Yes-associated Protein Activation Research Articles

Simulated microgravity attenuates skin wound healing by inhibiting dermal fibroblast migration via F-actin/YAP signaling pathway.

Skin and its cell components continuously subject to extrinsic and intrinsic mechanical forces and are mechanical sensitive. Disturbed mechanical homeostasis may lead to changes in skin functions. Gravity is the integral mechanical force on the earth, however, how gravity contributes to the maintenance of skin function and how microgravity in space affects the wound healing are poorly understood. Here, using microgravity analogs, we show that simulated microgravity (SMG) inhibits the healing of cutaneous wound and the accumulation of dermal fibroblasts in the wound bed. In vitro, SMG inhibits the migration of human foreskin fibroblast cells (HFF-1), and decreases the F-actin polymerization and YAP (yes-associated protein) activity. The SMG-inhibited migration can be recovered by activating YAP or F-actin polymerization using lysophosphatidic acid (LPA) or jasplakinolide (Jasp), suggesting the involvement of F-actin/YAP signaling pathway in this process. In SMG rats, LPA treatment improves the cutaneous healing with increased dermal fibroblasts in the wound bed. Together, our results demonstrate that SMG attenuates the cutaneous wound healing by inhibiting dermal fibroblast migration, and propose the crucial role of F-actin/YAP mechano-transduction in the maintenance of skin homeostasis under normal gravity, and YAP as a possible therapeutic target for the skin care of astronauts in space.

THU497 Elucidating The Role Of The IQGAP1-YAP Axis In ECM Stiffness-Mediated Hepatocellular Carcinoma Progression

Abstract Disclosure: S.E. Hobbs: None. H. Kimmel: None. G. Underhill: None. S. Anakk: None. Hepatocellular Carcinoma (HCC), the primary form of liver cancer, is the second leading cause of cancer associated-associated deaths worldwide. Complex signaling mechanisms and several cellular processes are coordinated using scaffolding proteins. IQ motif-containing GTPase Activating Protein 1 (IQGAP1) is a scaffolding protein that is overexpressed in multiple epithelial cancers, including ovarian, breast, and liver. Previous work has shown that overexpression of IQGAP1 activates Yes-associated Protein (YAP) to promote HCC in mice. Extracellular matrix (ECM) stiffness, due to liver fibrosis in HCC, can activate YAP to promote tumor progression. Remarkably, our preliminary data show that IQGAP1 has increased localization at the plasma membrane when HepG2 cells (an HCC cell line) are seeded onto a stiff ECM (25 kPa). YAP is modulated by actin dynamics, which can be altered by changes in the plasma membrane. Therefore, IQGAP1’s movement to the plasma membrane can potentially activate YAP by modulating actin dynamics. We also found that the anti-HCC drug sorafenib, can induce the expression IQGAP1 and activate YAP in HepG2 cells grown on plastic. These sorafenib-mediated changes are counterproductive and can contribute to drug resistance of HCC. Finally, we found that inhibition of YAP in HepG2 cells significantly decreases IQGAP1 expression, indicating that there is bidirectional crosstalk between the two proteins. Future directions will elucidate if loss of IQGAP1 decreases ECM stiffness-induced YAP activation, proliferation, and migration in HCC cells. In addition, we will determine if ECM stiffness alters sorafenib-mediated IQGAP1 upregulation, as ECM stiffness has been linked to drug resistance in HCC. Overall, this work will shed light on the potential role of the IQGAP1-YAP axis in ECM stiffness-mediated HCC progression. Presentation: Thursday, June 15, 2023

ANNEXIN A2 FACILITATES NEOVASCULARIZATION TO PROTECT AGAINST MYOCARDIAL INFARCTION INJURY VIA INTERACTING WITH MACROPHAGE YAP AND ENDOTHELIAL INTEGRIN Β3.

Cardiac macrophages with different polarization phenotypes regulate ventricular remodeling and neovascularization after myocardial infarction (MI). Annexin A2 (ANXA2) promotes macrophage polarization to the repair phenotype and regulates neovascularization. However, whether ANXA2 plays any role in post-MI remodeling and its underlying mechanism remains obscure. In this study, we observed that expression levels of ANXA2 were dynamically altered in mouse hearts upon MI and peaked on the second day post-MI. Using adeno-associated virus vector-mediated overexpression or silencing of ANXA2 in the heart, we also found that elevation of ANXA2 in the infarcted myocardium significantly improved cardiac function, reduced cardiac fibrosis, and promoted peri-infarct angiogenesis, compared with controls. By contrast, reduction of cardiac ANXA2 exhibited opposite effects. Furthermore, using in vitro coculture system, we found that ANXA2-engineered macrophages promoted cardiac microvascular endothelial cell (CMEC) proliferation, migration, and neovascularization. Mechanistically, we identified that ANXA2 interacted with yes-associated protein (YAP) in macrophages and skewed them toward pro-angiogenic phenotype by inhibiting YAP activity. In addition, ANXA2 directly interacted with integrin β3 in CMECs and enhanced their growth, migration, and tubule formation. Our results indicate that increased expression of ANXA2 could confer protection against MI-induced injury by promoting neovascularization in the infarcted area, partly through the inhibition of YAP in macrophages and activation of integrin β3 in endothelial cells. Our study provides new therapeutic strategies for the treatment of MI injury.

New Insights into the Roles of the Hippo Signaling Pathway in Stem Cell Biology

: A highly conserved kinase cascade, including mammalian STE20-like protein kinases (MSTs), large tumor suppressors (LATSs), and downstream transcription coactivators containing YES-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are involved in the Hippo signaling pathway. As two critical transcriptional coactivators, TAZ and YAP are adversely regulated by the Hippo signaling pathway. They lead to the induction of organ regeneration and the expression of cell growth and division-promoting genes through binding to transcription factors, especially TEA domain transcription factors (TEADs). Previous studies highlight the role of the Hippo pathway in organ size control, homeostasis, cell proliferation, and tumor development. Research has shown this pathway's vital role in cancer stem cell biology, including self-renewal and drug resistance. Improper activation of YAP and TAZ through the Hippo pathway dysregulation or its increased expression can accelerate tumorigenesis. Thus, pharmacological inhibition of YAP and TAZ is suggested as a promising approach in the treatment of tumors with high YAP/TAZ activity.

YAP mediates resistance to EGF-induced apoptosis in EGFR-mutated non-small cell lung cancer cells

Mechanisms underlying the growth and survival of non-small cell lung cancer (NSCLC) cells positive for activating mutations of the epidermal growth factor receptor gene (EGFR) have remained unclear. We here examined the functional relation between such mutant forms of EGFR and Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway that regulates cell proliferation and survival. Under the condition of serum deprivation, epidermal growth factor (EGF) induced activation of YAP in NSCLC cell lines positive for mutated EGFR but not in those wild type (WT) for EGFR. Similar EGF-induced activation of YAP was apparent in A549 lung cancer cells forcibly expressing mutant EGFR but not in those overexpressing the WT receptor. Furthermore, EGF induced apoptotic cell death in serum-deprived A549 cells overexpressing the WT form of EGFR but not in those expressing mutant EGFR, and knockdown of YAP rendered the latter cells sensitive to this effect of EGF. Our results thus suggest that activation of YAP mediates resistance of EGFR-mutated NSCLC cells to EGF-induced apoptosis and thereby contributes specifically to the survival of such cells.

Different stimuli induce endothelial dysfunction and promote atherosclerosis through the Piezo1/YAP signaling axis

Vascular endothelial dysfunction is the initial step in atherosclerosis (AS). AS tends to occur at vascular bifurcations and curves, and endothelial cells(ECs) are highly susceptible to injury due to mechanical forces induced by disturbed flow (DF) with inconsistent blood flow directions. However, the pathogenesis of endothelial cell dysfunction in AS remains unclear and needs further study. Here, we found that Piezo1 expression was significantly increased in DF- and oxidized low-density lipoprotein(ox-LDL)-treated HUVECs in vitro and a model of atherosclerotic plaque growth in ApoE−/− mice fed a Western diet. Furthermore, Piezo1 upregulated autophagy levels in the HUVECs model, which was reversed by Piezo1 knockdown with a lentivirus-mediated shRNA system. Mechanistically, the level of Yes-associated protein (YAP), a transcriptional coactivator in the Hippo pathway, was significantly elevated in the DF- and ox-LDL-induced HUVECs model, and this effect was further inhibited by Piezo1 knockdown. Moreover, the Piezo1 agonist Yoda1 inhibited the protein level of microtubule-associated protein 1 light chain 3-II(LC3-II) and increased the protein level of sequestosome1(p62/SQSTM1) in a dose-dependent manner, while significantly promoting the protein expression and nuclear translocation of YAP. The YAP inhibitor CA3 weakened Yoda1-mediated inhibition of autophagy. Our results suggest that Piezo1 may regulate endothelial autophagy by promoting YAP activation and nuclear translocation, thereby contributing to vascular endothelial dysfunction.

Deletion of Endothelial TRPV4 Protects Heart From Pressure Overload-Induced Hypertrophy.

Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4ECKO) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, ECM gels of varying stiffness, unbiased RNA sequencing, siRNA, Western blot, qPCR, and confocal immunofluorescence techniques. We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4lox/lox mice, 28 days post-transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα NOS3, and FGF2) with concomitant increase in microvascular density in TRPV4ECKO hearts after transverse aortic constriction compared with TRPV4lox/lox. Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/LATS pathway. Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.

The TGFβ→TAK1→LATS→YAP1 Pathway Regulates the Spatiotemporal Dynamics of YAP1.

The Hippo kinase cascade functions as a central hub that relays input from the "outside world" of the cell and translates it into specific cellular responses by regulating the activity of Yes-associated protein 1 (YAP1). How Hippo translates input from the extracellular signals into specific intracellular responses remains unclear. Here, we show that transforming growth factor β (TGFβ)-activated TAK1 activates LATS1/2, which then phosphorylates YAP1. Phosphorylated YAP1 (p-YAP1) associates with RUNX3, but not with TEAD4, to form a TGFβ-stimulated restriction (R)-point-associated complex which activates target chromatin loci in the nucleus. Soon after, p-YAP1 is exported to the cytoplasm. Attenuation of TGFβ signaling results in re-localization of unphosphorylated YAP1 to the nucleus, where it forms a YAP1/TEAD4/SMAD3/AP1/p300 complex. The TGFβ-stimulated spatiotemporal dynamics of YAP1 are abrogated in many cancer cells. These results identify a new pathway that integrates TGFβ signals and the Hippo pathway (TGFβ→TAK1→LATS1/2→YAP1 cascade) with a novel dynamic nuclear role for p-YAP1.

Genetic and Pharmacological YAP Activation Induces Proliferation and Improves Survival in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocyte loss following myocardial infarction cannot be addressed with current clinical therapies. Cell therapy with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a potential approach to replace cardiomyocyte loss. However, engraftment rates in pre-clinical studies have been low, highlighting a need to refine current iPSC-CM technology. In this study, we demonstrated that inducing Yes-associated protein (YAP) by genetic and pharmacological approaches resulted in increased iPSC-CM proliferation and reduced apoptosis in response to oxidative stress. Interestingly, iPSC-CM maturation was differently affected by each strategy, with genetic activation of YAP resulting in a more immature cardiomyocyte-like phenotype not witnessed upon pharmacological YAP activation. Overall, we conclude that YAP activation in iPSC-CMs enhances cell survival and proliferative capacity. Therefore, strategies targeting YAP, or its upstream regulator the Hippo signalling pathway, could potentially be used to improve the efficacy of iPSC-CM technology for use as a future regenerative therapy in myocardial infarction.

YAP co-localizes with the mitotic spindle and midbody to safeguard mitotic division in lung-cancer cells.

YES-associated protein (YAP) is a part of the Hippo pathway, with pivotal roles in several developmental processes and dual functionality as both a tumor suppressor and an oncogene. In the present study, we identified YAP activity as a microtubular scaffold protein that maintains the stability of the mitotic spindle and midbody by physically interacting with α-tubulin during mitotic progression. The interaction of YAP and α-tubulin was evident in co-immunoprecipitation assays, as well as observing their co-localization in the microtubular structure of the mitotic spindle and midbody in immunostainings. With YAP depletion, levels of ECT2, MKLP-1, and Aurora B are reduced, which is consistent with YAP functioning in midbody formation during cytokinesis. The concomitant decrease in α-tubulin and increase in acetyl-α-tubulin during YAP depletion occurred at the post-transcriptional level. This suggests that YAP maintains the stability of the mitotic spindle and midbody, which ensures appropriate chromosome segregation during mitotic division. The increase in acetyl-α-tubulin during YAP depletion may provide a lesion-halting mechanism in maintaining the microtubule structure. The depletion of YAP also results in multinuclearity and aneuploidy, which supports its role in stabilizing the mitotic spindle and midbody.

Neuromedin U contributes to radiation resistance in colorectal cancer via YAP/TAZ signaling activation.

Resistance to radiation therapy remains a treatment obstacle for patients with high‑risk colorectal cancer. Neuromedin U (NMU) has been identified as a potential predictor of the response to radiation therapy by RNA sequencing analysis of colorectal cancer tissues obtained from patients. However, the role of NMU in colorectal cancer remains unknown. In order to investigate role of NMU in colorectal cancer, NMU expression was regulated using small interfering RNA or an NMU‑expression pCMV3 vector, and cell counting, wound‑healing and clonogenic assays were subsequently performed. NMU knockdown decreased colorectal cancer cell proliferation and migration, and sensitized the cells to radiation. Conversely, NMU overexpression increased radiation resistance, proliferation and migration of colorectal cancer cells. Furthermore, by western blotting and nuclear fractionation experiments, NMU knockdown inhibited the nuclear translocation of yes‑associated protein (YAP) and transcriptional co‑activator with PDZ‑binding motif (TAZ), resulting from the phosphorylation of these proteins. By contrast, the nuclear translocation of YAP and TAZ was increased following NMU overexpression in colorectal cancer cells. Recombinant NMU regulated YAP and TAZ activity, and the expression of the YAP and TAZ transcriptional target genes AXL, connective tissue growth factor and cysteine‑rich angiogenic inducer 61 in an NMU receptor 1 activity‑dependent manner. These results suggested that NMU may contribute to the acquisition of radioresistance in colorectal cancer by enhancing the Hippo signaling pathway via YAP and TAZ activation.

P4HA2-induced prolyl hydroxylation of YAP1 restricts vascular smooth muscle cell proliferation and neointima formation

Vascular smooth muscle cell (VSMC) proliferation and neointima formation play significant roles in atherosclerosis development and restenosis following percutaneous coronary intervention. Our team previously discovered that TEA domain transcription factor 1 (TEAD1) promotes vascular smooth muscle differentiation, which is necessary for vascular development. Conversely, aberrant YAP1 activation upregulates the platelet-derived growth factor receptor beta to encourage VSMC proliferation and neointima formation. In this study, we aimed to investigate the molecular mechanisms of YAP1/TEAD signaling during neointima formation. Our research focused on the prolyl 4-hydroxylase alpha 2 (P4HA2) and its downstream target, Yes-associated protein 1 (YAP1), in regulating VSMC differentiation and neointima formation. Our results indicated that P4HA2 reduction leads to VSMC dedifferentiation and promotes neointima formation after injury. Furthermore, we found that P4HA2-induced prolyl hydroxylation of YAP1 restricts its transcriptional activity, which is essential to maintaining VSMC differentiation. These findings suggest that targeting P4HA2-mediated prolyl hydroxylation of YAP1 may be a promising therapeutic approach to prevent injury-induced neointima formation in cardiovascular disease.

Abstract P2061: The Intersection Of O-GlcNAc And Yap - A Novel Molecular Axis In Heart Failure

Introduction: Heart failure (HF) and pathological myocardial hypertrophy continue to grow world-wide and are strongly tied to obesity, hyperglycemia, and ischemia. These stressors lead to increased levels of O-beta-N-Acetylglucosamine modified proteins (OGN) in the heart. OGN levels are determined by the net activity of two enzymes, OGT and OGA. Our recent work showed excessive OGN was sufficient to cause HF and pathological myocardial hypertrophy. Here we test the relationship between OGN and Yes Associated Protein (YAP), a known promoter of cardiac hypertrophy. Objectives: Test if OGN modifies YAP in the heart and evaluate the role of OGN modified YAP in the development of pathologic cardiac hypertrophy and heart failure. Methods: We have shown that mouse models with myocardial OGT overexpression (OGT TG) developed pathological cardiac hypertrophy and HF. We found these animals have increased YAP activity. Myocardial OGA overexpression (OGA TG) attenuated OGN following transaortic constriction(TAC) and were protected against cardiac hypertrophy and HF. Interbreeding OGT TG with OGA TG rescued animals from pathological hypertrophy, HF and normalized YAP activity . We quantified levels of activated YAP protein/gene expression in hearts from WT, OGT, OGA and OGT x OGA mice. We evaluated YAP/TEAD1 target gene expression levels in OGA TG mice protected against TAC. We used an adenoviral YAP/TEAD expression reporter to quantify YAP/TEAD1 activity in cardiac myocytes, in the presence and absence of increased O-GlcNAcylation. Results: We found WT levels of activated (nuclear) YAP in OGA TG, increased active YAP in OGT TG and normal levels in OGT x OGA mice. YAP/TEAD1 target genes are upregulated in OGT TG hearts but interbred OGT x OGA animals have normal cardiac function and YAP/TEAD1 target gene expression. OGA TG mice who are protected against TAC induced heart failure/hypertrophy and have decreased myocardial levels of YAP/TEAD1 gene targets involved in pathological hypertrophy. Increased O-GlcNAcylation in cardiac myocytes activates YAP/TEAD activity and YAP/TEAD downstream gene expression. Conclusion: These preliminary data suggest a novel pathway for promoting pathological hypertrophy and HF via activation of the O-GlcNAc -YAP/TEAD1 axis.

Abstract P3091: Suppression Of Myeloid Yap Antagonizes Adverse Cardiac Remodeling During Pressure Overload Stress

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) subjected to PO stress had preserved systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Studies in RAW264.7 cells demonstrated the effectiveness of pharmacological YAP inhibition using verteporfin to attenuate pro-inflammatory gene expression, and augment VEGFA levels. Finally, we leveraged nanoparticle-mediated delivery of verteporfin and observed attenuated PO-induced pathological remodeling and inflammation compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Ehrlichia Wnt SLiM ligand mimic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Musculoskeletal defects associated with myosin heavy chain‐embryonic loss of function are mediated by the YAP signaling pathway

Mutations in MYH3, the gene encoding the developmental myosin heavy chain‐embryonic (MyHC‐embryonic) skeletal muscle‐specific contractile protein, cause several congenital contracture syndromes. Among these, recessive loss‐of‐function MYH3 mutations lead to spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis. We find that Myh3 germline knockout adult mice display SCTS phenotypes such as scoliosis and vertebral fusion, in addition to reduced body weight, muscle weight, myofiber size, and grip strength. Myh3 knockout mice also exhibit changes in muscle fiber type, altered satellite cell numbers and increased muscle fibrosis. A mass spectrometric analysis of embryonic skeletal muscle from Myh3 knockouts identified integrin signaling and cytoskeletal regulation as the most affected pathways. These pathways are closely connected to the mechanosensing Yes‐associated protein (YAP) transcriptional regulator, which we found to be significantly activated in the skeletal muscle of Myh3 knockout mice. To test whether increased YAP signaling might underlie the musculoskeletal defects in Myh3 knockout mice, we treated these mice with CA3, a small molecule inhibitor of YAP signaling. This led to increased muscle fiber size, rescue of most muscle fiber type alterations, normalization of the satellite cell marker Pax7 levels, increased grip strength, reduced fibrosis, and decline in scoliosis in Myh3 knockout mice. Thus, increased YAP activation underlies the musculoskeletal defects seen in Myh3 knockout mice, indicating its significance as a key pathway to target in SCTS and other MYH3‐related congenital syndromes.

The role of PKN1 in glioma pathogenesis and the antiglioma effect of raloxifene targeting PKN1.

PKN1 (protein kinase N1), a serine/threonine protein kinase family member, is associated with various cancers. However, the role of PKN1 in gliomas has rarely been studied. We suggest that PKN1 expression in glioma specimens is considerably upregulated and positively correlates with the histopathological grading of gliomas. Knocking down PKN1 expression in glioblastoma (GBM) cells inhibits GBM cell proliferation, invasion and migration and promotes apoptosis. In addition, yes-associated protein (YAP) expression, an essential effector of the Hippo pathway contributing to the oncogenic role of gliomagenesis, was also downregulated. In contrast, PKN1 upregulation enhances the malignant characteristics of GBM cells and simultaneously upregulates YAP expression. Therefore, PKN1 is a promising therapeutic target for gliomas. Raloxifene (Ralo), a commonly used selective oestrogen-receptor modulator to treat osteoporosis in postmenopausal women, was predicted to target PKN1 according to the bioinformatics team from the School of Mathematics, Tianjin Nankai University. We showed that Ralo effectively targets PKN1, inhibits GBM cells proliferation and migration and sensitizes GBM cells to the major chemotherapeutic drug, Temozolomide. Ralo also reverses the effect of PKN1 on YAP activation. Thus, we confirm that PKN1 contributes to the pathogenesis of gliomas and may be a potential target for Ralo adjuvant glioma therapy.

Matrix stiffness-induced α-tubulin acetylation is required for skin fibrosis formation through activation of Yes-associated protein.

Skin fibrosis, a pathological process featured by fibroblast activation and extracellular matrix (ECM) deposition, makes a significant contribution to morbidity. Studies have identified biomechanics as the central element in the complex network of fibrogenesis that drives the profibrotic feedback loop. In this study, we found that the acetylation of α-tubulin at lysine 40 (K40) was augmented in fibrotic skin tissues. Further analysis showed that α-tubulin acetylation is required for fibroblast activation, including contraction, migration, and ECM deposition. More importantly, we revealed that biomechanics-induced upregulation of K40 acetylation promotes fibrosis by mediating mechanosensitive Yes-associated protein S127 dephosphorylation and its cytoplasm nucleus shuttle. Furthermore, we demonstrated that the knockdown of α-tubulin acetyltransferase 1 could rescue the K40 acetylation upregulation caused by increased matrix rigidity and ameliorate skin fibrosis both in vivo and in vitro. Herein, we highlight the critical role of α-tubulin acetylation in matrix stiffness-induced skin fibrosis and clarify a possible molecular mechanism. Our research suggests α-tubulin acetylation as a potential target for drug design and therapeutic intervention.

Verteporfin induces YAP-dependent cell cycle arrest and caspase-mediated cellular apoptosis in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) subtype endows distinctive biological features, including aberrant proliferation, and deducing the molecular mechanism harmonizing the TNBC characteristics is crucial for a greater understanding and prognosis of the disease. The aim of the present study was to analyze the anti-tumorigenic effects of verteporfin (VP) in TNBC in vitro. We evaluated the tumorigenic properties of TNBC cells on VP treatment to assess the cell viability, apoptosis, cell cycle, cell survival, and protein/mRNA expressions in MDA-MB-231 and SUM-159 breast cancer cells. Transient silencing of yes-associated protein (YAP) was performed to validate the data. TNBC cells exhibited a comparatively higher active YAP downstream signaling, and VP resulted in the nuclear exclusion of YAP to a significant extent. VP inhibits the proliferation of TNBC cells by altering cyclin-dependent kinase inhibitors, thereby rendering cellular arrest at the G0/G1 phase. In a dose-dependent manner, VP induces the apoptotic machinery in TNBC cells. Moreover, transient silencing of YAP in TNBC cells exhibited a similar pattern of antiproliferative effects. Elevated YAP nuclear activity and downstream signaling in TNBC are associated with sustaining the proliferative capacity of the cells by inhibiting cellular apoptosis. VP induces antiproliferative effects on TNBC through cytoplasmic retention of YAP. Consequently, cells rewire the course of the cell cycle and stimulate cellular death. We suggest YAP signaling as a prerequisite for TNBC cell progression, and VP without light activation exerts anti-tumorigenic effects on TNBC cells.

ECSIT Is a Critical Factor for Controlling Intestinal Homeostasis and Tumorigenesis through Regulating the Translation of YAP Protein.

The intestinal epithelium is the fastest renewing tissue in mammals and its regenerative process must be tightly controlled to minimize the risk of dysfunction and tumorigenesis. The orderly expression and activation of Yes-associated protein (YAP) are the key steps in driving intestinal regeneration and crucial for intestinal homeostasis. However, the regulatory mechanisms controlling this process remain largely unknown. Here, it is discovered that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a multi-functional protein, is enriched along the crypt-villus axis. Intestinal cell-specific ablation of ECSIT results in the dysregulation of intestinal differentiation unexpectedly accompanied with enhanced YAP protein dependent on translation, thus transforming intestinal cells to early proliferative stem "-like" cells and augmenting intestinal tumorigenesis. Loss of ECSIT leads to metabolic reprogramming in favor of amino acid-based metabolism, which results in demethylation of genes encoding the eukaryotic initiation factor 4F pathway and their increased expression that further promotes YAP translation initiation culminating in intestinal homeostasis imbalance and tumorigenesis. It is also shown that the expression of ECSIT is positively correlated with the survival of patients with colorectal cancer. Together, these results demonstrate the important role of ECSIT in regulating YAP protein translation to control intestinal homeostasis and tumorigenesis.