Yes-associated Protein Research Articles

Salsolinol as an RNA m6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy.

JOURNAL/nrgr/04.03/01300535-202503000-00032/figure1/v/2024-06-17T092413Z/r/image-tiff Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, Sal) is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an environmental toxin that causes Parkinson's disease. However, the mechanism by which Sal mediates dopaminergic neuronal death remains unclear. In this study, we found that Sal significantly enhanced the global level of N6-methyladenosine (m6A) RNA methylation in PC12 cells, mainly by inducing the downregulation of the expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway. The m6A reader YTH domain-containing family protein 2 (YTHDF2) promoted the degradation of m6A-containing Yes-associated protein 1 (YAP1) mRNA, which is a downstream key effector in the Hippo signaling pathway. Additionally, downregulation of YAP1 promoted autophagy, indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity. These findings reveal the role of Sal on m6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy. Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis

ObjectivesThis study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DesignA time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. ResultsThe ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. ConclusionsThese findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

The role of YAP/TAZ on joint and arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common forms of arthritis with undefined etiology and pathogenesis. Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ), which act as sensors for cellular mechanical and inflammatory cues, have been identified as crucial players in the regulation of joint homeostasis. Current studies also reveal a significant association between YAP/TAZ and the pathogenesis of OA and RA. The objective of this review is to elucidate the impact of YAP/TAZ on different joint tissues and to provide inspiration for further studying the potential therapeutic implications of YAP/TAZ on arthritis. Databases, such as PubMed, Cochran Library, and Embase, were searched for all available studies during the past two decades, with keywords "YAP," "TAZ," "OA," and "RA."

YAP and TAZ differentially regulate postnatal cortical progenitor proliferation and astrocyte differentiation.

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of β1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.

Leveraging the Fragment Molecular Orbital and MM-GBSA Methods in Virtual Screening for the Discovery of Novel Non-Covalent Inhibitors Targeting the TEAD Lipid Binding Pocket.

The Hippo pathway controls organ size and homeostasis and is linked to numerous diseases, including cancer. The transcriptional enhanced associate domain (TEAD) family of transcription factors acts as a receptor for downstream effectors, namely yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which binds to various transcription factors and is essential for stimulated gene transcription. YAP/TAZ-TEAD facilitates the upregulation of multiple genes involved in evolutionary cell proliferation and survival. TEAD1-4 overexpression has been observed in different cancers in various tissues, making TEAD an attractive target for drug development. The central drug-accessible pocket of TEAD is crucial because it undergoes a post-translational modification called auto-palmitoylation. Crystal structures of the C-terminal TEAD complex with small molecules are available in the Protein Data Bank, aiding structure-based drug design. In this study, we utilized the fragment molecular orbital (FMO) method, molecular dynamics (MD) simulations, shape-based screening, and molecular mechanics-generalized Born surface area (MM-GBSA) calculations for virtual screening, and we identified a novel non-covalent inhibitor-BC-001-with IC50 = 3.7 μM in a reporter assay. Subsequently, we optimized several analogs of BC-001 and found that the optimized compound BC-011 exhibited an IC50 of 72.43 nM. These findings can be used to design effective TEAD modulators with anticancer therapeutic implications.

The reversal of PXR or PPARα activation-induced hepatomegaly

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

The cellular localization and oncogenic or tumor suppressive effects of angiomiotin-like protein 2 in tumor and normal cells.

Angiomiotin (AMOT) family comprises three members: AMOT, AMOT-like protein 1 (AMOTL1), and AMOT-like protein 2 (AMOTL2). AMOTL2 is widely expressed in endothelial cells, epithelial cells, and various cancer cells. Specifically, AMOTL2 predominantly localizes in the cytoplasm and nucleus in human normal cells, whereas associates with cell-cell junctions and actin cytoskeleton in non-human cells, and locates at cell junctions or within the recycling endosomes in cancer cells. AMOTL2 is implicated in regulation of tube formation, cell polarity, and shape, although the specific impact on tumorigenesis remains to be conclusively determined. It has been shown that AMOTL2 enhances tumor growth and metastasis in pancreatic, breast, and colon cancer, however inhibits cell proliferation and migration in lung, hepatocellular cancer, and glioblastoma. In addition to its role in cell shape and cytoskeletal dynamics through co-localization with F-actin, AMOTL2 modulates the transcription of Yes-associated protein (YAP) by binding to it, thereby affecting its phosphorylation and cellular sequestration. Furthermore, the stability and cellular localization of AMOTL2, influenced by its phosphorylation and ubiquitination mediated by specific proteins, affects its cellular function. Additionally, we observe that AMOTL2 is predominantly downregulated in some tumors, but significantly elevated in colorectal adenocarcinoma (COAD). Moreover, overall analysis, GSEA and ROC curve analysis indicate that AMOTL2 exerts as an oncogenic protein in COAD by modulating Wnt pathway, participating in synthesis of collagen formation, and interacting with extracellular matrix receptor. In addition, AMOTL2 potentially regulates the distribution of immune cells infiltration in COAD. In summary, AMOTL2 probably functions as an oncogene in COAD. Consequently, further in-depth mechanistic research is required to elucidate the precise roles of AMOTL2 in various cancers.

Association of low Angiomotin-p130 and high YAP1 nuclear expression with adverse prognosis in epithelial ovarian cancer.

The aim of our study was to examine the association of Angiomotin (Amot-p130) and Yes-associated protein 1 (YAP1) expressions and their prognostic significance in epithelial ovarian cancer (EOC). A total of 100 primary EOC samples were obtained for immunohistochemical analysis of Amot-p130 and YAP1 expressions. Correlation analysis was performed between Amot-p130 or YAP1 and clinical factors. The overall survival time was calculated. Low Amot-p130 and high YAP1 nuclear expression were identified in 34 and 56 of 100 EOC tissues, respectively. Both low Amot-p130 and high YAP1 nuclear expression were associated with advanced tumor stage, high-grade carcinoma, and non-response to chemotherapy (p<0.05). They were also associated with shorter overall survival time (p<0.05) by log-rank test. A marker of low Amot-p130 and high YAP1 expression was associated with high-grade ovarian carcinoma, late-stage disease, non-response to chemotherapy, and shorter overall survival time (p<0.05). Low Amot-p130 and high YAP1 nuclear expression can provide additional prognostic information for patients with EOC. A marker of low Amot-p130 and high YAP1 expression may be a potent predictor of poor prognosis in patients with epithelial ovarian cancer.

HAX1-Overexpression Augments Cardioprotective Efficacy of Stem Cell-Based Therapy Through Mediating Hippo-Yap Signaling

Although stem/progenitor cell therapy shows potential for myocardial infarction repair, enhancing the therapeutic efficacy could be achieved through additional genetic modifications. HCLS1-associated protein X-1 (HAX1) has been identified as a versatile modulator responsible for cardio-protective signaling, while its role in regulating stem cell survival and functionality remains unknown. In this study, we investigated whether HAX1 can augment the protective potential of Sca1+ cardiac stromal cells (CSCs) for myocardial injury. The overexpression of HAX1 significantly increased cell proliferation and conferred enhanced resistance to hypoxia-induced cell death in CSCs. Mechanistically, HAX1 can interact with Mst1 (a prominent conductor of Hippo signal transduction) and inhibit its kinase activity for protein phosphorylation. This inhibition led to enhanced nuclear translocation of Yes-associated protein (YAP) and activation of downstream therapeutic-related genes. Notably, HAX1 overexpression significantly increased the pro-angiogenic potential of CSCs, as demonstrated by elevated expression of vascular endothelial growth factors. Importantly, implantation of HAX1-overexpressing CSCs promoted neovascularization, protected against functional deterioration, and ameliorated cardiac fibrosis in ischemic mouse hearts. In conclusion, HAX1 emerges as a valuable and efficient inducer for enhancing the effectiveness of cardiac stem or progenitor cell therapeutics.Graphical

Amplification of Hippo Signaling Pathway Genes Is Governed and Implicated in the Serous Subtype-Specific Ovarian Carcino-Genesis.

Among women, ovarian cancer ranks as the fifth most common cause of cancer-related deaths. This study examined the impact of Hippo signaling pathway on ovarian carcinogenesis. Therefore, the signatures related to Hippo signaling pathway were derived from the molecular signatures database (MSigDB) and were used for further analysis. The Z score-based pathway activation scoring method was employed to investigate the expression patterns of these signatures in the mRNA expression profiles of ovarian cancer cohorts. Compared to other subtype tumors, the results of this study show that the Hippo signaling pathway signatures are dysregulated prominently in serous subtype-specific ovarian carcinogenesis. A receiver operating characteristic (ROC) curve-based results of the Hippo gene set, yes-associated protein 1 (YAP1), and mammalian sterile 20-like kinases 1 (MST1) genes can predict the serous subtype tumors by higher specificity and sensitivity with significant areas under the curve values also further reconfirmed these signaling dysregulations. Moreover, these gene sets were studied further for mutation analysis in the profile of high-grade serous ovarian adenocarcinoma in the cBioPortal database. The OncoPrint results reveal that these Hippo signaling pathway genes are amplified highly during the grade three and stage third or fourth of serous type ovarian tumors. In addition, the results of the Dependency Map (DepMap) plot also clearly show that these genes are amplified significantly across the ovarian cancer cell lines. Finally, overall survival (OS) curve plot investigations also revealed that these gene expressions show poor survival patterns linked to highly expressed conditions in serous subtypes of ovarian cancer patients with significant p-values (p < 0.05). Thus, the current finding would help to develop the targeted therapies treatment for serous subtype ovarian carcinogenesis.

Discovery of potent small molecule ubiquitin-specific protease 10 inhibitors with anti-hepatocellular carcinoma activity through regulating YAP expression

High expression of ubiquitin-specific protease 10 (USP10) promote the proliferation of hepatocellular carcinoma (HCC), thus the development of USP10 inhibitors holds promise as a novel therapeutic approach for HCC treatment. However, the development of selective USP10 inhibitor is still limited. In this study, we developed a novel USP10 inhibitor for investigating the feasibility of targeting USP10 for the treatment of HCC. Due to high USP10 inhibition potency and prominent selectivity, compound D1 bearing quinolin-4(1H)-one scaffold was identified as a lead compound. Subsequent research revealed that D1 significantly inhibits cell proliferation and clone formation in HCC cells. Mechanistic insights indicated that D1 targets the ubiquitin pathway, facilitating the degradation of YAP (Yes-associated protein), thereby triggering the downregulation of p53 and its downstream protein p21. Ultimately, this cascade leads to S-phase arrest in HCC cells, followed by cell apoptosis. Collectively, our findings highlight D1 as a promising starting point for USP10-positive HCC treatment, underscoring its potential as a vital tool for unraveling the functional intricacies of USP10.

The Hippo signalling pathway in bone homeostasis: Under the regulation of mechanics and aging.

The Hippo signalling pathway is a conserved kinase cascade that orchestrates diverse cellular processes, such as proliferation, apoptosis, lineage commitment and stemness. With the onset of society ages, research on skeletal aging-mechanics-bone homeostasis has exploded. In recent years, aging and mechanical force in the skeletal system have gained groundbreaking research progress. Under the regulation of mechanics and aging, the Hippo signalling pathway has a crucial role in the development and homeostasis of bone. We synthesize the current knowledge on the role of the Hippo signalling pathway, particularly its downstream effectors yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), in bone homeostasis. We discuss the regulation of the lineage specification and function of different skeletal cell types by the Hippo signalling pathway. The interactions of the Hippo signalling pathway with other pathways, such as Wnt, transforming growth factor beta and nuclear factor kappa-B, are also mentioned because of their importance for modulating bone homeostasis. Furthermore, YAP/TAZ have been extensively studied as mechanotransducers. Due to space limitations, we focus on reviewing how mechanical forces and aging influence cell fate, communications and homeostasis through a dysregulated Hippo signalling pathway.

Gut Microbiota Affects Mouse Pregnane X Receptor Agonist Pregnenolone 16α-Carbonitrile-Induced Hepatomegaly by Regulating Pregnane X Receptor and Yes-Associated Protein Activation.

Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis, and the gut microbiota is closely linked to liver physiologic and pathologic status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota contributes to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist pregnenolone 16α-carbonitrile (PCN) for 5 days. Depletion of gut microbiota was achieved using broad-spectrum antibiotics (ABX) and fecal microbiota transplantation (FMT) was performed to restore the gut microbia. The composition of gut microbiota was analyzed by 16S rRNA sequencing, while the expression of PXR, YAP, and their downstream target genes and proteins were assessed. The results indicated that PCN treatment altered the composition and abundance of specific bacterial taxa. Furthermore, depletion of gut microbiota using ABX significantly attenuated PCN-induced hepatomegaly. FMT experiments further demonstrated that the fecal microbiota from PCN-treated mice could induce liver enlargement. Mechanistic studies revealed that ABX treatment impeded the PXR and YAP activation induced by PCN, as evidenced by decreased expression of PXR, YAP, and their downstream targets. Moreover, alterations in PXR and YAP activation were likely contributing to hepatomegaly in recipient mice following FMT from PCN-treated mice. Collectively, the current study demonstrated that gut microbiota is involved in PCN-induced hepatomegaly via regulating PXR and YAP activation, providing potential novel insights into the involvement of gut microbiota in PXR-mediated hepatomegaly. SIGNIFICANCE STATEMENT: This work describes that the composition of gut microbiota is altered in mouse pregnane X receptor (PXR) agonist pregnenolone 16α-carbonitrile (PCN)-induced hepatomegaly. Treatment with an antibiotic cocktail depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Additionally, fecal microbiota transplantation from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and yes-associated protein activation.

YAP prevents senescence of dermal fibroblast and inhibits melanogenesis via paracrine effect of DKK1.

Senile skin hyperpigmentation displays remarkable histopathological features of dermal aging. The crosstalk between melanocytes and dermal fibroblasts plays crucial roles in aging-related pigmentation. While senescent fibroblasts can upregulate pro-melanogenic factors, the role of anti-melanogenic factors, such as dickkopf1 (DKK1), and the upstream regulatory mechanism during aging remain obscure. This study investigated the roles of yes-associated protein (YAP) and DKK1 in the regulation of dermal fibroblast senescence and melanogenesis. Our findings demonstrated decreased YAP activity and DKK1 levels in intrinsic and extrinsic senescent fibroblasts. YAP depletion induced fibroblast senescence and downregulated the expression and secretion of DKK1, whereas YAP overexpression partially reversed the effect. The transcriptional regulation of DKK1 by YAP was supported by dual-luciferase reporter and chromatin immunoprecipitation assays. Moreover, YAP depletion in fibroblasts upregulated Wnt/β-catenin in melanocytes and stimulated melanogenesis, which was partially rescued by the re-supplementation of DKK1. Conversely, overexpression of YAP in senescent fibroblasts decreased Wnt/β-catenin levels in melanocytes and inhibited melanogenesis. Additionally, reduced levels of YAP and DKK1 were verified in the dermis of solar lentigines. These findings suggest that, during skin aging, epidermal pigmentation may be influenced by YAP in the dermal microenvironment via the paracrine effect of DKK1.

Yes-associated Protein Mediates Acute Lung Injury in Adult but not Juvenile Mice

Objective: Adults with The Acute Respiratory Distress Syndrome (ARDS) have worse outcomes than children with Pediatric ARDS. This finding can be reproduced in animal models, suggesting a difference in Acute Lung Injury (ALI) mechanisms. Understanding these mechanistic differences could inform age-specific treatment. Hypothesis: Since Yes-associated protein (YAP) is important for lung development and reactive to mechanical stretch, we hypothesized that YAP is important for ALI mechanisms. Methods: To induce ALI, we intranasally instilled mice with Pseudomonas aeruginosa strain K (2.5 x 105 colony forming units (c.f.u.)) in juvenile (3-week-old) and adult (10-week-old) C57BL/6J mice. 24 h later, we collected bronchoalveolar lavage (BAL) fluid and measured protein, cell count, and c.f.u. To knockdown YAP expression, we injected siRNA 24 h before P. aeruginosa instillation. We removed the lungs and enriched for CD45+ and CD41+ fraction, and CD31+ fraction using Dynabeads sheep anti-rat IgG (Invitrogen). In separate experiments, lungs were separated into nuclear and cytosolic fractions for immunoblot analysis. For immunoprecipitation, the cytosolic fraction was added to beads coated with anti-YAP antibody (rabbit monoclonal). We washed the beads and eluted bound proteins, which were identified by label-free mass spectrometry. Data and results: While both juvenile and adult mice had significantly increased BAL cell counts and protein 24 h after P. aeruginosa instillation, adult mice had significantly greater cell counts and protein than juveniles. BAL c.f.u. were similar in both groups, suggesting equal instillation of P. aeruginosa. P. aeruginosa instillation increased whole lung YAP expression in both cytosolic and nuclear fractions in adult mice by &gt;3-fold, but YAP expression was not increased in P. aeruginosa-treated juvenile mice. YAP protein increases occurred in CD31+ fraction (endothelial cells), but no YAP was quantified in CD45+/CD41+ fraction (leukocytes and platelets). Vascular knockdown of YAP expression by ~75% in adult mice blocked P. aeruginosa-induced BAL cell count increases. Proteomics analysis revealed 62 proteins that had significantly altered YAP binding upon P. aeruginosa treatment (47 with increased YAP binding). The complement factors H and I had a log2-fold-change for YAP binding of 4.56 and 2.26, respectively, upon P. aeruginosa treatment. Conclusions: Adult mice exhibited YAP-dependent Acute Lung Injury; juveniles did not. YAP-dependent mechanisms of injury may be secondary to YAP binding to the complement inhibitory proteins H and I. More work is needed to establish the cell-specificity of YAP-complement factor binding and to determine the location of complement signaling. HL148403, Parker B Francis, and Columbia University Department of Pediatrics Children’s Health Innovation Nucleation Fund to RFH; Ernest E Just Summer Scholarship to AMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3142: Pik3c3 Safeguards Smooth Muscle Identity By Promoting Lysosome-mediated Yap1 Degradation

Background: Mature vascular smooth muscle cells (VSMCs) are quiescent and geared for maintaining vascular homeostasis by expressing a unique repertoire of contractile genes. However, key factors in controlling the contractile phenotype of VSMCs are poorly defined. Class III PI3K, PIK3C3 (Phosphatidylinositol 3-Kinase Catalytic Subunit Type 3) is a critical regulator of vesicular trafficking. Although PIK3C3 is known to play an essential role in cardiac homeostasis, its role in VSMCs remains elusive. Methods: To elucidate the role of Pik3c3 in regulating SMC fate and plasticity, we generated smooth muscle-specific Pik3c3 inducible knock out (KO) mice with SMC-lineage tracing. Proteomic mass spectrometry, next-generation RNA sequencing, SMC fate mapping coupled with single-cell RNA sequencing were applied to determine the phenotype and trajectories of SMCs after Pik3c3 ablation. Results: Specific ablation of Pik3c3 in SMCs results in severe gastrointestinal pseudo-obstruction and premature death. Analysis of aorta from the KO mice revealed dramatic vascular remodeling including dilatation, thickening of the vascular wall, spontaneous neointimal formation that was largely contributed by cells of SMC origin, and complete loss of contractility. Proteomics, bulk RNA-seq, and scRNA-seq studies of SMC-restricted Pik3c3 KO mouse aorta showed near complete loss of smooth muscle identity and the acquisition of an intermediate smooth muscle progenitor-like cell phenotype that underwent re-programming into multiple cell types including macrophages, T cells and osteoblasts. Mechanistically, we found that Pik3c3 deletion in VSMCs induces the accumulation of YAP1 (Yes associated protein 1) and its downstream target genes; this elevated YAP1 program appears to result from a blockade of autophagic lysosome-mediated degradation of YAP1, leading to phenotypic modulation of SMCs. Importantly, pharmacological inhibition of YAP by Verteporfin or genetic deletion of Yap1 rescues the vascular phenotype of Pik3c3 SM-specific KO mice. Conclusion: Our study reveals PIK3C3 as a master regulator safeguarding VSMC identity, and perturbation of the PIK3C3-YAP axis may be a novel mechanism underlying VSMC-driven vascular diseases in humans.

CD47, a novel YAP target gene, contributes to hepatic stellate cell activation and liver fibrosis induced by high-fat diet

Activated hepatic stellate cells (HSCs) have been widely recognized as a primary source of pathological myofibroblasts, leading to the accumulation of extracellular matrix and liver fibrosis. CD47, a transmembrane glycoprotein expressed on the surface of various cell types, has been implicated in non-alcoholic fatty liver disease. However, the precise role of CD47 in HSC activation and the underlying regulatory mechanisms governing CD47 expression remain poorly understood. In this study, we employed single-cell RNA sequencing analysis to investigate CD47 expression in HSCs from mice subjected to a high-fat diet. CD47 silencing in HSCs markedly inhibited the expression of fibrotic genes and promoted apoptosis. Mechanistically, we found that Yes-associated protein (YAP) collaborates with TEAD4 to augment the transcriptional activation of CD47 by binding to its promoter region. Notably, disruption of the interaction between YAP and TEAD4 caused a substantial decrease in CD47 expression in HSCs and reduced the development of high-fat diet-induced liver fibrosis. Our findings highlight CD47 as a critical transcriptional target of YAP in promoting HSC activation in response to a high-fat diet. Targeting the YAP/TEAD4/CD47 signaling axis may hold promise as a therapeutic strategy for liver fibrosis.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

Abstract 104: Endothelial Restoration Of CAD GWAS Gene PLPP3 By Nanomedicine Suppresses Mechano-sensitive YAP/TAZ Activity And Reduces Atherosclerosis In Vivo

Genome-wide association studies (GWAS) have suggested new molecular mechanisms driving vascular complications such as coronary artery disease (CAD). Nevertheless, a major challenge to developing new therapeutic approaches is to spatiotemporally manipulate these GWAS-identified genes in specific vascular tissues of interest in vivo . Atherosclerosis largely occurs in arterial sites where local disturbed flow (DF) activates vascular endothelial cells. The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) have merged as critical transcriptional regulators responding to biomechanical stimuli. The molecular mechanisms by which DF activates while unidirectional flow (UF) inactivates YAP/TAZ remain incompletely understood. Our recent studies demonstrated that DF and genetic predisposition at the CAD locus 1p32.2 cooperatively suppress endothelial phospholipid phosphatase 3 (PLPP3) transcription by inactivating a mechano-sensitive endothelial enhancer; reduced PLPP3 and consequent activation of lipid lysophosphatidic acid (LPA)-receptor signaling, drive endothelial dysfunction. Here we devised a nanomedicine strategy employing VCAM-1-targeting liposome to preferentially deliver chemically-modified PLPP3 mRNA to inflamed endothelial cells in vivo , effectively suppressing endothelial YAP/TAZ activity and reducing DF-induced atherosclerosis in mice. We discovered a molecular paradigm by which PLPP3 inactivates YAP/TAZ by reducing LPA-induced myosin II and ROCK1. These results highlight a new mechanistic link between GWAS and YAP/TAZ mechano-regulation and moreover, establish the proof of concept of vascular wall-based therapies employing targeted mRNA-based nanomedicine.

Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms.

Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.