: Untreated; DEX (0.01): Treated with 0.01 μmol / L DEX; DEX (1): Treated with 1 μmol / L DEX. G: Effect of DEX and Yoda1 on Hes1 gene expression. Human femoral cortical bone organ cultures were treated with DEX and Yoda1 simultaneously. RNA was extracted and qPCR was performed 6 hours later. N=3. All graphs are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. *p<0.05, **p<0.01, ***p<0.001. Ctrl: Untreated; DEX: DEX 1 μmol / L; DEX + Yoda1: DEX 1 μmol / L, Yoda1 10 μmol / L.H: Effect of DEX and Yoda1 on Hes1 phosphorylation. MLOY4 cells were treated with DEX for 24 hours, Yoda1 was added, and cells were harvested 2 hours later. Proteins were extracted and analyzed by Western blotting. Ctrl: Untreated; Yoda1: Yoda1 10 μmol / L; DEX: DEX 1 μmol / L; DEX + Yoda1: DEX 1 μmol / L, Yoda1 10 μmol / L. Comprehensive analysis of the mechanism behind the DEX-dependent decrease in mechanical loading response was performed by integrating RNA sequencing analysis of bone tissue from GIOP and non-GIOP patients with RNA sequencing analysis of mouse bone loading. A: Number and age of patients who underwent human bone tissue RNA sequencing. B: Comparative analysis of bone tissue variation between GIOP and non-GIOP patients. OSTN: osteocrin. C: GO analysis based on genes with variation between GIOP and non-GIOP patients. D: Extraction of genes associated with the DEX-dependent decrease in mechanical loading response. Conditions: Combination and purpose of gene variation dataset conditions used for filtering; Genes: Number of genes meeting the conditions; Criteria: Criteria for filtering by the desired conditions. E: Expression changes of 10 genes associated with DEX-dependent mechanical load response reduction in mouse and human RNA sequencing. Symbol: Gene symbol; log2 FC: Expression change ratio during mouse bone loading; padj Mouse, Human: Significant difference after multiple testing correction; log2 F: Expression change ratio between GIOP and non-GIOP patients. F: RNA sequencing results of Acan, Sox9, and Sfrp2 during mouse bone loading. G: Expression changes of ACAN (Aggrecan), SOX9 (SRY-box9), and SFRP2 (Secreted Frizzled Related Protein 2) between GIOP and non-GIOP patients. The effects of DEX and Yoda1 on Piezo1 expression and osteoblast differentiation in human periosteal cells are shown. A: Changes in Piezo1 protein expression during periosteal cell differentiation induction.Periosteal cells obtained by collagenase treatment from periosteum harvested from human knee joints were cultured and induced to differentiate using differentiation-inducing medium (STEM PRO Osteogenesis kit (Thermo Fisher Scientific, A1007201)). After 3 days, the medium was changed and DEX was added, followed by 24 hours of culture. Yoda1 was added, and 4 hours later, protein was extracted and Western blotting was performed. Undiff.: Periosteal cells in undifferentiated medium. Diff.: Periosteal cells after differentiation induction. DEX: Cells treated with 1 μmol / L DEX after differentiation induction. DEX + Yoda1: Cells treated with 1 μmol / L DEX after differentiation induction, followed 24 hours later by 10 μmol / L Yoda1. B: MC3T3-E1 cells were induced to differentiate using ascorbic acid and β-glycerophosphate. After 3 days, DEX and Yoda1 were added under the same conditions as for periosteal cells, and protein was extracted and Western blotting was performed. C: Human periosteal cells were treated with DEX for 24 hours, followed by Yoda1 treatment. RNA was collected 4 hours later and subjected to qPCR for various genes. DEX: 1 μmol / L DEX; DEX + Yoda1: 1 μmol / L DEX, 10 μmol / L Yoda1. SMOC1: SPARC-related modular calcium binding 1; COL14A1: Collagen Type XIV Alpha 1 Chain. Quantification was performed using the ΔΔCt method with Hprt as the internal standard. N=4. All graphs are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.001. D, E: Human periosteal cells were induced to differentiate into bone. 24 hours later, DEX and Yoda1 were added simultaneously. Medium was changed every 3 days, and alkaline phosphatase staining or ALP assay was performed 14 days later. Undifferentiated: undifferentiated conditions. Differentiated: osteoblast differentiation induction. DEX: 1 μmol / L, Yoda1: 1 μmol / L. The scale bar in the low-magnification images indicates 2 mm and was observed at 1.2x magnification. The scale bar in the high-magnification images indicates 500 μm and was observed at 4x magnification. E: ALP assay results. F: WST assay results.Undifferentiated periosteal cells were treated with 1 μmol / L DEX and Yoda1, and after 24 hours, a WST assay was performed and absorbance at 450 nm was measured. G, H: Alizarin red staining. Human periosteal cells were induced to differentiate into bone, and after 24 hours, DEX and Yoda1 were added simultaneously. The medium was changed every three days, and after 21 days, Alizarin red staining was performed. The drug addition conditions and microscopic observation conditions were the same as for ALP staining. H: After alizarin staining, cells were treated with 5% formic acid at room temperature for 10 minutes, and the absorbance at 415 nm was measured and plotted.

[0019] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0020] "Identity" of an amino acid sequence refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin, Altschul S. F., "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin, Altschul S. F., "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on the BLAST algorithm. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0021] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0022] As used herein, the terms "nucleic acid" and "polynucleotide" are not particularly limited and encompass both natural and artificial nucleic acids. Specifically, in addition to DNA, RNA, and the like, known chemical modifications may be used, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include those in which the phosphate moiety or hydroxy moiety is modified with, for example, biotin, an amino group, a lower alkylamino group (e.g., having 1 to 8 carbon atoms), an acetyl group, etc. Furthermore, BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, may also be used.

[0023] As used herein, "enhancement of Piezo1 and / or Hes1" refers to activation of Piezo1 protein and / or Piezo1 mRNA, or Hes1 protein and / or Hes1 mRNA, resulting in an improved quality and / or quantity.

[0024] The enhancement of the protein or mRNA may be qualitatively and / or quantitatively improved compared to the normal state, or may be qualitatively and / or quantitatively improved compared to a state in which the function or response is reduced, for example, by steroid administration.

[0025] An agent that brings about this state is called an enhancer.

[0026] 2. The active ingredient, a Piezo1 and / or Hes1 enhancer, is not particularly limited as long as it can enhance Piezo1 and / or Hes1, and can be, for example, an enhancer of the expression or function of Piezo1 and / or Hes1, more specifically, an enhancer of the expression of Piezo1 protein and / or Piezo1 mRNA, an enhancer of the function of Piezo1 protein, an enhancer of the expression of Hes1 protein and / or Hes1 mRNA, an enhancer of the function of Hes1 protein, etc.

[0027] Piezo1 is an ion channel that senses mechanical stress and is sometimes referred to as piezo-type mechanosensitive ion channel component 1. The species from which Piezo1 is derived is not particularly limited, and includes animals, such as various mammals, including humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0028] Piezo1 genes from various species and their sequences (amino acid sequences of the protein and nucleotide sequences of the mRNA) are known. Specifically, the human Piezo1 gene is a gene designated by NCBI Gene ID: 9780. Examples of human Piezo1 proteins include proteins consisting of the amino acid sequence set forth in SEQ ID NO: 1 (NCBI Reference Sequence: NP_001136336.2), and examples of human Piezo1 mRNA include mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 (NCBI Reference Sequence: NM_001142864.4). Furthermore, the Piezo1 protein and Piezo1 mRNA may also encompass the aforementioned splicing variants.

[0029] The function of the Piezo1 protein is ion channel activity that senses mechanical stress. As long as the Piezo1 protein retains this function, it may have amino acid mutations such as substitutions, deletions, additions, and insertions. From the viewpoint of less likely impairment of activity, preferred mutations include substitutions, and more preferably conservative substitutions.

[0030] Piezo1 mRNA may also have base mutations such as substitutions, deletions, additions, and insertions, as long as the protein translated from the mRNA has its original function, i.e., ion channel activity that senses mechanical stress. Preferred mutations are those that do not result in amino acid substitutions in the protein translated from the mRNA or those that result in conservative amino acid substitutions.

[0031] Preferred examples of Piezo1 proteins include at least one protein selected from the group consisting of the proteins described in (a) below and the proteins described in (b) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, and (b) a protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having ion channel activity that senses mechanical stress.

[0032] In the above (b), the identity is more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0033] An example of the protein described in (b) above is (b') a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 1, and which has ion channel activity that senses mechanical stress.

[0034] In the above (b'), "plurality" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.

[0035] Preferred examples of Piezo1 mRNA include at least one selected from the group consisting of the mRNA described in (c) below and the mRNA described in (d) below: (c) mRNA comprising the nucleotide sequence shown in SEQ ID NO: 2, and (d) mRNA consisting of a nucleotide sequence having 85% or more identity with the nucleotide sequence shown in SEQ ID NO: 2 and encoding a protein having ion channel activity that senses mechanical stress.

[0036] In the above (d), the identity is more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0037] An example of the mRNA described in (d) above is (d') an mRNA consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence shown in SEQ ID NO: 2, and encoding a protein having ion channel activity that senses mechanical stress.

[0038] In the above (d'), "plurality" means, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, and even more preferably 2 to 10.

[0039] The activity of ion channels sensitive to mechanical stress can be measured by the following method. Cells expressing a target protein (test cells) and cells not expressing the target protein (or with reduced expression) (control cells) are prepared, and Ca ions are then transferred to these cells. 2+ The influx is measured with and without mechanical stress using Fluo-8 Calcium Flux Assay Kit - No Wash (Abcam, ab112129). Measurements can be performed according to the method described in the Examples below. 2+ In control cells, mechanical stress-induced Ca influx was increased, whereas 2+ If there is no increase in influx or the increase is smaller than that in the test cells, it can be determined that the target protein has ion channel activity that is sensitive to mechanical stress.

[0040] Hes1 is a transcription factor sometimes referred to as hes family bHLH transcription factor 1. The species from which Hes1 is derived is not particularly limited, and includes various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer.

[0041] Hes1 genes and their sequences (amino acid sequences of the protein and nucleotide sequences of the mRNA) derived from various biological species are known. Specifically, the human Hes1 gene is designated NCBI Gene ID: 3280. Examples of human Hes1 proteins include the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 (NCBI Reference Sequence: NP_005515.1), and examples of human Hes1 mRNA include the mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 (NCBI Reference Sequence: NM_005524.4). The Hes1 protein and Hes1 mRNA may also include the splicing variants described above.

[0042] The function of the Hes1 protein is transcription factor activity. As long as the Hes1 protein retains this function, it may have amino acid mutations such as substitutions, deletions, additions, and insertions. Mutations are preferably substitutions, more preferably conservative substitutions, from the viewpoint of less likely loss of activity.

[0043] Hes1 mRNA may also have nucleotide mutations such as substitutions, deletions, additions, and insertions, so long as the protein translated from the mRNA retains its original function, i.e., transcription factor activity. Preferred mutations are those that do not result in amino acid substitutions in the protein translated from the mRNA or those that result in conservative amino acid substitutions.

[0044] Preferred specific examples of Hes1 proteins include at least one selected from the group consisting of the proteins described in (e) below and the proteins described in (f) below: (e) a protein comprising the amino acid sequence shown in SEQ ID NO: 3, and (f) a protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having transcription factor activity.

[0045] In the above (f), the identity is more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0046] An example of the protein described in (f) above is (f') a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 3, and which has transcription factor activity.

[0047] In the above (f'), "plurality" means, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, and even more preferably 2.

[0048] Preferred specific examples of Hes1 mRNA include at least one selected from the group consisting of the mRNA described in (g) below and the mRNA described in (h) below: (g) mRNA comprising the base sequence shown in SEQ ID NO: 4, and (h) mRNA consisting of a base sequence having 85% or more identity to the base sequence shown in SEQ ID NO: 4 and encoding a protein having transcription factor activity.

[0049] In the above (h), the identity is more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0050] An example of the mRNA described in (h) above is (h') an mRNA consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence shown in SEQ ID NO: 4, and encoding a protein having transcription factor activity.

[0051] In the above (h'), "plurality" means, for example, 2 to 50, preferably 2 to 30, more preferably 2 to 10, and even more preferably 2 to 5.

[0052] Transcription factor activity can be measured according to the following method. A reporter vector containing a Piezo1 promoter placed upstream of a luciferase coding sequence and an effector vector (test vector) containing an expression cassette for a protein of interest or an empty vector (control vector) are introduced into cells, and a luciferase assay is performed. The assay can be performed according to the method described in the Examples below. If luciferase activity is higher when the test vector is introduced than when the control vector is introduced, the protein of interest can be determined to have transcription factor activity.

[0053] More specifically, Piezo1 and / or Hes1 enhancers include Piezo1 activators (including Piezo1 agonists), LSD1 inhibitors, polynucleotides containing a Piezo1 coding sequence, polynucleotides containing a Hes1 coding sequence, Piezo1 proteins, and Hes1 proteins.

[0054] A Piezo1 activator is a molecule that can enhance the function of a Piezo1 protein and / or Piezo1 mRNA by binding to and activating them, and is not particularly limited thereto. In one embodiment, a Piezo1 activator can also enhance the expression of a Piezo1 protein and / or Piezo1 mRNA.

[0055] Piezo1 activators can be, for example, small molecules, polynucleotides (such as aptamers), or polypeptides, but are preferably small molecules. The molecular weight of a small molecule compound is, for example, 1000 or less, 800 or less, 700 or less, 600 or less, or 500 or less, and the molecular weight is, for example, 100 or more, 150 or more, or 200 or more.

[0056] Numerous Piezo1 activators have been reported in various publications. An exemplary Piezo1 activator is Yoda1. Yoda1 (2-((2,6-Dichlorophenyl)methylsulfanyl)-5-pyrazin-2-yl-1,3,4-thiadiazole) is a small molecule activator developed for the mechanosensitive ion channel Piezo1. Derivatives of Yoda1 (Yoda1 derivatives) can be used in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are used in some embodiments. Exemplary activators are disclosed in U.S. Patent No. 5,629,499 and British J. of Pharmacology 175(1744-1759): 2018. Specific structural examples of Yoda1 and Yoda1 derivatives are shown below (U.S. Patent No. 5,629,499 and British J. of Pharmacology 175(1744-1759): 2018).

[0057]

[0058]

[0059]

[0060] Further examples of Piezo1 activators include Jedi1 and / or Jedi2, or their derivatives (Nature Communications (2018) 9:1300). Jedi1 and Jedi2 have a 3-methylfuran carboxylate structural motif.

[0061] EC of Piezo1 activators against human Piezo1 50 (measured according to the method described in Elife. 2015 May 22:4:e07369) is, for example, 500 μmol / L or less, 300 μmol / L or less, 200 μmol / L or less, 100 μmol / L or less, 50 μmol / L or less, 40 μmol / L or less, or 30 μmol / L or less.

[0062] LSD1 inhibitors are molecules that can inhibit the function of LSD1 (lysine-specific histone demethylase 1A) (demethylation of the fourth methylated lysine residue in histone H3 (H3K4)) or suppress LSD1 expression, but are not particularly limited thereto. It has been reported that LSD1 inhibitors activate the NOTCH signaling pathway, which includes Hes1 (Sci Signal. 2019 Feb 5;12(567):eaau2922.).

[0063] LSD1 inhibitors may be, for example, low molecular weight compounds, polynucleotides (aptamers, siRNAs, antisense polynucleotides, ribozymes, etc.), polypeptides (antibodies, etc.), etc., but are preferably low molecular weight compounds. The molecular weight of low molecular weight compounds is, for example, 1000 or less, 800 or less, 700 or less, 600 or less, or 500 or less, and, for example, 100 or more, 150 or more, or 200 or more.

[0064] Many LSD1 inhibitors have been reported in various literatures. LSD1 inhibitors that are low molecular weight compounds are described in, for example, WO2011 / 131697, WO2012 / 135113, WO2013 / 057322, WO2010 / 143582, WO2011 / 131576, WO2013 / 022047, WO2013 / 025805, WO2014 / 058071, WO2014 / 084298, WO2014 / 085613, WO20 14 / 086790, WO2014 / 164867, WO2014 / 194280, WO2014 / 205213, WO2015 / 021128, WO2015 / 031564, WO201 5 / 089192, WO2015 / 120281, WO2015 / 123465, WO2015 / 123437, WO2015 / 123424, WO2015 / 123408, WO2015 / 134973, WO2015 / 156417, WO2015 / 168466, WO2015 / 181380, WO2015200843, WO2016003917, WO2016 / 00 4105, WO2016 / 007722, WO2016 / 007727, WO2016 / 007731, WO2016 / 007736, WO2016 / 034946, WO2016 / 037 005, CN105541806, WO2016 / 123387, WO2016 / 130952, CN105924362, CN105985265, WO2016 / 161282, CN106045862, CN106045881, WO2016 / 172496, WO2016 / 177656, WO2017 / 004519, and WO2017 / 027678. Specific examples of LSD1 inhibitors that are low molecular weight compounds include TCP, ORY-1001, GSK-2879552, IMG-7289, INCB059872, CC-90011, ORY-2001, and RO7051790. LSD1 inhibitors that are polynucleotides or polypeptides are disclosed, for example, in JP-A-2019-534317.

[0065] IC of LSD1 inhibitors 50is, for example, 500 nmol / L or less, 300 nmol / L or less, 200 nmol / L or less, 100 nmol / L or less, 50 nmol / L or less, 40 nmol / L or less, or 30 nmol / L or less.

[0066] The polynucleotides containing the Piezo1 coding sequence and the Hes1 coding sequence are not particularly limited, as long as they are capable of expressing the Piezo1 protein and the Hes1 protein in the target animal. Examples of such polynucleotides include mRNA and expression cassettes. Typically, the expression cassette contains a polynucleotide containing a promoter sequence and the Piezo1 protein coding sequence or the Hes1 protein coding sequence (and, if necessary, a transcription termination signal sequence). The expression cassette can also be in the form of a vector.

[0067] The expression vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; and viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus.

[0068] The promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter, etc. In addition, various promoters that can be induced by drugs and various promoters that can be induced in a tissue-specific manner (for example, bone tissue-specific manner) can also be used.

[0069] In addition to the above, the expression vector may contain other elements that an expression vector can contain. Examples of other elements include an origin of replication and a drug resistance gene. The drug resistance gene is not particularly limited, but examples include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, and a puromycin resistance gene.

[0070] The polynucleotides can be easily obtained by known genetic engineering techniques, such as PCR, restriction enzyme digestion, and DNA ligation.

[0071] The Piezo1 protein and Hes1 protein may be chemically modified as long as they retain their functions.

[0072] Piezo1 protein and Hes1 protein have a C-terminus containing a carboxyl group (-COOH) or a carboxylate (-COO － ), amide (-CONH2) or ester (-COOR).

[0073] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C including α-naphthylmethyl 1－2 C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0074] The Piezo1 protein and Hes1 protein may have a carboxyl group (or carboxylate) other than that at the C-terminus amidated or esterified. In this case, the ester may be, for example, the C-terminal ester described above.

[0075] Furthermore, in the Piezo1 protein and Hes1 protein, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a formyl group, an acetyl group, or the like). 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., hydroxyl group, sulfanyl group, amino group, imidazolyl group, indolyl group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.);1－6 C such as alkanoyl group 1－6 Also included are those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are bound.

[0076] The Piezo1 protein and Hes1 protein may be tagged with a known protein tag, such as a histidine tag, a FLAG tag, or a GST tag, as long as they have anticancer activity.

[0077] The Piezo1 protein and Hes1 protein may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0078] The Piezo1 protein and Hes1 protein may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0079] The Piezo1 protein and Hes1 protein can be obtained according to known methods, such as chemical synthesis, purification from mammalian cells or tissues (e.g., serum), or purification from a transformant containing a polynucleotide encoding the Piezo1 protein or Hes1 protein. In a preferred embodiment of the present invention, the Piezo1 protein and Hes1 protein are chemically synthesized proteins. When obtained by purification from a transformant, the transformant is not particularly limited as long as it is a cell that can express the Piezo1 protein or Hes1 protein from a polynucleotide containing the Piezo1 protein or Hes1 protein-encoding sequence. Various cells, such as bacteria such as Escherichia coli, insect cells, and mammalian cells, can be used.

[0080] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM Examples of Sf cells that can be used include Sf9 cells (ATCC CRL1711) and Sf21 cells. Examples of animal cells that can be used include monkey COS-7 cells, monkey Vero cells, Chinese hamster CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells.

[0081] The above-mentioned active ingredients can enhance Piezo1 and / or Hes1 in bone tissue (preferably bone cells).

[0082] The above-mentioned active ingredients can be used alone or in combination of two or more.

[0083] 3. Uses A Piezo1 and / or Hes1 enhancer can be used as an active ingredient in an agent for preventing or treating steroid-induced osteoporosis. In one aspect, the present invention relates to an agent for preventing or treating steroid-induced osteoporosis, comprising a Piezo1 and / or Hes1 enhancer. In another aspect, the present invention relates to an agent for use in combination with a steroid or for administration to a subject who has previously received steroids, comprising a Piezo1 and / or Hes1 enhancer. Hereinafter, these may be collectively referred to as the "agent of the present invention."

[0084] Glucocorticoid-induced osteoporosis is osteoporosis caused by the administration of steroids, which leads to bone fragility (reduction in bone mass, reduction in bone microstructure, etc.) and increases the risk of fragility fractures.

[0085] In one embodiment, the agent of the present invention can be used to suppress weakening of not only cancellous bone but also cortical bone (particularly, to suppress weakening of cortical bone), thereby enabling it to exert a preventive effect against fractures such as vertebral fractures and hip fractures.

[0086] The steroid drug that causes steroid-induced osteoporosis is not particularly limited, and examples thereof include corticosteroids, more specifically, prednisolone, methylprednisolone, betamethasone, dexamethasone, triamcinolone, hydrocortisone, clobetasone, fluorometholone, triamcinolone, triamcinolone acetonide, alclometasone, fluocinolone acetonide, diflucortolone, fludroxycortide, fluocinonide, budesonide, diflorasone, amcinonide, mometasone furan, beclomethasone, fluticasone, fluticasone furan, difluprednate, clobetasol, cortisone, ciclesonide, deprodone, fludrocortisone, etc. In one embodiment, the steroid drug is an oral medication, and in another embodiment, it is an external medication.

[0087] A subject with a history of steroid administration is, for example, a subject who has received steroids to such an extent that steroid administration is suspected of causing or potentially causing steroid-induced osteoporosis. For example, the subject may be a subject who has received steroids for, for example, one week or more, two weeks or more, one month or more, two months or more, three months or more, four months or more, five months or more, or six months or more. The subject may also be a subject who is currently receiving steroids or who has been receiving the last steroid for, for example, one week or more, two weeks or more, one month or more, two months or more, three months or more, four months or more, five months or more, or six months or more since the last administration of steroids.

[0088] As used herein, "treatment" can include concepts such as cure, remission, alleviation, mitigation, and suppression of progression of symptoms. Furthermore, "prevention" can include concepts such as not only preventing the onset of a disease, but also delaying the onset of the disease and suppressing symptoms once the disease has occurred.

[0089] The content of the active ingredient in the agent of the present invention can be appropriately determined taking into consideration the type of target disease, the desired therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. For example, the content of the active ingredient in the agent of the present invention can be about 0.0001 to 100 parts by weight, assuming that the total amount of the agent of the present invention is 100 parts by weight.

[0090] The administration form of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Parenteral administration is preferred. Dosage forms for oral and parenteral administration and their preparation methods are well known to those skilled in the art, and can be prepared according to conventional methods by mixing the active ingredient with a pharmaceutically acceptable carrier, etc.

[0091] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions, creams, and gels), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, and LNP (Lipid Nano Particle) preparations. For example, injectable preparations are prepared by dissolving the active ingredient in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. The agent of the present invention can also be prepared as a lyophilized preparation for immediate use.

[0092] The agent of the present invention may further contain other drugs that are effective in treating or preventing diseases.

[0093] The agent of the present invention can contain any carrier or additive, for example, a pharmaceutically acceptable carrier or additive.

[0094] Pharmaceutically acceptable carriers and additives include, but are not limited to, excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; flavorings such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersing agents such as surfactants; diluents such as water and physiological saline; base waxes, etc.

[0095] The dosage of the agent of the present invention can be determined based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological findings such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the agent is administered as part of a combination of other drugs. The dosage of the agent of the present invention can be, for example, approximately 1 μg / kg (body weight) to 10 g / kg (body weight) per day. The administration schedule of the agent of the present invention can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered 1 to 5 times per day to 1 month.

[0096] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0097] In this specification, room temperature refers to 20 to 30°C.

[0098] (1) Materials and Methods <Cell Culture> MLOY4 cells (Karafest, CVCL_M098) were cultured in a medium containing αMEM (Nacalai Tesque, 21444-05), 5% FBS (Hyclone, SH30396.03), 5% FCS (Gibco, 16010-159), and 1% antifungal antibiotic solution (Sigma, A5955), and were cultured on a 10 cm Rat Collagen Type I-coated dish (Iwaki, 4020-010).

[0099] <Cell imaging> 2 × 10 MLOY4 cells were plated on a type I collagen-labeled 96-well plate (Iwaki, 4860-010). 3Cells were seeded per well, and after 24 hours, dexamethasone (DEX) (Sigma, D1756-100MG, final concentration 1 μmol / L) and Yoda1 (Cayman, 21904, final concentration 1 or 3 μmol / L) were added. After 72 hours, the supernatant was collected and washed with PBS. The cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing once with PBS, the cells were permeabilized with 0.1% triton in PBS for 2 minutes and then blocked with 3% BSA in PBS (30 minutes at room temperature). Rhodamine-labeled phalloidin (Thermo Fisher Scientific, R415) was diluted 200-fold in 0.2% BSA in PBS, and 50 μL of the solution was added per well. The cells were incubated for 2 hours at room temperature or overnight at 4°C, after which they were washed three times with PBS. 50 μL of Hoechst 33258 (Dojindo Chemical Co., H341) was added and incubated at room temperature for 15 minutes to stain the nuclei. After washing with PBS, 200 μL of PBS was added to each well, and image analysis was performed using an In cell Analyzer 6000 (GE Healthcare) (40x magnification).

[0100] <Cell image analysis> Using the analysis software (In Cell Developer) attached to the In Cell Analyzer, the morphological changes of the observed cells (number of actin intersections) were randomly calculated. The cells detected for calculation were counted only for nucleated live cells stained with Hoechst 33258.

[0101] <Ca 2+Influx Measurement: MLOY4 cells were electroporated into a Rat Collagen Type I-coated 96-well plate (Iwaki, 4860-010) with Piezo1 siRNA (Thermo Fisher silencer select siRNA, Piezo1 s107968) or negative control RNA (Thermo Fisher silencer select 14390843). 24 hours later, DEX (Sigma, D1756-100MG) was diluted with medium to a final concentration of 0.1 or 1 μmol / L, and the medium was replaced with a new one for DEX treatment. 72 hours later, the medium was removed and 100 μL of 1% FBS was added. Following the instructions of the Fluo-8 Calcium Flux Assay Kit - No Wash (Abcam, ab112129), 100 μL of Fluo-8 dye loading solution was added and incubated for 30 minutes in an incubator. After leaving the plate at room temperature for 30 minutes, 3.10 μmol / L of Yoda1 (Cayman, 21904) was added, and measurements were performed immediately for 5 minutes using a fluorescence plate reader (TriStar5, Berthold).

[0102] <Electroporation> MLOY4 cells were cultured and detached with TrypLE express (Gibco). After mixing with OptiMEM (Gibco), the cells were counted and centrifuged to obtain a cell count of 0.5-1 x 10 6 The cells were suspended in OptiMEM (Gibco) at a concentration of 100 μL. 4 pmol of siRNA (Thermo Fisher silencer select siRNA, Hes1 s67461, Piezo1 s107968, siRNA negative control #14390843) was added to the cell mixture, mixed, and transferred to a NEPA21 (Nepa Gene) cuvette. Electroporation was performed at a voltage of 150 V, with a pulse width of 7.5 ms and a pulse interval of 50 ms. The cells were then diluted 10-fold with αMEM 5% FCS 5% FBS and plated at 5 × 10 cells per 96-well plate. 4After seeding at 1000 cells / well and culturing at 37°C for 48 hours, RNA was recovered, followed by reverse transcription and qPCR according to the instructions for the CellAmp Direct TB Green RT-qPCR kit (Takara Bio, 3735S).

[0103] <RNA extraction from cultured cells> After washing various cultured cells twice with PBS, 800 μL of TRIzol (Ambion, 15596018) was added and the cells were collected with a scraper. 160 μL of chloroform was added, mixed well, and left to stand for 15 minutes. After centrifugation at 15,000 g, 4°C, for 5 minutes, the aqueous layer was collected and mixed with an equal volume of ethanol. RNA was extracted and purified using the Direct-zol micro kit (Zymoresearch, R2052) according to the manufacturer's instructions.

[0104] <RT Reaction / qPCR> RT reactions (12.5 μL) were performed using 100 ng of RNA extracted from cells and tissues according to the instructions for the ReverTra Ace® qPCR RT Kit (TOYOBO, FSQ101). qPCR was performed using 2 μL of the RT reaction mixture and Fast SYBR™ Green Master Mix (Thermo Fisher Scientific, 4385612). The Step One Real-Time PCR System (Thermo Fisher Scientific) was used for measurement. Analysis was performed using the instrument's software based on the ΔΔCt method using the HPRT gene as a standard.

[0105] <RNA and protein extraction from mouse and human femoral cortical bone> Mouse and human bones stored at -80°C were ground to powder in liquid nitrogen using a freezer mill (SPEX, 6775). 800 μL of TRizol (Ambion, 15596018) was added to dissolve the bone tissue. 160 μL of chloroform was added, mixed well, and allowed to stand for 15 minutes. After centrifugation at 15,000 g for 5 minutes at 4°C, the aqueous layer was collected and mixed with an equal volume of ethanol. RNA was extracted and purified using a Direct-zol micro kit (Zymoresearch, R2052) according to the manufacturer's instructions. For protein extraction, bones were ground to powder in liquid nitrogen, and 800 μL of protein extraction buffer (50 mmol / L Tris pH 7.5, 0.1% SDS, 250 mmol / L NaCl, 2 mmol / L DTT, 0.5% NP40) was added per well and incubated at 4°C for 30 minutes. After centrifugation at 12,000 g for 10 minutes, the supernatant was collected as the protein extract and quantified using Pierce Bradford Plus Protein Assay Kits (Thermo Fisher Scientific, 23236).

[0106] <Cortical bone organ culture from human femoral neck> Femoral heads and necks from patients undergoing total hip arthroplasty were preserved in PBS immediately after surgery and washed with PBS on the same day. The femoral necks were then cross-sectioned using a mini band saw (PROXXON). The cancellous bone was removed using a Luer tube. The cortical bone was separated into 1-2 cm cubes and cultured overnight in 10 cm dishes (coated with type I collagen) in αMEM 10% FBS. The bones were then transferred to 6-well plates and cultured in medium supplemented with Yoda1 (final concentration 10 μmol / L), DEX (final concentration 1 μmol / L), or both DEX and Yoda1. The bones were then cultured at 37°C for 6 hours, frozen in liquid nitrogen, and stored frozen at -80°C until RNA extraction.

[0107] <Collection and culture of human bone tissue-derived periosteal cells> Periosteum cells were collected from bone tissue obtained during total knee replacement surgery, removing soft tissue. They were washed twice with PBS and then finely cut in DMEM / F-12, GlutaMAX supplement (Gibco, 10565018), 10% FBS, and 0.2% Type 2 collagenase (Worthington, LS004176). They were then incubated overnight at 37°C. After passing through a 70 μm filter, they were centrifuged at 500×G for 3 minutes. The entire volume was seeded onto a collagen-coated 6 cm dish (Iwaki, 4010-010). They were cultured in DMEM / F-12, GlutaMAX supplement, and 10% FBS. The medium was replaced after 4 days. The medium was changed every 3 days, and when the cells reached confluence, they were detached using TrypLE (Gibco 12605010) and seeded onto collagen-coated 10 cm dishes (Iwaki, 4020-010). The cells were cultured until P3 and then cryopreserved in a STEM Cell Banker (Zenyaku Kogyo, CB045). Periosteal cells were used for experiments until P13.

[0108] <Bone differentiation induction, Alizarin red staining, ALP staining, ALP activity (human periosteal cells)> Periosteal cells were collected from the distal femur of a human and cultured at 3 x 10 4Cells were seeded onto collagen-coated 24-well plates (Iwaki, 4820-010) at 100 cells / well. After 24 hours, the medium was replaced with osteogenic differentiation medium (STEM PRO Osteogenesis kit (Thermo Fisher Scientific, A1007201)). For the DEX-containing condition, DEX and Yoda1 were diluted in osteogenic differentiation medium to a final concentration of 1 μmol / L and 1 μmol / L, respectively. The medium was replaced every 3 days. For ALP staining, after 12 days, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, washed three times with water, and then stained with ALP staining solution (6.4% NBT, 3.2% BCIP (Promega, S3771), 20 mmol / L Tris-HCl pH 9.0, 40 mmol / L NaCl, 1 mmol / L MgCl2) at 37°C for 10 minutes. For ALP activity, after 12 days of differentiation, cells were washed twice with PBS and then protein was extracted using MPER (Thermo Fisher Scientific, 78501) and measured according to the instructions for the Lab Assay ALP (Fujifilm, 633-51021). The incubation time was 30 minutes, and ALP activity was converted to units. Protein concentration was measured and normalized using the Pierce Rapid Gold BCA Protein assay (Thermo Fisher Scientific, A53226). For alizarin staining, after 20 days of differentiation, cells were washed twice with PBS, fixed with 4% paraformaldehyde, washed three times with water, and stained with 1% alizarin red solution, pH 6.3-6.4 (Muto Chemical, 17972), for 10 minutes. After rinsing with water, cells were observed under a stereomicroscope (Nikon, AZ-100, 0.5x magnification) and an inverted microscope (Nikon, Eclipse Ts2, 20x magnification). After alizarin staining, the sample was extracted with 5% formic acid for 10 minutes, and then absorbance was measured at a wavelength of 415 nm.

[0109] <RNA sequence analysis> RNA extracted from mice was used to construct a library for sequence analysis using the SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara Bio, 634412), and sequence analysis was then performed using Hi Seq 3000 (Illumina) with 30 million paired reads.

[0110] <Data analysis> Gene variation analysis was performed on each Fastq file sent by the Research Institute for Microbial Diseases, Osaka University, using RaNA seq (https: / / ranaseq.eu / home). Statistical analysis was performed using DEseq2 with a Wald test and a p < 0.05 value considered significant. When analyzing multiple RNA seq data in combination, multiple testing was corrected using the false discovery ratio (FDR). Gene Set Enrichment Analysis (GESA) analysis was performed using the R (Ver. 4.3.1) program.

[0111] <Dexamethasone and Yoda1 Administration> Three-month-old male C57BL / 6J (Jackson) mice received subcutaneous administration of DEX (SIGMA, D1756-100MG) at 0.2 mg / mL in injection water at 5 mL / kg (1 mg / kg) once daily, 5 days a week, for 28 days. On day 29, mice were necropsied and femurs were collected. Yoda1 (TOCRIS, 5586) was dissolved in DMSO to prepare a 40 mmol / L stock solution, which was stored in aliquots at -30°C. On the day of administration, the solution was diluted to 0.5 mmol / L with 5% ethanol and administered intraperitoneally at 10 mL / kg (5 μmol / kg) once daily, 5 days a week. Vehicle mice received subcutaneous and intraperitoneal administration of injection water and 5% ethanol at the same doses and frequencies as DEX and Yoda1. In addition, the DEX-only treatment group received intraperitoneal administration of 5% ethanol at the same dose and frequency as Yoda1. For bone morphometry, alizarin complexone (Dojindo Chemical Industries, Ltd., 348-00093) was dissolved in 2% sodium bicarbonate (Fujifilm Wako Pure Chemical Industries, Ltd.) at 6 mg / mL and administered subcutaneously at 5 mL / kg on day 22. After four days of labeling, calcein (SIGMA, C0875) was diluted with saline at 2 mg / mL and administered at 5 mL / kg on day 27. After one day of labeling, on day 29, blood was collected from the inferior vena cava under isoflurane anesthesia, and femurs and tibias were collected and used for various experiments.

[0112] <Bone Loading> Mice under isoflurane anesthesia were vertically fixed in the tibia, and a load ranging from -1 N to -13.5 N was applied for 0.1 seconds every 10 seconds using an Electroforce 5500 (EA) for 40 cycles. Bone loading was performed 2-3 times per week, with 2-3 days between sessions, for a total of 5 sessions over a 2-week period. Bone loading was performed only on the left paw of each mouse (the loading group), while the right paw served as the control and was used for micro-CT analysis. For bone morphometry, tetracycline hydrochloride (SIGMA T-7660) was dissolved in saline at 2 mg / mL and administered subcutaneously at 10 mL / kg on day 6. After 2 days of labeling, calcein (SIGMA, C0875) was diluted in saline at 2 mg / mL and administered at 5 mL / kg on day 9. After one day of labeling, on the 11th day, blood was collected from the inferior vena cava under isoflurane anesthesia, and then femurs and tibiae were collected and used for various experiments.

[0113] <Micro-CT analysis> Tibiae harvested from mice were fixed in 70% ethanol and bone structure analysis was performed using a SkyScan 1272 (Bruker). Trabecular bone volume (BV / TV) was calculated by 3D analysis of the area 1 mm from the epiphyseal line of the proximal tibia. Cortical bone width was calculated by 3D analysis of the area 4-5 mm from the epiphyseal line of the proximal tibia and 1 mm distal to it. Images were acquired at 8 microns per pixel, with each image captured at 1.2 degrees. The images were reconstructed using the analysis software provided with the SkyScan and analyzed automatically.

[0114] <Biomechanics analysis (three-point bending test)> Femurs collected from mice were placed in PBS and stored at -80°C until use in the experiment. Prior to the three-point bending test, micro-CT analysis was performed to measure the surface area and diameter of the femur. For the three-point bending test, the femur was fixed at two points at 8 mm and moved at 0.1 mm / s from the midpoint to 2 mm using an Electroforce5500 (TA). A load-displacement curve was created and various parameters were calculated. The jig used for the three-point bending test was custom-made by Square Medical.

[0115] <Preparation and sectioning of paraffin-embedded cortical bone blocks from human femoral necks> Femoral heads and necks from patients undergoing total hip arthroplasty were stored in PBS immediately after surgery and washed with PBS on the same day. The femoral necks were then cut into cross sections using a mini band saw (PROXXON). The tissue surrounding the calcar was infiltrated in 10% formalin solution and fixed for at least one day. The sections were then treated with water, 70, 80, and 90% ethanol for one hour each, followed by dehydration overnight in 100% ethanol. If necessary, sections were degreased with 50% ethanol and 50% chloroform for two hours, followed by further dehydration and degreasing overnight in 100% ethanol. The sections were then treated with 90, 80, and 70% ethanol, and water for one hour each, followed by solution replacement. The sections were then treated with 0.5 mol / L EDTA. The tissue was decalcified using M-60 (SOFTEX), which was changed every three days for the first three days, followed by X-ray imaging. After decalcification was complete, the tissue was treated with running water for more than two hours, followed by treatment with 70, 80, and 90% ethanol for 30 minutes each. Paraffin infiltration was performed using a specimen pretreatment device, Tissue-Tek VIP 6 AI (Sakura Fine Tech). After infiltration was complete, the paraffin and bone tissue were placed in an embedding container, cooled, and tissue blocks were prepared.

[0116] A sliding microtome REM-710 (Yamato Koki) equipped with a microtome blade (Feather A35TYPE) was used to thinly slice the paraffin-embedded block tissue cooled to 4°C at 3.5 μm intervals. The tissue for immunostaining was spread on a non-peeling coated slide glass (Matsunami, CRE-01), and the tissue for HE staining was spread on a Fine Frost Blue slide glass (Matsunami, FF-004). The tissue was stretched on a hot plate at 40°C for approximately 1 hour and then dried overnight in a dryer at 50°C.

[0117] <Preparation and sectioning of paraffin-embedded cortical bone blocks from mouse femoral necks> Femurs were removed from euthanized mice and fixed in 10% formalin solution for at least one day. They were then treated with water, 70, 80, and 90% ethanol for one hour each, followed by overnight dehydration and defatting in 100% ethanol. The solution was then replaced with 90, 80, and 70% ethanol, and water for one hour each. This was followed by treatment with 0.5 mol / L EDTA. Decalcification was performed for one week, with the solution changed every three days thereafter. After decalcification, the bones were treated with running water for at least two hours, followed by treatment with 70, 80, and 90% ethanol for 30 minutes each. Paraffin infiltration was performed using a Tissue-Tek VIP 6 AI specimen pretreatment system (Sakura Fine Tech). After infiltration, the paraffin and bone tissue were placed in an embedding container and cooled to prepare tissue blocks.

[0118] A sliding microtome REM-710 (Yamato Koki) equipped with a microtome blade (Feather A35TYPE) was used to thinly slice the paraffin-embedded block tissue cooled to 4°C at 3.5 μm intervals. The tissue for immunostaining was spread on a non-peeling coated slide glass (Matsunami, CRE-01), and the tissue for HE staining was spread on a Fine Frost Blue slide glass (Matsunami, FF-004). The tissue was stretched on a hot plate at 40°C for approximately 1 hour and then dried overnight in a dryer at 50°C.

[0119] <Preparation and observation of specimens for bone morphometry> After autopsy, tibiae were trimmed and immersed in 70% ethanol in a 2 mL light-proof tube overnight, then replaced with a new solution. After 2-3 days, the solution was replaced with another 70% ethanol solution. The bones were dehydrated overnight in 30% sucrose. According to the instructions for the osteoresin embedding kit (Fujifilm Wako Pure Chemical Industries, Ltd., 297-56001), a methyl methacrylate monomer resin polymerization agent was prepared and the dehydrated tibiae were immersed in this solution. A Cryomold No. 3 (Sakura Finetech, 4557) was placed in isopentane cooled in liquid nitrogen, and the resin-embedded bone tissue was placed in an SCEM (section-lab, C-EM001) to prepare a frozen block. The block was then wrapped in aluminum foil and stored at -80°C. The bone specimens were sliced ​​at 5 μm using a cryostat (Leica, CM3050S), and then sliced ​​with 2.0 cm of Clyofilm Type 2C (9) onto frosted blue slides (Matsunami, FF-004). ProLong Diamond Antifade Mountant (Invitrogen, P36961) was added and the specimens were mounted. Fluorescence observation was performed using an FV3000 microscope (Evident).

[0120] <Tissue Immunostaining (Osteocalcin, Piezo1)> Thin tissue slides were immersed in xylene for 5 minutes, repeated four times. Then, they were immersed in 100% ethanol for 5 minutes, repeated twice. They were immersed in 95, 80, and 70% ethanol for 2 minutes, and then washed in running water for 5 minutes. Proteinase K was added dropwise and activated at room temperature for 6 minutes. Three 3-minute washes were repeated in PBST. Endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide for 10 minutes, followed by three 3-minute washes in PBST. The tissue sections were surrounded by a blocking pen and blocked with Blocking One Histo (Nacalai Tesque, 06349-64) for 1 hour at room temperature. Osteocalcin primary antibody (Takara Bio, M188) diluted 200-fold in PBS containing 5% Blocking One was added dropwise and incubated at room temperature for 1 hour. The sections were washed three times for 3 minutes with PBST. Simple Stain Mouse MAX-PO (Nichirei Biosciences, H2205) was added as a secondary antibody and incubated at room temperature for 30 minutes. The sections were washed three times for 3 minutes with PBST. Simple Stain DAB solution (Nichirei Biosciences, 415171) was added and incubated until the tissue developed color (approximately 2-5 minutes). The reaction was stopped by immersion in water, and the nuclei were stained by immersion in hematoxylin for 10 seconds and washed with water for 5 minutes. The sections were then immersed in 70, 80, 95, and 100% ethanol for 1 minute. They were then immersed again in 100% ethanol for 1 minute, followed by immersion in xylene for 5 minutes three times. The sections were then mounted on slides and allowed to dry for at least 30 minutes. Images of the slides were captured using an Aperio scans cope CS2 (Leica). For Piezo1 immunostaining, cells were treated with Tris-EDTA buffer, pH 9.0 (Abcam, ab93684) at 80°C for 30 minutes. The dilution conditions for the Piezo1 primary antibody (Protein Tech, 15939-1) were the same as for osteocalcin.

[0121] <Tissue Immunostaining (SOST)> Thin tissue slides were immersed in xylene for 5 minutes, repeated four times. Next, they were immersed in 100% ethanol for 5 minutes, repeated twice. They were then immersed in 95, 80, and 70% ethanol for 2 minutes, followed by 5-minute rinses in running water. Endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide for 10 minutes, and then washed with PBST for 5 minutes. The tissue sections were surrounded by a blocking pen, and normal goat serum included with ABC System VECTASTAIN ABC-AP (VECTOR, PK4005) was added dropwise for 20 minutes at room temperature for blocking. After washing with PBST for 5 minutes, SOST primary antibody (R&D Systems, AF1589) diluted 20-fold in PBST was added dropwise and incubated for 1 hour at room temperature. The slides were then washed with PBST for 5 minutes. The biotin-labeled goat IgG secondary antibody provided with the kit was added as a secondary antibody and incubated for 30 minutes at room temperature. The sample was then washed with PBST for 5 minutes. The ABC reagent provided with the kit was added and incubated for 30 minutes at room temperature. After washing with DW for 5 minutes, Simple Stain DAB solution (Nichirei Biosciences, 415171) was added and incubated until the tissue developed color (approximately 10 minutes). The reaction was stopped by immersion in water, and the nuclei were stained by immersion in hematoxylin for 10 seconds and then washed with water for 5 minutes. The sample was then immersed in 70, 80, 95, and 100% ethanol for 1 minute. After immersion in 100% ethanol for 1 minute, the sample was then immersed in xylene for 5 minutes, three times. The sample was then mounted on a slide and allowed to dry for at least 30 minutes. Images of the slides were captured using an Aperio scanscope CS2 (Leica).

[0122] <Ploton silver staining> Paraffin-embedded bone sections were deparaffinized with xylene and ethanol, washed with water, and then incubated in silver staining solution (33% silver nitrate, 0.7% gelatin type B (Nitta Gelatin, 211116), 0.4% formic acid) for 55 minutes at room temperature in the dark. After three 5-minute washes with water, sections were incubated in 5% sodium thiosulfate solution at room temperature for 10 minutes. After three 5-minute washes with water, nuclei were stained with hematoxylin for 10 seconds. After a 5-minute wash with running water, sections were immersed in 70, 80, 95, and 100% ethanol for 1 minute. After a 5-minute immersion in 100% ethanol, sections were immersed in xylene for 5 minutes three times. The sections were then mounted on slides and dried for at least 30 minutes. Images of the slides were captured using an Aperio scanscope CS2 (Leica).

[0123] <Bone phalloidin staining> Femurs were collected from euthanized mice and fixed in 4% paraformaldehyde for 1-2 days, then washed in PBS for 1 hour. Decalcified with 0.5 mol / L EDTA for 14 days, changing the solution every 3-4 days. Washing with PBS for 1 hour was repeated three times at 4°C. The epiphysis was cut, and the tip of the 0.6 mL tube was cut and centrifuged at 8,000 G for 1 minute to flush out the bone marrow. The bones were immersed in 10% sucrose in PBS and left at room temperature for 15 minutes. The bones were immersed in 15% sucrose in PBS and left at room temperature for 15 minutes. The bones were immersed in 30% sucrose in PBS and left at 4°C overnight. A cryomold No. 3 (Sakura Finetech, 4557) was placed in isopentane cooled in liquid nitrogen, and the femur was placed in OCT compound (Sakura Finetech, 4583) to prepare a frozen block. The block was then wrapped in aluminum foil and stored at -80°C. Sections were cut at 40 μm thickness using a cryostat and collected in 24-well plates containing PBS. The OCT was removed by washing three times for 5 minutes with PBS. Blocking was performed overnight at 4°C with Blocking One Histo (Nacalai Tesque, 06349-64). After three 5-minute washes with PBST, 200 μL of Alexa Fluor 488 Phalloidin (Invitrogen, A12379) diluted 400-fold with 5% Blocking One Histo was added and incubated overnight at 4°C. After washing three times for 5 minutes with PBST, 200 μL of DAPI (Dojindo Chemical Industries, Ltd., 340-07971) diluted 250-fold in PBS was added and incubated at room temperature for 30 minutes. Five 5-minute washes with PBS were repeated five times. The bone was collected with a brush, placed on a slide, and mounted with a drop of ProLong Diamond Antifade Mountant (Invitrogen, P36961). Fluorescence observation was performed using an FV3000 (Evident).

[0124] <TRAP staining> Paraffin-embedded thin sections were immersed in xylene for 5 minutes, repeated four times. Next, they were immersed in 100% ethanol for 5 minutes, repeated twice. They were then immersed in 95, 80, and 70% ethanol for 2 minutes, and then washed in running water for 5 minutes. TRAP staining was performed according to the instructions for the TRAP staining kit (Cosmo Bio, AK04F). After staining, the reaction was stopped with water, and the sections were mounted in Glycergel Mounting Medium (DAKO, C0563) and dried for at least 30 minutes. Images of the slides were captured using an Aperio scanscope CS2 (Leica).

[0125] <HE staining> The sliced ​​tissue slides were immersed in xylene for 5 minutes, repeated four times. Next, they were immersed in 100% ethanol for 5 minutes, repeated twice. They were immersed in 95, 80, and 70% ethanol for 2 minutes and then washed in running water for 5 minutes. They were immersed in hematoxylin for 10 minutes and then washed in running water for 10 minutes. They were immersed in eosin for 1 minute and then washed in water for 1 minute. They were immersed in 70, 80, 95, and 100% ethanol for 1 minute. They were then immersed again in 100% ethanol for 5 minutes, followed by immersion in xylene for 5 minutes, repeated three times. They were then mounted on a slide and allowed to dry for at least 30 minutes. Images of the slides were captured using an Aperio scans cope CS2 (Leica).

[0126] <TUNEL staining> Thin-sectioned tissue slides were immersed in xylene for 5 minutes, repeated four times. Next, they were immersed in 100% ethanol for 5 minutes, repeated twice. They were then immersed in 95, 80, and 70% ethanol for 2 minutes each, and rinsed with running water for 5 minutes. Apoptotic cells were detected according to the instructions of the MEBSTAIN Apoptosis TUNEL Kit Direct (MBL, 8445). PBS was added to the tissue and heated at 37°C for 30 minutes. Proteinase K solution was added and incubated at 37°C for 30 minutes, followed by four 2-minute washes with water. TdT buffer II was added to the tissue and incubated at room temperature for 10 minutes, followed by TdT solution and incubation at 37°C for 60 minutes. The slides were immersed in TB solution, incubated at room temperature for 15 minutes, and then washed four times with purified water for 2 minutes each. After mounting with a fluorescent anti-fading mounting medium and placing a cover glass, the sections were observed under a confocal laser microscope (Evident, FV3000).

[0127] <CUT & RUN Assay> MLOY4 cells were cultured and detached with TrypLE express (Gibco, 12605010). The cells were mixed with OptiMEM (Gibco, 31985062), counted, centrifuged, and collected at 1 x 10 6 The cells were suspended at a concentration of 100 μL. 4 pmol of siRNA (Thermo Fisher silencer select siRNA, Hes1 s67461, Piezo1 s107968) was added to the cell mixture, mixed, and transferred to a NEPA21 (Nepa Gene) cuvette. Electroporation was performed at a voltage of 150 V, a pulse width of 7.5 ms, and a pulse interval of 50 ms. The cells were then diluted 10-fold with a solution diluted with αMEM to 5% FCS and 5% FBS, and plated at 1 × 10 cells per 10 cm collagen-coated dish plate. 6 Cells were seeded on a dish. Control, DEX, and Yoda1-treated groups were electroporated with negative control siRNA. After 24 hours, cells were treated with 1 μmol / L DEX or 10 μmol / L Yoda1 and cultured for another 24 hours. Cells under each condition were harvested and cultured at 4 × 10 5The cells / condition were used for the CUT & RUN assay. The CUT & RUN assay was performed according to the instructions for the CUT & RUN Assay Kit (Cell Signaling, 86652S). Anti-Hes1 antibody (Adipogen, AG-20T-0400) was used at a 50-fold dilution for enrichment. The enrichment reaction was performed at 4°C for 2 hours. The enriched chromatin was extracted with phenol-chloroform and ethanol-precipitated according to the kit's instructions, and dissolved in 50 μL of TE 8.0. qPCR was performed in a 20 μL reaction volume using 2 μL per condition. Two primers were designed within the predicted Hes1 binding region (Primer 1 and 2).

[0128] <Protein extraction and protein quantification> MLOY4 cells were cultured in a collagen-coated 6-well plate (Iwaki, 4810-010) at 3 x 10 ５ Cells were seeded per well and cultured for 24 hours, then treated with 1 μmol / L DEX. 24 hours later, they were treated with 5 μmol / L Yoda1. For CaM kinase II inhibition, KN93 (CAYMAN, 21472) was diluted in medium to a final concentration of 1 or 3 μmol / L and added 2 hours before Yoda1 addition. After washing with PBS, cells were added to 70 μL / well of a 1:100 solution of RIPA buffer (Thermo Fisher Scientific, 89901) supplemented with Protease / Phosphatase Inhibitor Cocktail (100x) (Cell signaling, #5872). For Piezo1, cells were harvested by adding 70 μL / well of 50 mmol / L Tris pH 7.5, 0.1% SDS, 250 mmol / L NaCl, 2 mmol / L DTT, and 0.5% NP40. After sonication on ice for 10 minutes using a Bioruptor II (BM Instruments), the cells were centrifuged at 12,000 g for 5 minutes. The supernatant was collected as a protein extract and quantified using the Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, A53225).

[0129] <Luciferase assay> MLOY4 cells were cultured in a collagen-treated 10 cm dish (Iwaki, 4020-010) until confluent, and then detached with TrypLE express (Gibco, 12605010). The cells were mixed with OptiMEM (Gibco, 31985062), counted, centrifuged, and collected at 1 × 10 6 The cells were suspended at a concentration of 100 μL. Ten μg of either the pNL3.1[Nluc / minP] Vector (Promega, N1031) carrying the predicted Hes1 binding domain (pNL3.1 / Hes1) or the empty vector (pNL3.1) was added to the cell suspension. For normalization with firefly luciferase, 1 μg of the pGL4.53[Luc2 / PGK] Vector (Promega, E5011) was added to the cell suspension. After transfection by electroporation, 1 μmol / L DEX was added. After another 24 hours, the cells were starved in serum-free medium and treated with Yoda1 (80 μL / well). After 4 hours, 80 μL of ONE-Glo EX solution (included with the Nano-Glo Dual-Luciferase Reporter Assay System, Promega, N1610) was added, mixed by pipetting, and incubated for 10 minutes at room temperature. Firefly luciferase was measured using a luminescence plate reader (Berthold, Centro XS3 LB960). 80 μL of Nano DLR Stop & Glo solution was added, mixed by pipetting, and incubated for 10 minutes. NanoLuc-derived luminescence was measured using a plate reader. Firefly luciferase was used as an internal standard in each well.

[0130] Western blotting: Bolt LDS Sample Buffer (Thermo Fisher Science, B0007) was added to the protein solution and heated to 95°C for 5 minutes. 30 μg of protein was loaded per well onto a Bolt 4-12% Bis-Tris Plus gel (Thermo Fisher Science, NW04122BOX) and electrophoresed using MOPS SDS Running Buffer (Invitrogen NP0001). After electrophoresis, the gel was transferred at 20 V for 1 hour in a semi-dry condition and then blocked with 5% skim milk for 10 minutes at room temperature. Piezo1 was blocked overnight with 5% skim milk in PBS at 4°C. The gel was washed three times for 5 minutes with 1% skim milk in PBST. Each antibody was diluted with Can Get Signal Solution 1 (TOYOBO, NKB-101) and incubated overnight at 4°C. The dilution ratios were as follows: Piezo1 antibody (Proteintech, 15939-I-Ap) 1:300; Akt (Cell Signaling, 4691); Phospho-Akt (Ser473) (Cell Signaling, 4060); p44 / 42 MAPK (Erk1 / 2) (Cell Signaling, 4698); Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (Cell Signaling, 4370S); CaM kinase II (Abcam, ab52476); CaM kinase II (pThr 286) (R&D Systems, PPS002); Hes1 (Adipogen, AG-20T-0400; Phospho-HES1 (Ser 37) (Invitrogen, PA5-105350); β-Actin (Cell Signaling, 4691); Signaling, 4970S) was used at a 1000-fold dilution. The cells were washed three times for 5 minutes with PBST solution containing 1% skim milk.Anti-rabbit IgG, HRP-linked antibody (Cell Signaling, 7074S) was used as the secondary antibody, diluted 2000-fold with Can get signal solution 2 (TOYOBO, NKB-101), and incubated for 1 hour at room temperature. For Piezo1, the secondary antibody was diluted 10,000-fold with Can get signal solution 2 and incubated for 1 hour at room temperature. After washing three times for 5 minutes with TBST and twice for 5 minutes with TBS, detection was performed with ECL Prime Western Blotting Detection Reagents (Cytiva, RPN2232) and the images were captured using a ChemiDoc Imaging System (Bio-Rad).

[0131] Statistical analysis: All data are expressed as mean ± SD. Student t test, one-way ANOVA, and Tukey-Kramer test were performed using EXSUS10.0 (CAC Clore). Significant differences were considered p < 0.05.

[0132] (2) Results To clarify the events occurring in bone tissue in patients with steroid-induced osteoporosis (GIOP) and non-steroid-induced osteoporosis, we prepared and examined thin sections of bone tissue near the calcar of the femoral neck of patients who had undergone total hip arthroplasty (Fig. 1). Visualization of the canalicular network in bone tissue using Ploton silver staining revealed that the canalicular network in GIOP patients was attenuated compared with non-GIOP patients (Fig. 1A, B). Comparison of empty lacunae in osteocytes using HE staining revealed an increase in empty lacunae in GIOP patients. TUNEL-positive cells were also more abundant in GIOP patients than in non-GIOP patients, indicating increased apoptosis of osteocytes (Fig. 1C, F). Interestingly, when Piezo1 expression in bone tissue was compared between GIOP and non-GIOP patients, immunohistochemistry and Western blotting revealed decreased Piezo1 expression in osteocytes (Fig. 1G, I). No difference was observed in the number of TRAP-positive osteoclasts in bone tissue between GIOP and non-GIOP patients.

[0133] Because Piezo1 expression was found to be reduced in bone tissue from GIOP patients, we suspected that Yoda1, a Piezo1 activator, might be effective in an animal model of steroid-induced osteoporosis, and performed an in vivo administration experiment (Fig. 2). DEX and Yoda1 were administered for 4 weeks, and bone structure in steroid-induced osteoporosis was compared (Fig. 2A). Micro-CT analysis of femoral bone structure revealed significant decreases in trabecular bone mass (BV / TV), trabecular number (Tb. N), and cortical bone width (Ct. Th.) in the DEX-treated group, whereas Yoda1 administration maintained these values ​​at levels comparable to those in the vehicle group (Fig. 2B, C). Yoda1 also suppressed DEX-induced changes in cortical bone porosity and trabecular structure in trabecular bone. Analysis of bone strength revealed that maximum load, energy absorption, and stiffness were all significantly decreased in the DEX-treated group, whereas Yoda1 administration maintained bone strength (Fig. 2D). Bone morphometry of trabecular and cortical bone was performed (Fig. 2EJ). Osteoblast surface (osteoid surface) (Ob. S / OS), osteoid volume (osteoid surface) (OV / OS), cortical bone width (Ct. Wi), and cortical bone surface (Ct. Ar.) were significantly decreased in the DEX-treated group, whereas these values ​​were maintained by Yoda1 administration (Fig. 2EJ). Bone formation rate was also significantly decreased in the DEX-treated group, whereas these values ​​were maintained by Yoda1 administration (Fig. 2KM).

[0134] Further immunochemical analysis of bone tissue was performed (Fig. 3). Piezo1 expression in bone tissue was significantly reduced in the DEX-treated group, whereas it was comparable in the Yoda1-treated group to that in the vehicle-treated group (Fig. 3A, B). SOST expression in osteocytes was significantly increased in the DEX-treated group compared with the vehicle-treated group, but was suppressed by Yoda1 (Fig. 3C, D). Platon silver staining of osteocytes revealed that osteocyte dendrites were reduced in the DEX-treated group compared with the vehicle-treated group, but were maintained by Yoda1 (Fig. 3E, F). Similar results were observed when osteocytes were stained with phalloidin (Fig. 3G, H). The number of osteocalcin-positive osteoblasts on the bone surface of cancellous bone was reduced by DEX treatment compared with the vehicle-treated group, whereas the number of osteoblasts was maintained by Yoda1 treatment (Fig. 3I, J). No significant differences were observed in the number of TRAP-positive cells on the trabecular surface and the erosion surface (ES / BS) in the DEX-treated group compared with the vehicle-treated group, but a significant decrease was observed in the DEX and Yoda1 co-administration group (Fig. 3 KM).

[0135] To more directly demonstrate that DEX attenuates the mechanical loading response of bone, we investigated the effects of periodic loading on bone structure in mouse tibiae for 10 days (Fig. 4A). Micro-CT analysis of bone structure revealed that loading significantly increased cancellous bone mass (BV / TV), trabecular number (Tb. N), and cortical bone width (Ct. Th) in the vehicle-treated and DEX and Yoda1-treated groups, but no significant differences were observed in the DEX-only group (Fig. 4B, C). Cortical bone porosity (Po. tot) significantly decreased in the vehicle-treated and DEX and Yoda1-treated groups, but no change was observed in the DEX-treated group (Fig. 4C). Bone formation rate also significantly increased in the vehicle-treated and DEX and Yoda1-treated groups, but no change was observed in the DEX-treated group (Fig. 4D, F). Immunostaining revealed an increase in osteocalcin on the periosteal surface in the vehicle-treated group and the DEX and Yoda1-treated group, both of which were mechanically loaded (Fig. 4G, H). The number of TRAP-positive cells in the trabecular bone was significantly increased in the DEX-treated group regardless of mechanical loading, but was comparable to that in the DEX and Yoda1-treated group (Fig. 4I, J). When we examined the effects of DEX and Yoda1 on osteoclast differentiation of bone marrow macrophages, we found that DEX promoted osteoclast differentiation, whereas Yoda1 had only a limited inhibitory effect on osteoclast differentiation of bone marrow macrophages.

[0136] Based on previous data, we suspected that DEX administration had a strong effect on bone cells, so we performed organ culture of human cortical bone from which soft tissue and bone marrow-derived tissue had been removed (Fig. 5A-D). PIEZO1 expression was significantly reduced by DEX treatment, but expression was maintained by DEX and Yoda1 treatment (Fig. 5A). SOST and the RANKL / OPG ratio were unchanged by DEX and Yoda1 treatment, but significantly increased by DEX treatment (Fig. 5B-D). WNT16 expression was strongly increased by DEX and Yoda1 treatment (Fig. 5C). Western blotting showed that Piezo1 expression was reduced by DEX treatment in MLOY4 cells, but increased by Yoda1 treatment (Fig. 5E). Analysis of downstream signals of Piezo1 revealed that Yoda1 activated the phosphorylation of Akt and ERK, and that DEX strongly inhibited the phosphorylation of Akt and ERK. Treatment with Yoda1 improved the inhibition of Akt and ERK phosphorylation by DEX (Fig. 5 F, G). 2+ To clarify the effect on ion influx, analysis was performed using Fluo8. Treatment with 3 μmol / L of Yoda1 resulted in Ca ions entering MLOY4 cells. 2+ Ion influx increased rapidly, and Ca in cells treated with 1 μmol / L of DEX for 24 hours. 2+ The influx of ions was inhibited to the same extent as in cells knocked down by Piezo1 siRNA (Fig. 5H). 2+ It was shown that ion influx was able to respond to the same level as the control even in the presence of DEX 1 μmol / L by increasing Yoda1 to 10 μmol / L. 2+This showed that DEX enhanced the phosphorylation of CaM kinase II, which functions downstream of ion influx (Fig. 5I). Interestingly, Yoda1-induced Akt phosphorylation was suppressed by pretreatment with KN93, a CaM kinase II inhibitor (Fig. 5J). Analysis of morphological changes in MLOY4 cells using an in cell analyzer revealed that DEX significantly increased actin crossing points, which were improved to the same level as control by Yoda1 (Fig. 5K, L).

[0137] To further elucidate the mechanism of mechanical loading response in mouse bone tissue affected by DEX administration, we subjected mice to short-term loading for 5 days, then harvested tibiae and performed RNA sequencing of the bone tissue to comprehensively analyze gene changes (Fig. 6A). After repeated loading, 130 genes were significantly altered in the bone tissue of mouse tibiae in the vehicle-treated group, but only 22 genes were significantly altered in the DEX-treated group (Fig. 6B, C). Comparing the gene changes in bone tissue under loading between the vehicle-treated and DEX-treated groups, we found that, as in the in vitro results, Piezo1 and Tnfrsf11b (Opg) were downregulated, while Tnfrsf11a (Rank) was upregulated in the DEX-treated group (Fig. 6D). Pathway analysis was performed on genes that responded normally to loading in the vehicle-treated group but showed no changes after DEX administration. The results showed that extracellular remodeling and osteoblast differentiation regulatory mechanisms were downregulated in the DEX-treated group, with the lowest significant difference (Figure 6E). To identify the molecules regulating Piezo1 expression using mouse RNA sequencing, we extracted 145 molecules that could potentially bind to the Piezo1 promoter region from the public database, ChIP Atlas. Overlapping the 2,084 genes whose expression was significantly downregulated in bone tissue treated with DEX for 5 days resulted in 14 candidate genes (Figure 6F). To narrow down the 14 candidate genes to those whose expression is increased in response to mechanical stress in bone cells, we mapped the results of gene changes when MLOY4 cells were subjected to mechanical stress using ultrasound stimulation (LIPUS) based on previously reported data (Shimizu, et al., Scientific Reports, 11(1), 1-15, 2021) against the 14 candidate genes. As a result, we found that only Hes1 (hairy and enhancer of split 1) was a gene whose expression was increased when MLOY4 cells were subjected to mechanical stress (Figure 6 G).

[0138] We sought to determine whether Hes1, identified by mouse RNA sequencing analysis, functions as a transcription factor for Piezo1 (Fig. 7). Hes1 knockdown experiments in MLOY4 cells revealed a significant decrease in Piezo1 gene expression (Fig. 7A). Western blotting analysis of protein expression revealed that Hes1 knockdown suppressed Piezo1 expression (Fig. 7B). To demonstrate Hes1 binding to the Piezo1 promoter, we designed multiple primers from the predicted Hes1 binding region identified by the ChIP Atlas and performed a CUT & RUN assay (Fig. 7C, D). Gene amplification of the Hes1 binding site was significantly greater when using a Hes1 antibody in Yoda1-treated MLOY4 cells than when using an IgG antibody, but not in DEX-pretreated or Hes1-knockdown cells (Fig. 7D). To clarify the effect of Piezo1 on transcription, we inserted the Hes1-binding domain into a NanoLuc vector and performed a luciferase assay. We found that Hes1-induced Piezo1 transcription was significantly reduced in cells pretreated with DEX, but was restored by treatment with Yoda1 (Fig. 7E). qPCR also demonstrated that Hes1 expression was reduced in a DEX concentration-dependent manner and increased by Yoda1 (Fig. 7F, G). Western blotting revealed that, although the total amount of Hes1 protein was not reduced by DEX, Hes1 phosphorylation was suppressed by DEX and activated by Yoda1 (Fig. 7H).

[0139] We further explored mechanisms affecting the mechanical loading response of mouse and human bone tissue by steroid administration, in addition to PIEZO1 in osteocytes (Fig. 8). RNA was extracted from the cortical neck bone tissue of age- and sex-matched GIOP and non-GIOP patients who underwent total hip arthroplasty, and RNA sequencing analysis was performed (Fig. 8A). OSTN (osteocrin) expression was significantly reduced in GIOP patients compared with non-GIOP patients (Fig. 8B). Pathway analysis revealed significantly reduced bone development, bone remodeling, mineralization, and joint tissue regulation in GIOP patients compared with non-GIOP patients (Fig. 8C). To combine the results of the mouse bone loading RNA sequencing and the human bone tissue RNA sequencing analysis, we performed FDR multiple testing correction to narrow down the genes commonly affected by steroid administration during mechanical loading in mouse and human bone tissue, and found 10 genes (Fig. 8D, E). In mouse bone tissue, Acan, Sox9, and Sfrp2 were significantly increased by bone loading in the vehicle-treated group, but the responsiveness to loading was lost in the DEX-treated group, and expression in bone tissue was higher in non-GIOP patients than in GIOP patients (Fig. 8F, G).

[0140] RNA sequencing analysis of mouse and human bone tissue revealed that the group of mechanical stress-responsive genes adversely affected by DEX included many chondrocyte-related marker genes. Therefore, we hypothesized that DEX might be damaging cell groups involved in osteoblast differentiation or membranous ossification during mechanical loading. We therefore compared the changes in Piezo1 expression during differentiation induction in MC3T3E1 cells and periosteal cells cultured from human bone tissue with those in osteoblasts (Fig. 9A). In periosteal cells, Piezo1 expression increased upon differentiation induction, and was suppressed by DEX, whereas Yoda1 enhanced Piezo1 expression (Fig. 9A). In osteoblasts, Piezo1 expression did not change upon differentiation induction, and was not affected by DEX (Fig. 9B). Focusing on the effects of DEX and Yoda1 on periosteal cells, we performed qPCR on the gene cluster identified by RNA sequencing and found that the expression of six genes (ACAN, SOX9, SFRP1, SFRP2, SMOC1, and COL14A1) was decreased by DEX and significantly increased by Yoda1 (Figure 9C). WST assays confirmed that DEX treatment affected periosteal cells, but the combined use of Yoda1 did not affect the cells (Figure 9F). Periosteal cells were induced to differentiate into osteoblasts, and ALP staining and Alizarin Red staining revealed that DEX inhibited osteoblast differentiation, while Yoda1 improved it (Figure 9D).

Claims

1. A preventive or therapeutic agent for steroid-induced osteoporosis, comprising a Piezo1 and / or Hes1 enhancer.

2. The preventive or therapeutic agent according to claim 1, wherein the Piezo1 and / or Hes1 enhancer is an enhancer of the expression or function of Piezo1 and / or Hes1.

3. The preventive or therapeutic agent according to claim 1, wherein the Piezo1 and / or Hes1 enhancer is at least one selected from the group consisting of a Piezo1 activator, an LSD1 inhibitor, a polynucleotide comprising a Piezo1 coding sequence, a polynucleotide comprising a Hes1 coding sequence, a Piezo1 protein, and a Hes1 protein.

4. The preventive or therapeutic agent according to claim 1, which contains a Hes1 enhancer.

5. A preventive or therapeutic agent according to any one of claims 1 to 4, wherein the Piezo1 and / or Hes1 enhancer is an agent that enhances Piezo1 and / or Hes1 in bone tissue.

6. A preventive or therapeutic agent according to any one of claims 1 to 4 for use in inhibiting weakening of cortical bone and cancellous bone.

7. The preventive or therapeutic agent according to any one of claims 1 to 4, which is used in combination with a steroid drug or for administration to a subject who has previously received a steroid drug.

8. An agent containing a Piezo1 and / or Hes1 enhancer for use in combination with a steroid or for administration to a subject who has previously received steroids.

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