Yes-associated Protein Dephosphorylation Research Articles

Macrophage-Derived Type I IFN, Renal Tubular Epithelial Cell Polyploidization, and AKI-to-CKD Transition.

Acute kidney injury (AKI) is recognized as a common risk factor for chronic kidney disease (CKD). Renal tubular epithelial cell polyploidization after AKI is closely associated with maladaptive repair, while the regulatory and molecular mechanisms remain poorly understood. Here, we set out to investigate the mechanism of tubular epithelial cell polyploidization and their role in the AKI-to-CKD transition. The change characters of polyploid tubular epithelial cells and macrophages after AKI were detected by flow cytometry and immunofluorescence. The underlying mechanism was explored by RNA-sequencing analysis, immunofluorescence and western blot. The role of tubular epithelial cell polyploidization in the AKI-to-CKD transition was evaluated by transgenic mice and drug interventions. We discovered that tubular epithelial cells underwent polyploidization after AKI and polyploid tubular epithelial cells exhibited greater fibrotic phenotypes than non-polyploid cells. Furthermore, we revealed an upregulated IFN-β response feature within tubular epithelial cells after AKI, and identified that macrophage-derived IFN-β bound to IFN-I receptor 1 (IFNAR1) of tubular epithelial cells and induced their polyploidization. Mechanistically, IFN-β, secreted by macrophages through activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, acted on tubular epithelial cells and facilitated inorganic pyrophosphatase binding to Yes-associated protein (YAP), which mediated YAP dephosphorylation and subsequent nuclear translocation, culminating in p21 expression and polyploidization. Importantly, delayed blockade of the IFN-β response and pharmacological inhibition of STING or YAP activation on day 4 after AKI significantly attenuated persistent tubular epithelial cell polyploidization and AKI-induced kidney fibrosis. Macrophage-derived IFN-β contributed to tubular epithelial cell polyploidization by regulating inorganic pyrophosphatase/YAP signaling pathway-mediated p21 expression and further promoted AKI-to-CKD transition.

NRP1 Induces Enhanced Stemness and Chemoresistance in Glioma Cells via YAP.

Neuropilin-1 (NRP1), a transmembrane glycoprotein, plays an important role in the malignant progression of gliomas; however, its role in chemoresistance is not fully understood. In this study, we observed the effects of NRP1 on the stemness and chemoresistance of glioma cells and the mediating role of Yes-associated protein (YAP). We constructed NRP1 overexpressing LN-229 glioma cells. Cells were treated with recombinant NRP1 protein (rNRP1) and the YAP inhibitor Super-TDU when necessary. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the sensitivity of cells to temozolomide (TMZ). Sphere and clone formation assays were performed to detect the sphere- and clone-forming abilities of cells. Western blotting was performed to detect cellular CD133, CD44, p-LATS1, and p-YAP protein expression. Immunofluorescence and flow cytometry were used to detect the subcellular localization of YAP and apoptosis, respectively. We found that both NRP1 overexpression and rNRP1 treatment enhanced self-renewal, TMZ resistance, and CD133 and CD44 protein expression in LN-229 cells. NRP1 overexpression and rNRP1 treatment also induced LATS1 and YAP dephosphorylation and YAP nuclear translocation. Super-TDU inhibits NRP1 overexpression-induced enhanced self-renewal and TMZ resistance in LN-229 cells. Our study suggests that NRP1 induces increased stemness in glioma cells, resulting in chemoresistance, and that this effect is associated with YAP activation.

Activation of skeletal muscle FAPs by LPA requires the Hippo signaling via the FAK pathway

Lysophosphatidic acid (LPA) is a lysophospholipid that signals through six G-protein coupled receptors (LPARs), LPA1 to LPA6. LPA has been described as a potent modulator of fibrosis in different pathologies. In skeletal muscle, LPA increases fibrosis-related proteins and the number of fibro/adipogenic progenitors (FAPs). FAPs are the primary source of ECM-secreting myofibroblasts in acute and chronic damage. However, the effect of LPA on FAPs activation in vitro has not been explored. This study aimed to investigate FAPs’ response to LPA and the downstream signaling mediators involved. Here, we demonstrated that LPA mediates FAPs activation by increasing their proliferation, expression of myofibroblasts markers, and upregulation of fibrosis-related proteins. Pretreatment with the LPA1/LPA3 antagonist Ki16425 or genetic deletion of LPA1 attenuated the LPA-induced FAPs activation, resulting in decreased expression of cyclin e1, α-SMA, and fibronectin. We also evaluated the activation of the focal adhesion kinase (FAK) in response to LPA. Our results showed that LPA induces FAK phosphorylation in FAPs. Treatment with the P-FAK inhibitor PF-228 partially prevented the induction of cell responses involved in FAPs activation, suggesting that this pathway mediates LPA signaling. FAK activation controls downstream cell signaling within the cytoplasm, such as the Hippo pathway. LPA induced the dephosphorylation of the transcriptional coactivator YAP (Yes-associated protein) and promoted direct expression of target pathway genes such as Ctgf/Ccn2 and Ccn1. The blockage of YAP transcriptional activity with Super-TDU further confirmed the role of YAP in LPA-induced FAPs activation. Finally, we demonstrated that FAK is required for LPA-dependent YAP dephosphorylation and the induction of Hippo pathway target genes. In conclusion, LPA signals through LPA1 to regulate FAPs activation by activating FAK to control the Hippo pathway.

Losartan attenuates upstream vasculopathy in a modified piglet model of pulmonary vein stenosis: contribution of the Hippo pathway.

Diffuse pulmonary vein stenosis (PVS) is an intractable congenital heart disease for which the underlying mechanism remains unclear. In this study, we investigated the effect of losartan and the role of the Hippo pathway in PVS. A total of 19 neonatal piglets were divided into 3 groups: a sham group (n=7), a banded group (n=6) with the left upper pulmonary vein and common trunk of both lower pulmonary veins banded, and a losartan group (n=6) with losartan treatment (1 mg/kg/d) after the banding operation. After 8 weeks, the piglets underwent hemodynamic measurement and harvesting. The upstream pulmonary veins were collected for histological staining and molecular biological analysis. Losartan and/or angiotensin II (stepwise concentrations from 0.1 to 100 µmol/L) were added to a human umbilical vein endothelial cell culture to investigate the potential mechanism in vitro. The modified model demonstrated the main characteristics of patients with PVS, including pulmonary hypertension and intimal hyperplasia in the upstream veins. Upregulation of yes-associated protein (YAP) and angiotensin II type 1 receptor was observed in the neointima (P<0.01). Losartan treatment improved the pathological changes in piglets and decreased YAP expression in the neointima (P<0.01). In vitro, losartan suppressed angiotensin II-induced cell proliferation by inhibiting dephosphorylation and nuclear translocation of YAP in human umbilical vein endothelial cells (P<0.05). Losartan treatment ameliorates intimal hyperplasia and inhibits YAP activation. The activation of the Hippo-YAP pathway is involved in the vasculopathy of progressive PVS. These findings may contribute to the development of new approaches for treating PVS.

Abstract 3938: Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex

Abstract Hippo pathway alterations in human cancers often result in dephosphorylation of yes-associated protein (YAP1) and its paralog TAZ (WWTR1), allowing the formation of an active complex with transcriptional enhanced associate domain transcription factors (TEADs). This complex formation results in the activation of pro-survival and pro-proliferative transcriptional programs in cancer cells. Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of tumors are driven by YAP1/TEAD activity. Although traditionally difficult to drug with small molecules, identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs necessary for YAP1 interaction has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TAZ-TEAD complex formation and transcriptional activity. We report the discovery and characterization of the novel YAP1/TAZ-TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TAZ-TEAD activity. In biochemical thermal shift assays, MRK-A caused a concentration-dependent melting temperature shift of 8-12.5 and 0.6-1.5 degrees for TEAD1 and TEAD2, respectively, indicating direct binding to TEAD protein. In cellular assays, MRK-A demonstrated inhibition of a TEAD-based reporter assay, with little to no activity in multiple orthogonal off-target reporter assays such as WNT, NF-KB, TGFB and PPARG (8.4 nM vs. &amp;gt;10000 nM), which is consistent with the exquisite selectivity profile of this molecule (&amp;gt;1000x selectivity against 350+ measured kinases and other common off-targets). In the NF2-deficient mesothelioma cell line H226, MRK-A suppressed the transcription of endogenous YAP/TAZ-TEAD target genes CYR61, ERBB3, ANKRD1 and CTGF (50-75% inhibition at 100 nM), but not LATS1, a non-TEAD regulated Hippo pathway gene. In co-immunoprecipitation assays, MRK-A disrupted the interaction of YAP1 and TEAD in H226 cells at concentrations consistent with inhibition of target genes. In addition, MRK-A potently blocked the clonogenic growth and viability of H226 cells in a dose-dependent manner (maximal response at 1 µM compound &amp;gt;90% growth inhibition), while sparing the Hippo wild-type mesothelioma cell line H28. Furthermore, structurally similar control compounds, MRK-B and MRK-C, without the ability to block TEAD-mediated transcription (TEAD reporter MCF7 assay IC50 &amp;gt; 10000 nM), did not impact the clonogenic growth of H226 cells. In vivo, MRK-A did not show acute tolerability signals in mice and demonstrated pharmacokinetics suitable for daily oral dosing in efficacy studies. In summary, we report the structure and characterization of MRK-A demonstrating potent and specific inhibition of YAP1/TAZ-TEAD mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling. Citation Format: Casey J. Moure, Christopher Sondey, Mangeng Cheng, My Mansueto, Rafael Fernandez, Sebastian E. Schneider, Julia V. Ramirez, Brian Long, Erin DiMauro, Brandon Vara, Charles Yeung, Abe Achab, Jongwon Lim, Ronald Kim, Cayetana Zarate, Jonathan Bennett, Rachel Palte, Robert Foti, Vladimir Simov, Evan Barry. Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3938.

YAP is critical to inflammation, endothelial-mesenchymal transition and subretinal fibrosis in experimental choroidal neovascularization

Subretinal fibrosis causes local damage to the retina and irreversible vision loss, as the final stage of neovascular age-related macular degeneration (nAMD). More recently, the endothelial-to-mesenchymal transition (EndoMT) has been considered one of the most significant sources of myofibroblasts in subretinal fibrosis, though the underpinning molecular mechanisms remain unclear. In this study, a series of experiments were performed to test the hypothesis that Yes-associated protein (YAP) may be involved in EndoMT and subretinal fibrosis. We demonstrated that transforming growth factor (TGF)-β2 stimulation induces YAP dephosphorylation (activated) and nuclear transcription in human umbilical vein endothelial cells (HUVECs) by increasing reactive oxygen species (ROS) levels. Moreover, TGF-β2-mediated EndoMT and proinflammatory cytokine production in HUVECs were reduced by ROS clearance or YAP knockdown. Furthermore, the severity of subretinal fibrosis was markedly relieved by intravitreal administration of a small interfering RNA targeting YAP in the mouse laser-induced choroidal neovascularization (CNV) model. Our findings provide novel insights into a previously unknown effect of YAP on the EndoMT process and reveal YAP as a potential target for suppressing CNV-related subretinal fibrosis and protect vision.

PDE4D binds and interacts with YAP to cooperatively promote HCC progression

The role of cAMP in the development of hepatocellular carcinoma (HCC) is controversial and the biological function of cAMP-hydrolysing enzyme phosphodiesterase 4D (PDE4D) in HCC remains unclear. In this study, we observed markedly higher PDE4D expression in HCC patients with poor survival. PDE4D bound to yes-associated protein (YAP), and PDE4D expression positively correlated with YAP expression in HCC. Overexpression of PDE4D increased YAP dephosphorylation and activity and promoted HCC cell growth in vitro and in vivo, which was attenuated by the YAP inhibitor verteporfin. In contrast, silencing PDE4D reduced YAP expression and HCC cell growth. Notably, forced expression of YAP promoted PDE4D and YAP target gene expression and cell growth, which were abrogated by the PDE4D inhibitor roflumilast. Mechanistically, silencing of YAP caused PDE4D downregulation and HCC cell apoptosis via extracellular signal-regulated kinase (ERK) activation. Roflumilast activated cAMP-PKA signaling and induced cAMP-PKA-dependent YAP phosphorylation at serine 127, resulting in YAP degradation and suppression of HCC growth, which were reversed by the PKA inhibitor PKI. Additionally, transfection of the YAP-S127A mutant reversed roflumilast-mediated suppression of YAP and cell growth. Taken together, our findings indicate that PDE4D binds to and interacts with YAP to promote HCC progression. Targeting the PDE4D-YAP interaction with roflumilast may be an effective strategy for HCC treatment.

PPP1CA/YAP/GS/Gln/mTORC1 pathway activates retinal Müller cells during diabetic retinopathy

Diabetic retinopathy (DR) is a vision-loss complication caused by diabetes with high prevalence. During DR, the retinal microvascular injury and neurodegeneration derived from chronic hyperglycemia have attracted global attention to retinal Müller cells (RMCs), the major macroglia in the retina contributes to neuroprotection. Protein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA) dephosphorylates the transcriptional coactivator Yes-associated protein (YAP) to promote the transcription of glutamine synthetase (GS). GS catalyzes the transformation of neurotoxic glutamate (Glu) into nontoxic glutamine (Gln) to activate the mammalian target of rapamycin complex 1 (mTORC1), which promotes the activation of RMCs. In this study, in vitro MIO-M1 cell and in vivo mouse high-fat diet and streptozotocin (STZ)-induced diabetic model to explore the role of the PPP1CA/YAP/GS/Gln/mTORC1 pathway on the activation of MRCs during DR. Results showed that PPP1CA promoted the dephosphorylation and nuclear translocation of YAP in high glucose (HG)-exposed MIO-M1 cells. YAP transcribed GS in HG-exposed MIO-M1 cells in a TEAD1-dependent and PPP1CA-dependent way. GS promoted the biosynthesis of Gln in HG-exposed MIO-M1 cells. Gln activated mTORC1 instead of mTORC2 in HG-exposed MIO-M1 cells. The proliferation and activation of HG-exposed MIO-M1 cells were PPP1CA/YAP/GS/Gln/mTORC1-dependent. Finally, RMC proliferation and activation during DR were inhibited by the PPP1CA/YAP/GS/Gln/mTORC1 blockade. The findings supplied a potential idea to protect RMCs and alleviate the development of DR.

Protein Phosphatase 2A Mediates YAP Activation in Endothelial Cells Upon VEGF Stimulation and Matrix Stiffness.

Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis.

LACTB suppresses melanoma progression by attenuating PP1A and YAP interaction.

Very limited progress has been made in the management of advanced melanoma, especially melanoma of uveal origin. Lactamase β (LACTB) is a novel tumor suppressor; however, its biological function in melanoma remains unknown. Herein we demonstrated markedly lower LACTB expression levels in melanoma tissues and cell lines. Overexpression of LACTB suppressed the proliferation, migration and invasion of melanoma cells in vitro. Mechanistically, LACTB inhibited the activity of yes-associated protein (YAP). We showed that the level of phospho-YAP (Serine 127) was increased upon LACTB overexpression, which prevented the translocation of YAP to the nucleus. Further, LACTB could directly bind to PP1A and attenuate the interaction between PP1A and YAP, resulting in decreased YAP dephosphorylation and inactivation in a LATS1-independent manner. Additionally, transfection of phosphorylation-defective YAP mutants reversed LACTB-induced tumor suppression. Upstream, we demonstrated that SOX10 binds to the LACTB promoter and negatively regulates its transcription. Overexpression of LACTB also suppressed the tumorigenicity and lung metastasis of MUM2B uveal melanoma cells in vivo. Taken together, our findings indicate a novel SOX10/LACTB/PP1A signaling cascade that renders YAP inactive and modulates melanoma progression, offering a new therapeutic target for melanoma treatment.

Tribbles homolog 2 promotes hepatic fibrosis and hepatocarcinogenesis through phosphatase 1A-Mediated stabilization of yes-associated protein.

Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Tribbles homolog 2 (TRIB2) is an oncogene implicated in a variety of cancers, including liver cancer. However, the biological function and regulatory mechanism of TRIB2 in HSCs are poorly understood. In addition, little is known about its role in liver fibrosis progression to HCC. Here, we revealed the clinical significance of TRIB2 in liver fibrosis and HCC development. We investigated TRIB2 promoting liver fibrosis in vitro and in vivo. In mouse model of liver fibrosis and HCC, we measured hepatic fibrosis and HCC level through knockdown TRIB2 with shRNA. In addition, we performed western blotting, real-time quantitative PCR, immunofluorescence and co-immunoprecipitation assay to study TRIB2 function in LX-2 cells. TRIB2 expression was strongly upregulated in human fibrotic liver tissues and HCC tissues. TRIB2 colocalized with α-smooth muscle actin (α-SMA) in fibrotic and HCC liver tissues. Knockdown of TRIB2 inhibited HSC activation and liver fibrosis in vitro and in vivo. TRIB2 promoted Yes-associated protein (YAP) stabilization, nuclear localization, and subsequent fibrotic gene expression independent of the MST-LATS phosphorylation cascade in HSCs. TRIB2 interacted with YAP to recruit phosphatase 1A (PP1A), promoting PP1A-mediated YAP dephosphorylation. TRIB2 knockdown potently attenuated the development of fibrosis-associated liver cancer. TRIB2 is an attractive target for hepatic fibrosis and fibrosis-associated liver cancer treatment.

Activation of yes-associated protein mediates sphingosine-1-phosphate-induced proliferation and migration of pulmonary artery smooth muscle cells and its potential mechanisms.

The aims of the present study were to examine the molecular mechanisms underlying sphingosine-1-phosphate (S1P)-induced rat pulmonary artery smooth muscle cells (PASMCs) proliferation/migration and to determine the effect of yes-associated protein (YAP) activation on S1P-induced PASMCs proliferation/migration and its potential mechanisms. S1P induced YAP dephosphorylation and nuclear translocation, upregulated microRNA-130a/b (miR-130a/b) expression, reduced bone morphogenetic protein receptor 2 (BMPR2), and inhibitor of DNA binding 1(Id1) expression, and promoted PASMCs proliferation and migration. Pretreatment of cells with Rho-associated protein kinase (ROCK) inhibitor Y27632 suppressed S1P-induced YAP activation, miR-130a/b upregulation, BMPR2/Id1 downregulation, and PASMCs proliferation/migration. Knockdown of YAP using small interfering RNA also suppressed S1P-induced alterations of miR-130a/b, BMPR2, Id1, and PASMCs behavior. In addition, luciferase reporter assay indicated that miR-130a/b directly regulated BMPR2 expression in PASMCs. Inhibition of miR-130a/b functions by anti-miRNA oligonucleotides attenuated S1P-induced BMPR2/Id1 downregulation and the proliferation and migration of PASMCs. Taken together, our study indicates that S1P induces activation of YAP through ROCK signaling and subsequently increases miR-130a/b expression, which, in turn, downregulates BMPR2 and Id1 leading to PASMCs proliferation and migration.

Platelet‑derived growth factor‑BB mediates pancreatic cancer malignancy via regulation of the Hippo/Yes‑associated protein signaling pathway.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that serves a role in the development of several types of solid cancer, and abnormal PDGF activity has been reported in numerous human tumors. Tumor-derived PDGF ligands are considered to act in either a paracrine or autocrine manner, serving roles in the phosphorylation of receptors on tumor and stromal cells in the tumor microenvironment. Despite the well-established association between PDGF and tumor progression, the precise mechanisms of autocrine PDGF signaling in pancreatic tumor cells remain elusive. Therefore, the present study aimed to analyze the influence of PDGF-BB in pancreatic cancer. Pancreatic adenocarcinoma BxPC-3 cells were cultured and treated with recombinant human PDGF-BB in vitro. Cell proliferation was tested using an MTT assay. Cell apoptosis was measured using flow cytometry. Tumor cell migration and invasion were examined via wound-healing and Transwell assays, respectively. The expression and subcellular localization of Yes-associated protein (YAP) was determined using western blotting and immunofluorescence. The transcriptional activity of target genes was tested using a luciferase assay and reverse transcription-quantitative PCR. The present study revealed that PDGF-BB significantly promoted cell proliferation in pancreatic adenocarcinoma BxPC-3 cells and enhanced the aggressiveness of this cell line, as demonstrated by Transwell and wound-healing assays. Anoikis resistance is an important mechanism by which metastatic cells avoid apoptosis when detaching from adjacent cells or the extracellular matrix. PDGF-BB treatment inhibited anoikis under anchorage-independent conditions. Mechanistic experiments revealed that PDGF-BB promoted the upregulation and activation of the transcriptional coactivator YAP, an effector of the Hippo signaling pathway. RhoA or protein phosphatase-1 (PP-1) inhibition partially abolished the accumulation and activation of YAP, suggesting PDGF-BB-mediated YAP dephosphorylation and transactivation via the RhoA/PP-1 cascade. Pharmacologic inhibition of the PDGF receptor directly downregulated YAP activity and the expression levels of downstream genes. Furthermore, verteporfin, a small molecular inhibitor of the Hippo/YAP signaling pathway, partially reversed the effects of PDGF-BB on cell proliferation, anoikis resistance and cell migration. In conclusion, the present study revealed that the Hippo/YAP signaling pathway may be involved in the tumor-promoting activity of PDGF-BB in pancreatic cancer.

Inhibition of yes-associated protein dephosphorylation prevents aggravated periodontitis with occlusal trauma.

Occlusal trauma can aggravate periodontitis, but the mechanism remains unclear. Yes-associated protein (YAP), a mechanical stressor protein, may play an important role in this process. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to detect the expression of YAP and inflammatory factors in patients with periodontitis accompanied with or without occlusal trauma. Through local administration of Porphyromonas gingivalis and composite resin bonding on maxillary molars in mice, we established periodontitis and occlusal trauma models. Treatment with or without XAV939, to inhibit YAP activation, was performed in these models. Micro-computed tomography, immunofluorescence (IF), and qRT-PCR were used to explore the YAP pathway in periodontitis with occlusal trauma. Cyclic stress and lipopolysaccharide (LPS) stimuli were applied to the L929 mouse fibroblast cell line with or without XAV939. Western blot, IF, and qRT-PCR were used to verify the in vivo results. Activated dephosphorylated YAP and increased expression of inflammatory factors were observed in patients with periodontitis accompanied with occlusal trauma. In the mouse model of periodontitis with occlusal trauma, YAP transferred into the nucleus, resulting in Jun N-terminal kinases (JNK) related pro-inflammatory pathway up-regulation. L929 cell cyclic stress and LPS stimulation results confirmed the in vivo results. Application of XAV939 inhibited YAP protein dephosphorylation and reduced JNK pro-inflammatory pathway factor expression in vivo and in vitro. Occlusal trauma can activate YAP nuclear transfer, resulting in the up-regulation of the JNK pro-inflammatory pathway. This can be inhibited by the XAV939 YAP inhibitor.

Salvianolic acid B ameliorates atherosclerosis via inhibiting YAP/TAZ/JNK signaling pathway in endothelial cells and pericytes

Atherosclerosis (AS) is a chronic disease of the arterial wall where both innate and adaptive immunoinflammatory mechanisms are involved. Inflammation plays an important role in the pathological process of atherosclerosis at various stages. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also known as WWTR1) behave as a novel drug target against atherosclerosis. Therefore, the mechanism relationship of YAP/TAZ, inflammation and AS was explored in this study. Experiments demonstrated that serine dephosphorylation and nuclear translocation of YAP was increased in ECs and pericytes induced by oxidative low-density lipoprotein (ox-LDL), while the inhibition of YAP degraded the expression of downstream inflammatory factors. The expression of YAP/TAZ and inflammation proteins (JNK, NF-κB and TNF-α) in ECs and pericytes was suppressed through the application of Sal-B. Besides, Sal-B protects ECs and pericytes from oxidative stress and apoptosis. In vivo, Sal-B reduced en face and aortic root sinus lesions size, and decreased the expression of inflammation related factors (IL-6, IL-1β, TNF-α) and ox-LDL in serum sample of ApoE−/− mice fed a high fat diet. Therefore, our work provides a potential therapeutic strategy of using Sal-B to attenuate the development of atherosclerosis, the anti-atherosclerosis effects of Sal-B is related to regulate YAP/TAZ/JNK signaling pathway.

The crosstalk between AXL and YAP promotes tumor progression through STAT3 activation in head and neck squamous cell carcinoma.

Receptor tyrosine kinases (RTKs) and Yes‐associated protein (YAP) are critical driving factors in tumors. Currently, the regulation of RTKs in the Hippo‐YAP pathway has been recognized as an important issue. However, the relationship between AXL, one of the RTKs, and YAP in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, the crosstalk between AXL and YAP was thoroughly investigated in vitro and in vivo. We determined that there was a positive correlation between AXL and YAP in the HNSCC tissue samples and the Cancer Genome Atlas (TCGA) dataset, and high co‐expression was associated with poor prognosis. Inhibiting YAP decreased AXL expression in HNSCC cells, while YAP overexpression increased AXL. Moreover, ectopic expression of AXL reversed tumor suppressor phenotypes mediated by YAP silencing. This reversal effect was also confirmed in vivo. In addition, AXL overexpression and Gas6, a ligand of AXL, stimulated YAP dephosphorylation, nuclear translocation, and target gene transcription. AXL inhibition decreased YAP dephosphorylation and nuclear translocation. Mechanistically, Gas6 induced a competitive binding to phosphorylated signal transducers and activators of transcription 3 (STAT3) with large tumor suppressor kinase 1 (LATS1) and inhibited the Hippo pathway. This study revealed a novel non‐transcriptional effect of STAT3 in Gas6/AXL‐induced YAP activity, suggesting that STAT3 acted as a critical “molecular switch” during the mutual promotion between AXL and YAP, which might be a promising therapeutic target in HNSCC.

Osteoprotegerin promotes intimal hyperplasia and contributes to in-stent restenosis: Role of an αVβ3/FAK dependent YAP pathway

ObjectiveAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are related to in-stent-restenosis (ISR) following percutaneous coronary intervention (PCI). Osteoprotegerin (OPG) has been implicated in various vascular diseases. However, the effects of OPG on ISR and the underlying mechanism remained elusive. We here investigated the association between OPG and ISR, and to demonstrate the role and potential mechanisms of OPG in neointimal hyperplasia. Approach and resultsFrom 2962 patients who received coronary angiography and follow-up coronary angiography at approximately one year, 291 patients were diagnosed with ISR, and another 291 gender- and age- matched patients without ISR were selected as controls. Serum OPG levels were significantly increased in patients with ISR. Multivariable logistic regression analysis indicated that OPG level was independently associated with the increased risk of ISR. In a mouse femoral artery wire injury model, upregulated OPG was evidenced in vascular tissue after injury. OPG deletion attenuated the vascular injury-induced neointimal hyperplasia and related gene expression in mice. OPG promoted neointimal hyperplasia and human aortic smooth muscle cell (hASMC) proliferation and migration through activation of yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, whereas knockdown or inhibition of YAP in hASMCs blunted OPG-induced above effects. Moreover, we found that OPG, as a ligand for integrin αVβ3, mediated phosphorylation of focal adhesion kinase (FAK) and actin cytoskeleton reorganization, resulting in YAP dephosphorylation in hASMCs. OPG-dependent YAP and VSMC activation was prevented by treatment with αVβ3-blocking antibodies and inhibitors of FAK and actin stress fibers. ConclusionsIncreased serum OPG levels are associated with increased risk of ISR following PCI and OPG could promote neointimal hyperplasia in response to injury through integrin αVβ3 mediated FAK and YAP activation, indicating OPG/YAP inhibition might serve as an attractive novel target for the prevention of ISR after PCI.

Inhibition of Siah2 ubiquitin ligase ameliorates monocrotaline-induced pulmonary arterial remodeling through inactivation of YAP

AimsIt has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models. Main methodsIntraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit. Key findingsThe protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats. SignificanceSiah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH.

EWS-FLI1-mediated tenascin-C expression promotes tumour progression by targeting MALAT1 through integrin \u03b15\u03b21-mediated YAP activation in Ewing sarcoma

BackgroundThe extracellular matrix has been critically associated with the tumorigenesis and progression of Ewing sarcoma (ES). However, the regulatory and prognostic roles of tenascin-C (TNC) in ES remain unclear.MethodsTNC expression was examined in specimens by immunohistochemistry, and the association of TNC expression with ES patient survival was also analysed. TNC-knockout cell lines were constructed using CRISPR/Cas9 methods. In vitro experiments and in vivo bioluminescent imaging using BALB/c nude mice were conducted to evaluate the effect of TNC on ES tumour progression. RNA sequencing was performed, and the underlying mechanism of TNC was further explored.ResultsTNC was overexpressed in ES tissue and cell lines, and TNC overexpression was associated with poor survival in ES patients. TNC enhanced cell proliferation, migration and angiogenesis in vitro and promoted ES metastasis in vivo. The oncoprotein EWS-FLI1 profoundly increased TNC expression by directly binding to the TNC promoter region. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) upregulation induced by Yes-associated protein (YAP) activation was responsible for TNC-regulated ES tumour progression. Activated integrin α5β1 signalling might be correlated with YAP dephosphorylation and nuclear translocation.ConclusionsTNC may promote ES tumour progression by targeting MALAT1 through integrin α5β1-mediated YAP activation.

Mono (2-ethylhexyl) phthalate (MEHP) triggers the proliferation of hemangioma-derived endothelial cells via YAP signals

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.