Application of non-classical Hippo-based regulation method in diseases caused by RIPK4 inactivation mutation

Abstract

The invention relates to an application of a signal regulation method based on non-classical Hippo in diseases caused by RIPK4 inactivation mutation. YAP / TAZ is activated in a manner of knocking out RIPK4, phenotypes of diseases caused by RIPK4 inactivation mutation are successfully simulated, expression or activity of YAP / TAZ is respectively inhibited in a manner of knocking down, knocking out YAP / TAZ or overexpressing RIPK4, and drugs for treating or relieving diseases caused by RIPK4 inactivation mutation are developed by aiming at inhibiting YAP / TAZ gene expression or activity. In addition, abnormal expression of a group of cholesterol synthesis genes (Fdft1, Mvd, Cyp51 and Nsdhl) is found; therefore, YAP / TAZ with deregulated activity and cholesterol synthesis genes with abnormal expression can be used as diagnostic markers, and the YAP / TAZ gene can also be used as a therapeutic target.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular biology, and in particular to a method for regulating the non-classical Hippo signaling pathway based on RIPK4 and its application in diseases caused by RIPK4 inactivation mutations. Background Technology

[0002] Both humans and mice have a multi-layered epidermal structure, which gradually differentiates from a single layer of ectodermal cells during development. Basal cells, possessing stem cell-like properties, proliferate, differentiate, and migrate upwards, successively forming the spinous layer, granular layer, and stratum corneum. During terminal differentiation, granular layer cells flatten, and transglutaminase catalyzes the remodeling of various keratin fibers, forming a stable, tough, and hydrophobic keratinized capsule, ultimately acquiring the barrier function to resist harmful microorganisms and prevent fluid loss.

[0003] Dysregulation of key signals in skin development is not only closely related to developmental defects but also serves as a pathogenesis for some major diseases. For example, mutations in the RIPK4 gene can lead to Bartsocas-Papas syndrome, characterized by epidermal differentiation defects and adhesions. This syndrome is an autosomal recessive genetic disorder characterized by severe growth retardation, pterygium in the popliteal fossa, syndactyly, varying degrees of cleft lip and palate, and adhesions between the skin and body in the limbs. Although the prevalence of this syndrome is extremely low, most patients die before birth or in infancy. With the advancement of medical technology, more patients with Bartsocas-Papas syndrome may survive the neonatal period; however, repeated and often unsuccessful surgeries reduce their quality of life, and currently there are no specific drugs or clinical trials for potential treatments. Therefore, elucidating the pathogenesis of this syndrome, discovering new biomarkers, and developing appropriate drugs and therapies are crucial.

[0004] Exome sequencing of patients with Bartsocas-Papas syndrome revealed a large number of mutations concentrated in the kinase domain of RIPK4, leading to impaired kinase activity. Currently, only plakophilin-1 (PKP1) and IRF6 have been clearly studied in epidermal development. PKP1 is a desmosome protein, but PKP1 knockout mice exhibit largely normal epidermal thickness and barrier function. IRF6 mutations cause Van der Woude syndrome and pterygium syndrome, both of which are characterized by cleft lip and palate, but with much milder symptoms than Bartsocas-Papas syndrome. Systemic RIPK4 knockout mice mimic the phenotype of Bartsocas-Papas syndrome. Irf6 knockout mice are phenotyped similarly to Ripk4 knockout mice, but differences remain. First, Irf6 knockout results in the absence of the granular layer, while the granular layer in Ripk4 knockout mice is expanded, suggesting that other targets of RIPK4 may have a function in mediating granular layer differentiation. Secondly, Ripk4 knockout mice exhibit a significant barrier defect in the back, while Irf6 knockout mice primarily show this defect in the limbs and head. More importantly, the phosphorylation-mimicking mutation of Irf6 cannot rescue the abnormal skin differentiation caused by Ripk4 inactivation, suggesting that there may be other downstream mechanisms regulating epidermal differentiation from RIPK4.

[0005] Cocoon syndrome caused by CHUK (also known as a nuclear factor κ-B kinase subunit α inhibitor or IKKA) mutation is an extreme form of Bartsocas-Papas syndrome. The epidermal-specific Ripk4 transgene (K14-Ripk4) can rescue the epidermal phenotype in Ripk4 knockout mice but cannot promote epidermal differentiation in Chuk knockout mice. Furthermore, even when transplanted into normal recipient mice, the Ripk4 knockout skin phenotype cannot be rescued, while the Chuk knockout skin phenotype is restored. This indicates that RIPK4 regulates skin differentiation in a cell-autonomous manner, while CHUK regulates it in a non-cell-autonomous manner. Moreover, the CHUK gene is crucial for normal B cell development, while RIPK4 has no significant connection to B cell development. All of the above demonstrates that RIPK4 and CHUK do not belong to the same signaling pathway.

[0006] In summary, RIPK4 plays an important role in skin development and differentiation, but its specific regulatory mechanism is different from that of previously discovered pathways and remains unknown.

[0007] The Hippo signaling pathway plays an evolutionarily conserved role in organ size control, tissue regeneration, and tumorigenesis. In vitro studies have revealed that this pathway senses environmental mechanotransmitters and GPCR-mediated chemical signals, and regulates cell proliferation and apoptosis, stem cell self-renewal, and differentiation through the phosphorylation and inhibition of transcriptional coactivators YAP / TAZ via the core kinase cascade MST1 / 2 (MST)-LATS1 / 2 (LATS). However, the signal sources and mechanisms regulating Hippo pathway activity remain unclear. Epidermal tissue-specific YAP transgenesis leads to massive stem cell proliferation and inhibited differentiation, but knockout of the classical upstream kinase MST in the Hippo signaling pathway does not produce a significant phenotype. This suggests that MST is not the only regulatory signal, and other undiscovered signals exist. Summary of the Invention

[0008] This application reveals that RIPK4 kinase strongly activates LATS, a core member of the Hippo pathway, and inhibits downstream transcription factors YAP / TAZ to promote differentiation. Epidermal-specific RIPK4 knockout leads to abnormal activation of YAP / TAZ in the granular layer of mouse epidermis, resulting in inhibition of cholesterol biosynthesis. Further knockout of Yap / Taz can restore the skin barrier function on the back. This provides a non-classical Hippo signaling pathway regulation method based on RIPK4 and its application in diseases caused by RIPK4 inactivation mutations.

[0009] This application provides an application of a non-classical Hippo-based signal modulation method in diseases caused by RIPK4 inactivation mutations. By constructing a YAP / TAZ inhibitor to inhibit the expression or activity of YAP / TAZ, a drug for treating or alleviating diseases caused by RIPK4 inactivation mutations can be prepared.

[0010] The YAP / TAZ inhibitors include molecular inhibitors that knock down or eliminate the YAP / TAZ gene; the molecular inhibitors include interfering RNA, which includes siRNAs used to knock down YAP / TAZ as shown in SEQ ID NO. 1-4.

[0011] Preferably, the molecular inhibitor for knocking out the YAP / TAZ gene also includes the sgRNA used to knock out YAP / TAZ, as shown in SEQ ID NO. 5 and 6.

[0012] Preferably, the YAP / TAZ inhibitor includes an overexpression agent for the RIPK4 gene; the overexpression agent includes a vector for overexpressing RIPK4, and the primer sequences used to construct the RIPK4 overexpression vector are shown in SEQ ID NO.7 and 8.

[0013] Preferably, by constructing an RIPK4 inhibitor to inhibit the expression or activity of the RIPK4 gene and activate the expression or activity of YAP / TAZ, the phenotype of disease caused by RIPK4 inactivation mutation is simulated, and a disease model caused by RIPK4 inactivation mutation is created.

[0014] The RIPK4 inhibitor includes interfering RNA, which includes sgRNA used to knock out RIPK4 as shown in SEQ ID NO. 9 and 10.

[0015] Preferred diseases caused by RIPK4 inactivation mutations include Bartsocas-Papas syndrome.

[0016] A drug comprising, as an active ingredient, any one of the siRNA, the sgRNA, the RIPK4 overexpression vector, and the small molecule inhibitor.

[0017] Application of dysregulated YAP / TAZ and abnormally expressed cholesterol synthesis genes as diagnostic biomarkers for diseases caused by RIPK4 inactivation mutations.

[0018] The mRNA expression level of the abnormally expressed cholesterol synthesis gene was detected by real-time PCR, with primer sequences shown in SEQ ID NO.11-18.

[0019] Cholesterol synthesis genes include Fdft1, Mvd, Cyp51, and Nsdhl.

[0020] The beneficial effects of this invention are: by activating YAP / TAZ through RIPK4 knockout, the phenotype of Bartsocas-Papas syndrome is successfully mimicked; by knocking down, knocking out, or overexpressing RIPK4, the expression or activity of YAP / TAZ is inhibited, thereby developing drugs for the treatment or relief of Bartsocas-Papas syndrome with the aim of inhibiting YAP / TAZ gene expression or activity. Furthermore, abnormal expression of a group of cholesterol synthesis genes (Fdft1, Mvd, Cyp51, and Nsdhl) was discovered; therefore, dysregulated YAP / TAZ and abnormally expressed cholesterol synthesis genes can be used as diagnostic markers, and the YAP / TAZ gene can also be used as a therapeutic target. Attached Figure Description

[0021] Figure 1 Experimental results to identify the Hippo signaling pathway as a downstream target pathway of RIPK4.

[0022] In the figures: A, RIPK4 induces phosphorylation of the HM and AL sites of LATS1, suggesting LATS1 activation. HEK293T cells were transfected with the corresponding plasmid, and cell lysates were immunoprecipitated as shown. K51R represents a kinase-inactivating mutant of RIPK4. B, RIPK4 can directly phosphorylate the HM and AL sites of LATS1. LATS1 / 2DKO HEK293A cells were transfected as shown. Flag-RIPK4 was immunoprecipitated using anti-Flag, and in vitro kinase assays were performed using recombinant LATS1-602-C-KR as a substrate in the presence of ATP. C, RIPK4 promotes LATS1 kinase activity. HEK293T cells were transfected as shown, and LATS1 was immunoprecipitated using anti-HA antibody. In vitro kinase activity assays were then performed using recombinant GST-YAP protein as a substrate. K734R represents a kinase-inactivating mutant of LATS1. D, RIPK4 promotes YAP and TAZ phosphorylation in a LATS1 / 2-dependent manner. RIPK4 and YAP or TAZ were co-transfected into control and LATS1 / 2DKO HEK293A cells, and samples were detected by Western blotting and Phos-tag assays. RIPK4 inhibited the transcriptional activity of YAP / TAZ. The corresponding plasmids were co-transfected into HEK293T cells with 5xUAS-luciferase reporter gene, Gal4-TEAD4, and CMV-β-gal, and luciferase activity was detected, with the corresponding β-galactosidase activity used as an internal control. Data are expressed as mean ± SD.

[0023] Figure 2 To investigate the experimental results of RIPK4 regulating the activity of the Hippo signaling pathway in keratinocytes.

[0024] Figure: A, Schematic diagram of human RIPK4 protein and mutations in Bartsocas-Papas syndrome (red); KD, kinase domain; ID, intermediate domain; AR, ankylosing spondylogene repeat sequence. Numbers represent amino acid residues. R260fs represents a frameshift mutation at arginine position 260; S376X represents a nonsense mutation at serine position 376. B, The Bartsocas-Papas syndrome mutant of RIPK4 cannot induce LATS1 phosphorylation. HEK293A cells were transfected with the corresponding plasmid, and cell lysates were immunoprecipitated as shown in the figure. C, Ca 2+Conditional activation of RIPK4 was induced. HaCaT cells with RIPK4 C-terminal HA tag knock-in were treated with 3 mM calcium chloride for a specified time to induce differentiation. RIPK4 was immunoprecipitated with an anti-HA antibody, and in vitro kinase activity was detected in the presence of ATP-γ-S. Autophosphorylation of samples was detected by anti-thiophosphate antibody. D, RIPK4 knockout inhibits differentiation-induced YAP phosphorylation. RIPK4 expression in HaCaT cells was reinjected by retroviral infection, and the corresponding cells were treated with 3 mM calcium chloride for 24 hours to induce differentiation. E, HaCaT cells were treated with 3 mM calcium chloride for 4 days, and the transcriptome of the corresponding cells was analyzed by RNA-seq. Heatmaps depict the relative expression levels (Z-score) of differentially expressed genes (rows) in the samples (columns) (p<0.01), and the biological functions of differentially expressed genes were analyzed by MsigDB enrichment analysis. F. Venn diagram depicts the overlap between the 46 genes (blue) induced by RIPK4 knockout and suppressed by differentiation conditions and YAP / TAZ knockdown in Figure E, and the direct target genes of YAP / TAZ (yellow). G. Control and RIPK4 knockout NHEK cells were transfected with siRNA, treated with 1.5 mM calcium chloride for 3 days, and stained with anti-YAP / TAZ and anti-Involucrin (IVL) antibodies. H. Control cells and RIPK4 knockout NHEK cells were seeded on collagen gels containing NIH3T3 fibroblasts, immersed and cultured for 24 hours, then the culture medium at the top of the cells was removed, and the cells were cultured at the air-liquid interface for another week. The resulting organoid cultures were fixed and analyzed by immunofluorescence staining.

[0025] Figure 3 To investigate the experimental results of Ripk4 knockout leading to abnormal activation of Yap / Taz in mouse epidermal granular cells.

[0026] In the figure: A, control and Ripk4 - / - Appearance of E18.5 embryos. B, control and Ripk4. - / - C. Toluidine blue staining of E18.5 embryos. Histological and IHC analysis of skin sections from E18.5 embryos; dashed lines indicate the basement membrane. HE, hematoxylin-eosin staining. D. Control and Ripk4. - / -Immunofluorescence staining was performed directly on single cells isolated from the epidermis of E18.5 embryos after enzymatic digestion, revealing that Ripk4 knockout indeed led to aberrant activation of Yap / Taz in granular cells (Loricrin positive). E, UMAP analysis of single cells, with color coding according to their specified cell type. F, GSVA analysis of differentiation-related RIPK4-YAP / TAZ characteristic genes (adjusted p < 0.01). Boxes in the violin plot indicate interquartile range and median. ES, Wilcox effect size in independent two-sample test.

[0027] BAS, basal cells; ES / EDB, early spinous / early differentiated basal cells; SPN, spinous layer; GRN, granular layer; PAR, parakeratotic layer; MEL, melanocytes; MP, mononuclear phagocytes; T, T cells.

[0028] Figure 4 This is the experimental result of Ripk4 promoting cholesterol biosynthesis by inhibiting Yap / Taz.

[0029] Figure A shows the relative expression of differentially expressed genes (rows) in granular layer cell clusters (columns) (adjusted p < 0.05, log2FC ≥ 0.5, pct.1 > 0.2), with MsigDB enrichment analysis used to determine the biological function of these differentially expressed genes. Figure B shows primary keratinocytes from control and Ripk4 KO mice transfected with siRNA and treated with 1.5 mM calcium chloride for 24 hours. Target gene mRNA expression levels were detected by quantitative real-time PCR. Figure C shows primary keratinocytes from control and Ripk4 KO mice transfected with siRNA and treated with 1.5 mM calcium chloride for 24 hours. Cell expression was detected by immunofluorescence. Figure D shows free cholesterol levels detected by Filipin Ш staining using Nuclear Green staining. TM LCS1 was used for nuclear staining. E. Total cholesterol levels were measured using a commercial kit. Data are presented as mean ± SD.

[0030] Figure 5 The results show that Ripk4 promotes epidermal differentiation by inhibiting Yap / Taz.

[0031] In the figure: A, Toluidine blue staining of E18.5 embryos of the specified mice. B, Histological and IHC analysis of skin sections from E18.5 embryos of the specified mice; the dashed line indicates the basement membrane. HE, Hematoxylin-eosin staining. C, Quantitative analysis of the epidermis of E18.5 embryos of the specified mice. The mean epidermal thickness from eight random regions from two mice was used for each genotype. D, Schematic diagram of the Krt10-HA-Cre knock-in strategy. LHA, left homologous arm; RHA, right homologous arm; pA, polyA tail; PGK, human phosphoglycerate kinase 1 promoter; HygR, hygromycin resistance gene. E, Lysates of cultured primary keratinocytes (without air-liquid interface) and organoid skin cultures (with air-liquid interface) of the indicated genotypes examined by Western blotting. F, Histological and immunofluorescence analysis of organoid skin cultures of the specified genotypes. G, Quantitative analysis of epidermal thickness of organoid skin cultures of the specified genotypes. For each genotype, the average epidermal thickness was used from nine random fields of view from three organoids. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] I. Experimental Methods

[0034] 1. Experimental materials:

[0035] Animal experiments: All mouse research protocols were approved by the Animal Care and Use Committee of Zhejiang University. Standard laboratory diets for mice were purchased from Xietong Biotechnology and fed freely. The humidity in the SPF-grade animal room was 45%-60%, with a 12-hour light / dark cycle (7:00 AM - 7:00 PM). Mice were euthanized by cervical dislocation or carbon dioxide. Animal care was provided according to the regulatory standards of the Experimental Animal Center of Zhejiang University. All mouse alleles maintained a C57BL / 6 genetic background. The origin of the gene knockout mouse strains is shown in the table below.

[0036]

[0037]

[0038] Cellular experiments: HEK293T cells were derived from ATCC; HEK293A cells were derived from the University of California, San Diego (PMID: 30135582); HaCaT cells were derived from the German Cancer Research Center (PMID: 12771184); NHEK cells were derived from ScienCell; mouse primary keratinocytes were isolated from the dorsal epidermis of newborn mice. HEK293T, HEK293A, and HaCaT cells were cultured in DMEM containing 10% FBS and 50 μg / mL penicillin / streptomycin; NHEK cells were cultured in EpiLife medium (Gibco) supplemented with human keratinocyte growth additive (HKGS, Gibco) and 50 μg / mL penicillin / streptomycin; mouse primary keratinocytes were cultured on mitomycin C-treated NIH3T3 fibroblast feeder cells in a low-calcium medium containing 0.05 mM calcium. All cells were cultured in a 37°C humidified incubator with 5% CO2. Cell lines were validated at GeneDetection Biotechnology Co., Ltd. (Suzhou, China) using short tandem repeat (STR) analysis as described in the 2012 ANSI standard (ASN-0002) developed by the ATCC Standards Development Organization. Mycoplasma testing was performed monthly in tissue cultures using a mycoplasma detection kit. Cells used in the experiments were passaged 15-20 times after thawing.

[0039] 2. Plasmid construction:

[0040] All human genes originated from Ultimate. TM ORF LITE cloning. The RIPK4 gene was amplified by PCR (primer sequences shown in SEQ ID NO. 7-8), and cloned into the pRK7-Flag vector after double digestion with EcoRI and XbaI using a recombination method to obtain pRK7-Flag-RIPK4. BPS disease mutants and various truncated mutants of pRK7-Flag-RIPK4 were designed online using The The Primer Design Program, using pRK7-Flag-RIPK4 as a template, employed Pfu Turbo DNA Polymerase for PCR, followed by the addition of Dpn I and digestion at 37°C for 2 hours. The digestion products were then transformed into platings, and single clones were picked for sequencing identification. For the construction of the RIPK4 CRISPR / Cas9 plasmid, sgRNA primers were first designed online using the Benchling website. After primer dimerization, they were ligated into a PEP-KO or PEP-KI vector.

[0041] 3. Transfection and viral infection:

[0042] Transfection was performed using polyethyleneimine or liposomes according to the manufacturer's instructions. Stable cell lines were constructed using retroviral infection. HEK293T cells were seeded 18 hours prior to transfection, at a density of 30% to 40%. The viral plasmid and packaging plasmid were transfected into HEK293T packaging cells at a 1:1:1 ratio. 24 hours after transfection, the culture medium was replaced with fresh medium, and the target cells to be infected were seeded into culture dishes. Cell infection was performed 24 hours later: the supernatant of the packaging cells filtered through a 0.45 μm filter was mixed with fresh culture medium, and polyvinyl alcohol was added to a final concentration of 10 μg / ml. This mixture was then added to the target cells. Generally, two infections were performed, lasting two consecutive days. After the final infection, the target cells were screened with puromycin until all control cells died, yielding stably infected cells.

[0043] 4. Calcium-induced keratinocyte differentiation:

[0044] For HaCaT cell differentiation, dedifferentiation was induced according to a published protocol. In short, HaCaT cells were first dedifferentiated to a basal state by culturing in a low-calcium medium (4 mM L-glutamine, 0.03 mM calcium chloride, 10% dialyzed FBS, based on calcium-free DMEM) for 2 weeks. Cells were passaged when they reached 75-80% confluence to prevent high cell density-induced differentiation. Differentiation was then induced by adding 3 mM calcium chloride. For NHEK and mouse primary keratinocytes, differentiation was induced by adding 1.5 mM calcium chloride to the appropriate medium at specified time points.

[0045] 5. Skin organoid culture

[0046] For NHEK cells, the collagen gel was prepared by gently mixing 8 ml of ice-cold rat tail collagen I (4 mg / ml, 80%, v / v) and 1 ml of 10×PBS (10%, v / v), and adjusting the pH to 7.4 by adding approximately 60 μl of 2M NaOH. Then, under stirring on ice, a solution containing 5×10... 5 1 ml of FBS (10%, volume / volume) was added to the gel solution for NIH3T3 fibroblasts. The collagen gel mixture was pipetted into 12-well cell culture chambers and incubated at 37°C for 1 hour to solidify. Then, cloning cylinders (Sigma) were placed on the gel and gently pushed down to restrict cell growth, and the chambers were incubated at 37°C for 1 hour. After aspirating excess liquid, the gel was equilibrated in EpiLife medium (Gibco) containing 1.5 mM calcium chloride for 24 hours. After removing the medium, 2 × 10⁻⁶ cells were added to the gel. 5NHEK cells were seeded in glass loops and immersed for 24 hours. The glass loops were then removed, the culture medium was aspirated, and 300 μl of fresh EpiLife medium containing 1.5 mM calcium chloride was added to the lower chamber to expose the skin organoid cultures to the air / liquid interface. Subsequently, 300 μl of fresh EpiLife medium containing 1.5 mM calcium chloride was added to the lower chamber daily. After one week of exposure to the air / liquid interface, the cultures were fixed with 4% paraformaldehyde for 15 minutes, embedded in OCT, and sectioned, stained, and analyzed.

[0047] Decellularized dermis was prepared from newborn C57 mice using primary mouse keratinocytes. Newborn mouse skin was treated with dissociation buffer (20 mM EDTA, 8 μg / ml mitomycin C, PBS-based) at 37°C for 3 hours. The epidermis was then removed from the dermis with forceps, and the skin was rinsed three times with PBS. The dermis was transferred to cell culture chambers (6 wells), residual PBS was removed, and the dermis was allowed to stand in a biosafety cabinet for 30 minutes. Two million primary mouse keratinocytes were seeded onto the decellularized dermis. 1.5 ml of organoid culture medium (low-calcium medium supplemented with 1.5 mM calcium chloride, 1 ng / ml EGF, and 50 μg / ml vitamin C) was added to the upper chamber, and 2 ml of medium was added to the lower chamber. After immersion culture for 24 hours, the medium in both chambers was removed, and 1.5 ml of fresh organoid culture medium was added to the lower chamber to expose the organoid skin culture to the air-liquid interface. Subsequently, 1.5 ml of fresh organoid culture medium was added to the lower chamber daily. After being exposed to the gas-liquid interface for 5 days, the samples were fixed with 4% paraformaldehyde for 15 minutes, embedded by OCT, and then sectioned, stained, and analyzed.

[0048] 6. Protein blotting:

[0049] Western blotting was performed according to standard protocols. Tissue was homogenized with tissue lysis buffer (20 mM pH 7.5 Tris-HCl, 1 mM EDTA, 1 mM EGTA, 2% SDS, 150 mM NaCl, 0.1 mM DTT, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF and a mixture of protease inhibitors), and cells were collected with cell lysis buffer (50 mM pH 6.8 Tris-HCl, 1% SDS). Lysates were then separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% milk and incubated overnight at 4°C with the desired primary antibody. The membrane was then washed with 1×TBST and incubated for 1.5 h at room temperature with a secondary antibody bound to HRP. Proteins were detected using a self-made ECL assay kit or a commercial kit (Thermo-Scientific).

[0050] 7. Phos-tag detection of protein phosphorylation

[0051] When using Phos-tag gel electrophoresis, Phos-tag binds to the phosphate groups of proteins by chelating divalent metal cations, reducing the migration rate of phosphorylated proteins. Therefore, phosphorylated proteins can be separated from unphosphorylated or hypophosphorylated proteins. During electrophoresis, 15 μL of 5.0 mM Phos-tag and 15 μL of 10 mM MnCl2 are added to every 5 mL of separating gel. After electrophoresis, the membrane is washed for 10 minutes with 100 mL of transfer buffer containing 1 mM EDTA, then washed for 10 minutes with 100 mL of EDTA-free transfer buffer before transfer. The transfer program is 350 mA constant current for 120 minutes.

[0052] 8. Immunofluorescence staining and imaging

[0053] For frozen sections of skin organoid cultures, fix with 4% paraformaldehyde for 15 minutes and permeabilize with 0.1% Triton X-100 for 15 minutes; for cultured cells, fix with 4% paraformaldehyde for 15 minutes and permeabilize with 0.1% Triton X-100 for 5 minutes; for freshly isolated mouse primary keratinocytes, attach cells to coverslips at 1500 rpm for 5 minutes before fixation. All three tissues or cells were blocked with 2% BSA and 2% goat serum for 30 minutes, then incubated overnight with primary antibodies at 4°C. After rinsing with PBS, stain samples with fluorophore-conjugated secondary antibodies at room temperature for 2 hours, followed by further washing. Mount slides with DAPI-containing anti-fading mounting oil.

[0054] 9. HE staining and immunohistochemistry

[0055] Mouse embryos were fixed in 4% paraformaldehyde at room temperature for 24 hours and embedded according to standard protocol. Embryo sections were dewaxed using xylene and graded ethanol solutions. Tissue sections were stained with hematoxylin-eosin (HE). Immunohistochemical analysis: sections were boiled in extraction solution for 40 min, incubated in 3% H2O2 for 30 min, and blocked with 10% goat serum in PBS. Sections were stained overnight at 4°C with avidin-biotin complex system using specific antibodies. Signal was detected using the ABC kit with 3,3'-diaminobenzidine (DAB) as the chromogenic substrate. Cell nuclei were counterstained with hematoxylin.

[0056] 10. Gene knockout and knock-in based on CRISPR / Cas9

[0057] To knock out RIPK4, the sgRIPK4 (SEQ ID NO. 9, 10) strand was ligated into the PEP-KO vector digested with SapI. The resulting plasmid was transfected into designated cells. Forty-eight hours post-transfection, cells were selected with 2 μg / ml puromycin for another 48 hours. Remaining cells were collected and reseeded at low density to form colonies. Knockout of individual clones was examined by Western blotting and genomic DNA sequencing.

[0058] To perform differentiation-specific knockout of YAP / TAZ, we first started with E18.5Ripk4 - / - / Yap f / f / Taz f / f Primary keratinocytes were isolated from embryos, and HA-labeled Cre recombinase (HA-Cre) was knocked into the Krt10 promoter, a differentiation marker. To construct HA-Cre knock-in cells, a pair of sgRNAs (SEQ ID NO. 5, 6) targeting the 5' and 3' regions of the mouse Krt10 gene start codon were cloned into the PEP-KI vector (derived from the PEP-KO vector, with the wild-type Cas9 replaced by a D10A mutant). Genomic regions flanking the start codon were amplified by PCR to generate left and right homologous arms. These homologous arms were cloned into the pUC-GW-Amp vector and separated by the HA-Cre coding sequence, SV40 polyA signal, PGK promoter, hygromycin resistance gene, and β-Globin polyA signal. Target cells co-transfected with the three plasmids were screened with hygromycin and re-seeded at low density to form colonies. Successful knock-in of individual clones was verified by Western blotting and genomic DNA sequencing.

[0059]

[0060]

[0061] 11. Luciferase reporter gene assay

[0062] The corresponding plasmids, along with 5×UAS-luciferase reporter gene, Gal4-TEAD4, and CMV-β-gal, were co-transfected into HEK293T cells. After 24 hours, the culture medium was removed, and 1×Luciferase assay lysis buffer was added. Cells were lysed at 4°C in a shaker for 30 minutes. 20 μl of luciferase substrate and 20 μl of cell lysis buffer were added to each well of a white 96-well plate, and the luminescence intensity was quickly detected using a microplate reader. 150 μl of β-galactosidase substrate ONPG and 50 μl of cell lysis buffer were added to each well of a clear 96-well plate, and the plates were incubated at 37°C for 15 minutes. The absorbance was then read at 420 nm. The detected luminescence intensity was standardized using the corresponding β-galactosidase activity as an internal control, and the result was used as the final luciferase activity value for analysis.

[0063] 12. Immunoprecipitation and kinase activity assay

[0064] For immunoprecipitation, cells were lysed in lysis buffer (10 mM pH 7.5 Tris-HCl, 100 mM NaCl, 10 mM EDTA, 1% NP40, 50 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF) with an EDTA-free protease inhibitor added. Cell lysates were centrifuged at 12000 g for 15 min at 4 °C. The supernatant was collected and incubated with the desired antibody at 4 °C for 1.5 h by rotation. Protein A-sepharose beads were then added and incubated for another 1.5 h. The sample was then centrifuged and washed four times with ice-cold, mild lysis buffer. The sample was boiled in 1×SDS buffer.

[0065] To detect RIPK4-induced pLATS1 / 2-AL, cells were washed with PBS and lysed in lysis buffer supplemented with 1% SDS. After sonication, the lysates were diluted 10-fold with lysis buffer and centrifuged at 12000g for 15 minutes at 4°C. The supernatant was then immunoprecipitated with anti-Myc or anti-HA antibody and protein A-Sepharose. The immunoprecipitate was washed four times with lysis buffer containing 0.1% SDS and then boiled directly with SDS loading buffer.

[0066] For kinase activity assays, the immunoprecipitated kinase was washed three times with lysis buffer, once with kinase wash buffer (200 mM NaCl, 40 mM HEPES, pH 7.5), and once with kinase assay buffer (30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl2). The immunoprecipitate was then placed in kinase assay buffer containing 500 μM ATP and the specified substrate and vortexed at 30°C for 30 min. For the LATS1 kinase assay, 1 μg of GST-YAP purified from *E. coli* was used as the substrate. For the RIPK4 kinase assay, 1 μg of His-LATS1-602-C-K734R (co-purified with GST-MOB1A) purified from *E. coli* was used as the substrate. To detect the relative autophosphorylation level of the kinase, the immunoprecipitated kinase was placed in kinase assay buffer containing 500 μM ATP-γ-S and vortexed at 30°C for 30 min, then the reaction was terminated with 20 mM EDTA. Next, 2 mM PNBM was added to initiate alkylation, and the reaction was carried out at room temperature for 30 minutes. The reaction was terminated with SDS loading buffer and then boiled.

[0067] 13. Toluidine blue staining to detect mouse skin barrier function

[0068] Mouse embryos of E18.5 were placed in pre-chilled PBS on ice for 30 minutes. After euthanasia, the embryos were immersed sequentially in pre-chilled 50% and 100% methanol for 10 minutes each, washed once with PBS, and then stained with 0.1% toluidine blue solution for 5 minutes. After washing three times with PBS to remove excess dye, the embryos were photographed and analyzed.

[0069] 14. Cholesterol Level Measurement

[0070] Total cholesterol levels were determined using a biochemical assay kit (Beyotime, S0211S). Measurements were normalized to total protein levels and determined using a Pierce BCA protein assay kit (Thermo). To determine free cholesterol levels, cells on coverslips were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, and then stained with a solution containing 50 μg / ml Filipin Ш (for staining free cholesterol) and 1 μM ClearGreen. TM Incubate LCS1 (for staining nuclei) in PBS solution for 1 hour. After washing twice with PBS, fix the coverslip onto the slide and seal.

[0071] 15. Quantitative Real-Time PCR

[0072] Total RNA was extracted from cells using TRIzol reagent. cDNA was then synthesized using a first-strand synthesis system according to the manufacturer's instructions. Quantitative PCR analysis of the cDNA was performed using SYBR Green and gene-specific primers. Results were analyzed using the 2–ΔΔCt method. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as an internal control. Primer sequences are shown in SEQ ID NO. 11-18.

[0073] 16. Conventional transcriptome sequencing and analysis

[0074] Routine transcriptome sequencing was performed by Novogene. RNA quality was verified by the following methods: (1) monitoring RNA degradation and contamination on 1% agarose gel; (2) checking RNA purity using a NanoPhotometer spectrophotometer (IMPLEN); (3) measuring RNA concentration using a Qubit RNA Analysis Kit and a Qubit 2.0 fluorometer (Life Technologies); and (4) assessing RNA integrity using an RNA Nano 6000 Analysis Kit and a Bioanalyzer 2100 system (Agilent Technologies). A total of 1 μg of RNA was used as input material for each sample. The method was followed according to the manufacturer's recommendations. UltraTM RNALibrary Prep Kit for (NEB, USA) Sequencing libraries were generated, and index codes were added to attribute sequences to each sample. In short, mRNA was purified from total RNA using magnetic beads linked with poly-T oligonucleotides. Fragmentation was performed at high temperature using divalent cations in NEBNext first-strand synthesis reaction buffer (5×). First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H minus). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. In the reaction buffer, dNTPs containing dTTP were replaced with dUTPs. Remaining overhangs were converted to blunt ends by exonuclease / polymerase treatment. After 3′ adenylation, NEBNextAdaptors with hairpin loop structures were ligated to the DNA fragments for hybridization preparation. Library fragments were purified using the AMPure XP system (Beckman Coulter) to preferentially select 250–300 bp cDNA fragments. Size-selected, adapter-ligated cDNA was treated with 3 μl USER enzyme (NEB) at 37°C for 15 min, followed by 95°C for 5 min, and then PCR was performed. PCR was conducted using Phusion high-fidelity DNA polymerase, universal PCR primers, and Index(X) primers. Finally, the products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system. Following the manufacturer's instructions, the indexed samples were clustered on a cBot clustering system using the TruSeq PECluster Kit v3-cBot-HS (Illumina). After clustering, the library preparation was sequenced on the Illumina Novaseq platform, generating 150 bp paired-end reads. A reference genome (mm10) index was constructed using Hisat2 v2.0.5, and the clean paired-end reads were aligned to the reference genome using Hisat2 v2.0.5. Raw counts for each gene were calculated using featureCounts v1.5.0-p3. Then, the FPKM (Fragments Per Kilobase of Transcript per Million Mapper Reads) for each gene was calculated based on gene length and the number of reads mapped to that gene. Pseudo-FPKM values ​​were added to each element before statistical testing, and gene quantification was then performed using a log2 transformation. Differential expression analysis was performed on both cases using DESeq2 and edgeR (implemented in R software). A p-value of 0.01 and an absolute fold change of 1.5 were set as thresholds for significant differential expression. Heatmaps of clusters and labels were generated using the pheatmap R package. Gene Ontology (GO) enrichment analysis of differentially expressed genes was performed using the clusterProfiler R package.The Benjamini-Hochberg method was used to adjust the p-values. GO terms with adjusted p-values ​​less than 0.05 were considered to be significantly enriched for differentially expressed genes.

[0075] 17. Single-cell transcriptome sequencing and analysis

[0076] The dorsal skin of E18.5 embryos was washed three times with PBS. The skin was then flattened and floated derm-side down in 3 ml of 2.14 U / ml dispersant enzyme solution (EpiLife medium) at 4°C for 12 hours. The epidermis was then carefully peeled off from the dermis with forceps and the dermis was discarded. The epidermis was placed in 3 ml of trypsin-EDTA (0.05%) and incubated with shaking at 37°C for 15 minutes. 1 ml of FBS was added to stop trypsin digestion. Tissue debris was removed using a 40 μm filter. The cell suspension was centrifuged at 500 g for 5 minutes, and the cell pellet was incubated at 25°C. Resuspend the cells in Singleton erythrocyte lysis buffer for 10 minutes to remove red blood cells. Then centrifuge the cell suspension at 500g for 5 minutes and resuspend the cell pellet in PBS. Confirm cell viability is not less than 90% by trypan blue staining. Add 1×10⁻⁶ cells to the solution. 5 A single-cell suspension of 1 cell / mL is loaded onto the microfluidic device. Following the manufacturer's instructions, use... scRNA-seq libraries were constructed using reagents from the Singleleron Single-Cell RNA Library Kit. Each library was diluted to 4 nM and pooled. The pooled libraries were then sequenced using 150 bp paired-end reads on an Illumina HiSeq X.

[0077] Reads were aligned to the mouse genome (mm10) using CeleScope with default parameters. Only uniquely mapped reads without duplicates were retained. This provided a unique molecular identifier (UMI) count matrix containing 111,292 cells and 32,589 genes (two biological replicates per genotype, for a total of 4 samples). Environmental RNA was cleaned for each sample using the R package SoupX (v1.6.2) 86. The raw and filtered matrices were generated using CeleScope, following the basic workflow described in the SoupX guide. Known unique marker genes were provided as “unexpressed genes” as required by SoupX’s “manual” mode. During the filtering process, cells expressing fewer than 500 genes or fewer than 500 UMI counts, or cells expressing more than 6,000 genes or more than 20,000 UMI counts, with at least 10% being mitochondrial genes, were discarded. Finally, downstream analysis of the gene matrix of 103,121 cells and 32,589 genes was performed using Seurat (v5.0.1) in R (4.2.3). In short, the raw UMI counts were normalized to the total reads using log normalization and scaled by multiples of 10,000. The top 2,000 highly variable genes selected by variance-stabilized transformation (VST) were used for principal component analysis (PCA). Batch effects between samples were eliminated using the “RunHarmony” function in the R package harmony. Dimensionality reduction was achieved using the 1st to 17th dimensions generated by harmony, through uniform manifold approximation and projection (UMAP). Cell clusters were further identified using the 1st to 17th harmony dimensions based on Louvain clustering. Differentially expressed genes (DEGs) in each cluster were identified using the “FindAllMarkers” function in Seurat, and genes with an absolute log2 fold change greater than 0.5 and an expression percentage greater than 20% were identified as the final DEGs.

[0078] Cell type annotation was performed based on existing methods with some modifications. (Krt5) + / Krt14 + / Itga6 + The cells belong to the basal layer, Krt5 + / Krt14 + Krt1 + / Krt10 + The cells belong to early spiny / early differentiated basal cells, Krt1 + / Krt10 + / Dsg1a + The cells belong to the spinous layer, Flg + / Lor + / Ivl +Cells belonging to the granular layer, Krt6-positive cells belonging to the parakeratotic layer, Mitf + / Mlana + The cells belong to the melanocyte group. CD45 + In immune cells, CD3d + / CD3e + Cells were classified as T cells, and cells highly expressing CD14 and CD68 were classified as monocytes / phagocytes. Gene Ontology (GO) enrichment analysis was performed on DEG cells using the "compareCluster" function in clusterProfiler, with "pvalueCutoff" set to 0.05 and "ont" set to BP. Gene Set Variation (GSVA) ​​analysis was then performed using the "gsva" function in the GSVA package in R to calculate the enrichment of different gene sets associated with certain important pathways or factors. Finally, the GSVA scores of different cell groups were compared, and effect sizes were calculated using `wilcox_effsize(rstatix)`.

[0079] 18. Quantitative and statistical analysis

[0080] GraphPad Prism is used for graphical representation and statistical analysis. No statistical methods were used to estimate sample size. Unless otherwise stated, standard two-tailed unpaired t-tests were used for statistical analysis of both groups. Experimental replicates are depicted in the legend. Results are consistent across replicates.

[0081] II. Experimental Results and Discussion

[0082] 1. The Hippo signaling pathway is the downstream target pathway of RIPK4.

[0083] Kinase plasmids from a library containing 464 protein kinases (representing 86.2% of the human kinase genome) were individually co-transfected with LATS1 into HEK293T cells. New upstream kinases of LATS were identified by immunoblotting analysis of LATS hydrophobic motif phosphorylation (pLATS-HM) in cell lysates. The results revealed novel candidate kinases, including RIPK4. In classic mechanistic models in the field, upstream LATS kinases (e.g., MST) phosphorylate LATS-HM, leading to further autophosphorylation of the LATS activation cyclic phosphorylation (pLATS-AL). However, compared to MST2, RIPK4 induced stronger LATS1-AL phosphorylation. Figure 1 A). In in vitro phosphorylation assays, using a complex of recombinant LATS1-602-C-KR truncated mutant protein (containing the MOB binding region and the K734R mutant kinase domain) and MOB protein as a substrate, it was found that both HM and AL could be directly phosphorylated by RIPK4. Figure 1B). In vitro kinase activity assays revealed that RIPK4 promotes LATS1 kinase activity (B). Figure 1 C). LATS primarily exerts its biological function through phosphorylation of YAP / TAZ. Overexpression of RIPK4 induces YAP / TAZ phosphorylation in control cells, but has no inducing effect in LATS1 / 2 double knockout (DKO) cells. Figure 1 D). Furthermore, RIPK4 inhibits the activity of the YAP / TAZ luciferase reporter gene in a kinase-dependent manner ( Figure 1 E). In summary, RIPK4 is a novel upstream kinase of LATS, meaning that the Hippo signaling pathway is a downstream target pathway of RIPK4.

[0084] 2. RIPK4 regulates the activity of the Hippo signaling pathway in keratinocytes.

[0085] RIPK4 mutations lead to Bartsocas-Papas syndrome, characterized by epidermal differentiation disorder and barrier defects. Disease-derived mutants of RIPK4 (I81N, I121N, T184I, and R260fs) and kinase-inactivating mutants (K51R and D143N) all lose the ability to induce LATS phosphorylation. Figure 2 A and Figure 2 B). During keratinocyte differentiation, YAP phosphorylation increases along with elevated RIPK4 kinase activity. Figure 2 C). Under differentiation conditions, YAP phosphorylation is blocked in RIPK4 knockout cells, while RIPK4 reinjection restores YAP phosphorylation. Figure 2 D). Further RNA-seq analysis of the transcriptomes of control and RIPK4 knockout cells under basal or differentiation conditions revealed that epidermal differentiation characteristic genes were enriched in genes induced during differentiation (p<0.01), while these characteristic genes were suppressed in RIPK4 knockout cells. Figure 2 E). Knockdown of YAP / TAZ in RIPK4 knockout cells via siRNA induces constitutive expression of these genes, indicating that YAP / TAZ plays a crucial role in the differentiation process downstream of RIPK4. Furthermore, another group of 46 genes repressed during keratinocyte differentiation were induced by RIPK4 knockout and repressed after YAP / TAZ knockdown. Figure 2 E), of which 39 genes are in the previously reported reference list of YAP / TAZ direct target genes ( Figure 2 F). Immunofluorescence staining of keratinocyte differentiation markers Involucrin and KRT10 further confirmed that RIPK4 knockout terminates calcium-induced differentiation in NHEK cells. Figure 2G). However, when YAP / TAZ is suppressed by siRNA, differentiation markers are constitutively expressed and no longer regulated by RIPK4 (G). Figure 2 G). Furthermore, during the differentiation of NHEK cells into skin organoids, RIPK4 knockout leads to thickening of the basal layer, reduced expression of differentiation markers, and nuclear enrichment of YAP / TAZ, suggesting its overactivation. Figure 2 These results indicate that RIPK4 regulates the activity of the Hippo signaling pathway in keratinocytes, meaning that RIPK4 promotes keratinocyte differentiation by inhibiting YAP / TAZ.

[0086] 3. RIPK4 promotes terminal differentiation of epidermal granular layer cells by inhibiting YAP / TAZ.

[0087] To determine the role of YAP / TAZ downstream of RIPK4 in in vivo epidermal differentiation, a systemic Ripk4 knockout mouse was constructed. - / - Consistent with publicly available results, Ripk4 - / - The mice had fused external orifices and died shortly after birth, likely due to asphyxiation and excessive dehydration associated with skin defects. (E18.5 Ripk4) - / - The embryo has sticky and shiny skin with significantly fewer skin folds. Figure 3 A). Toluidine blue staining revealed skin barrier defects, most severe on the head, back, extremities, and tail. Figure 3 B). In-depth analysis of the epidermal tissue revealed that histological analysis of the back skin sections confirmed the presence of E18.5Ripk4. - / - The proliferation of the granular and spinous layers in the epidermis, as well as the loss of the outermost keratinized capsule, are replaced by a thick layer of parakeratosis. Figure 3 C). Consistent with published results, the expression of the proliferation marker Ki67 remains confined to the basal layer, and in Ripk4... - / - No significant differences were found in the epidermis. Further immunohistochemical (IHC) analysis revealed ectopic expression of K14 in parakeratotic cells. Conversely, the expression of the late differentiation marker K10 was significantly reduced in the suprabasal layer. Furthermore, staining for the granular layer marker Loricrin confirmed the expansion of the granular layer. These results are consistent with previous findings regarding Ripk4. - / - Reports of epidermal differentiation defects are consistent. However, IHC assays using antibodies against the non-phosphorylated active form of YAP and against YAP / TAZ (which recognizes both YAP and TAZ) revealed aberrant activation of YAP / TAZ in both the granular and parakeratotic layers, with the most pronounced activation in the granular layer. Figure 3 C). Immunofluorescence staining of freshly isolated mouse primary keratinocytes also confirmed an increase in nuclear YAP / TAZ in Loricrin-positive granular cells. Figure 3 D). However, nuclear localization and proliferation of YAP / TAZ in the basal layer were not affected, indicating a specific role of RIPK4 in the differentiation of the upper basal layer, suggesting that RIPK4 plays a key role in epidermal differentiation by inhibiting YAP / TAZ in the granular layer.

[0088] To further determine the effect of Ripk4 knockout on the epidermal differentiation process, E18.5 wild-type and Ripk4... - / - Single-cell transcriptomes of the dorsal epidermis were compared. Two-dimensional maps were generated using uniform manifold approximation and projection (UMAP), revealing 11 distinct cell populations. Figure 3 E). Using gene set variation analysis (GSVA), the characteristic gene set co-regulated by RIPK4-YAP / TAZ previously identified in vitro was visualized. Figure 2 E) in Ripk4 - / - The most significantly dysregulated gene in the epidermal granular layer (GRN) (with an effect size of 0.31 for the gene positively correlated with YAP / TAZ) was... Figure 3 F). Pathway analysis showed that gene expression related to skin development and fatty acid / cholesterol metabolism was also present in Ripk4. - / - Significantly reduced in the granular layer (GRN) of the epidermis ( Figure 4 A). Furthermore, the mRNA and protein levels of these cholesterol biosynthesis genes (Fdft1, Mvd, Cyp51, and Nsdhl) are also regulated by RIPK4 and YAP / TAZ. Figure 4 B and Figure 4 C). Total cholesterol and free cholesterol levels in mouse primary keratinocytes were measured using a biochemical kit and Filipin III staining. It was found that cholesterol levels were reduced in RIPK4 knockout cells, and further knockdown of YAP / TAZ could salvage cholesterol levels. Figure 4 D and Figure 4 E). Furthermore, Cldn4 is also regulated by RIPK4 and YAP / TAZ ( Figure 4 B and Figure 4 C), while Cldn4 is a component of cell tight junctions and is crucial for epidermal barrier function. Interestingly, the IRF6 target gene Ovlo1 is also regulated by RIPK4 and YAP / TAZ ( Figure 4 B). In summary, these results indicate that RIPK4 plays a specific role in cholesterol synthesis and granular layer differentiation by inhibiting YAP / TAZ.

[0089] 4. RIPK4 promotes epidermal differentiation by inhibiting YAP / TAZ.

[0090] If YAP / TAZ activation plays a role in defective epidermal differentiation, then their knockout should be able to reverse the skin phenotype of Ripk4 knockout mice. Systemic YAP knockout leads to early embryonic death, making skin phenotype analysis difficult. Therefore, a keratinocyte-specific Ripk4 knockout (Ripk4 knockout) was constructed by crossing with Krt14-Cre transgenic mice. cKO ) and Yap knockout (YAP cKO These mice were further crossed with fertile TAZ knockout strains to produce Ripk4. cKO / Yap cKO / Taz KO Mouse (cTKO). Ripk4 cKO With Ripk4 - / - The phenotypes are similar, including shiny skin and postpartum death. However, Ripk4 cKO The mouse's mouth remained open normally, likely due to the late occurrence of the gene deletion. (Compared to Ripk4) - / - Similarly, Ripk4 cKO Skin barrier defects in mice occur on the head, limb tips, and back, but are more limited to the midline on the back. Figure 5 A).

[0091] It is well known that YAP / TAZ plays a crucial role in maintaining the basal layer of the epidermis; therefore, keratinocyte-specific transgenic expression of active YAP induces extensive proliferation and expansion of the basal layer. In contrast, YAP... cKO Mice died during the perinatal period with fragile skin and a lack of epidermal tissue coverage at the limb tips. cTKO embryos exhibited more pronounced, macroscopically visible skin damage, primarily concentrated on the ventral lateral aspect of the trunk, confirmed by the loss of skin barrier function. Notably, skin barrier function along the dorsal midline from head to rump was significantly restored in cTKO mice. Figure 5 A), and histological analysis showed that the epidermal thickness of cTKO was similar to that of the wild type, although mild parakeratosis was still visible in some areas. Figure 5 B). However, normal coverage of the keratinocytes was found in cTKO, which may be the reason for the restoration of barrier function. Figure 5 B). Ripk4 was also observed at the same time. cKO The epidermis thickens, while the epidermis thickness of cTKO will decrease to some extent. Figure 5 C). As expected, with Ripk4 - / - Similarly, Ripk4 cKO Increased nuclear-localized YAP / TAZ was observed in the granular layer and parakeratotic layer of the dorsal epidermis, but no signal was found in the epidermis of cTKO mice, suggesting good antibody specificity. Figure 5B). However, although IRF6 has been identified as an important downstream target effector molecule mediated by RIPK4 in epidermal differentiation, keratinocyte-specific Irf6 knockout (Irf6 knockout) cKO The barrier function of the mouse's dorsal midline was intact, similar to that of Ripk4. cKO Significant differences were observed in mice.

[0092] Since YAP / TAZ also plays a crucial role in the basal layer, we further explored differentiation layer-specific knockout of YAP / TAZ in skin organoid models. First, from E18.5Ripk4... - / - / YAP f / f / TAZ f / f Primary keratinocytes were isolated from embryos, and HA-labeled Cre recombinase was knocked into the downstream of the differentiation marker Krt10 promoter. Figure 5 D), Immunoblotting revealed that Krt10 and HA-Cre were not expressed under basal conditions, but both were expressed when cells differentiated via an air / liquid interface method. Figure 5 E). Five days after induction of differentiation, active YAP was unaffected in the basal layer, but was knocked out in the Dsg1-labeled differentiation layer in the HA-Cre expression group. Figure 5 F). Similar to those observed in vivo, organoid thickness increased with RIPK4 KO and was rescued by Krt10-driven YAP / TAZ KO ( Figure 5 F and Figure 5 G). Furthermore, the lack of stratum corneum in the RIPK4 KO group was also salvaged by the differentiation layer YAP / TAZ KO ( Figure 5 These results indicate that RIPK4 promotes epidermal differentiation at least in part through YAP / TAZ inhibition.

[0093] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and not restrictive.

Claims

1. The application of a non-classical Hippo-based signal modulation method in diseases caused by RIPK4 inactivation mutations, characterized in that, By constructing YAP / TAZ inhibitors to inhibit the expression or activity of YAP / TAZ, drugs can be prepared to treat or alleviate diseases caused by RIPK4 inactivation mutations. The YAP / TAZ inhibitors include molecular inhibitors that knock down or eliminate the YAP / TAZ gene; the molecular inhibitors include interfering RNA, which includes siRNAs used to knock down YAP / TAZ as shown in SEQ ID NO. 1-4.

2. The application according to claim 1, characterized in that, The molecular inhibitors for knocking out the YAP / TAZ gene also include sgRNAs used to knock out YAP / TAZ, as shown in SEQ ID NO. 5 and 6.

3. The application according to claim 1, characterized in that, The YAP / TAZ inhibitor includes an overexpression agent for the RIPK4 gene; the overexpression agent includes a vector for overexpressing RIPK4, and the primer sequences used to construct the RIPK4 overexpression vector are shown in SEQ ID NO.7 and 8.

4. The application according to claim 1, characterized in that, The YAP / TAZ inhibitor also includes small molecule inhibitors; the small molecule inhibitors include pan-TEAD inhibitors.

5. The application according to claim 1, characterized in that, By constructing RIPK4 inhibitors to inhibit the expression or activity of the RIPK4 gene and activate the expression or activity of YAP / TAZ, the phenotype of disease caused by RIPK4 inactivation mutations can be simulated, thus creating a disease model caused by RIPK4 inactivation mutations. The RIPK4 inhibitor includes interfering RNA, which includes sgRNA used to knock out RIPK4, as shown in SEQ ID NO. 9 and 10.

6. The application according to claim 1 or 5, characterized in that, Diseases caused by the RIPK4 inactivation mutation include Bartsocas-Papas syndrome.

7. A drug, characterized in that, The active ingredient includes any one of the following: siRNA as described in claim 1, sgRNA as described in claim 2, RIPK4 overexpression vector as described in claim 3, and small molecule inhibitor as described in claim 4.

8. Application of dysregulated YAP / TAZ and abnormally expressed cholesterol synthesis genes as diagnostic biomarkers for diseases caused by RIPK4 inactivation mutations.

9. The application according to claim 8, characterized in that, The mRNA expression level of the abnormally expressed cholesterol synthesis gene was detected by real-time PCR, and the primer sequences are shown in SEQ ID NO.11-18.

10. The application according to claim 9, characterized in that, Cholesterol synthesis genes include Fdft1, Mvd, Cyp51, and Nsdhl.

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