Yes-associated Protein Target Genes Research Articles

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.

Regulation of YAP Promotor Accessibility in Endothelial Mechanotransduction.

Endothelial cells are constantly exposed to mechanical forces in the form of fluid shear stress, extracellular stiffness, and cyclic strain. The mechanoresponsive activity of YAP (Yes-associated protein) and its role in vascular development are well described; however, whether changes to transcription or epigenetic regulation of YAP are involved in these processes remains unanswered. Furthermore, how mechanical forces are transduced to the nucleus to drive transcriptional reprogramming in endothelial cells is poorly understood. The YAP target gene, AmotL2 (angiomotin-like 2), is a junctional mechanotransducer that connects cell-cell junctions to the nuclear membrane via the actin cytoskeleton. We applied mechanical manipulations including shear flow, stretching, and substrate stiffness to endothelial cells to investigate the role of mechanical forces in modulating YAP transcription. Using in vitro and in vivo endothelial depletion of AmotL2, we assess nuclear morphology, chromatin organization (using transposase-accessible chromatin sequencing), and whole-mount immunofluorescent staining of the aorta to determine the regulation and functionality of YAP. Finally, we use genetic and chemical inhibition to uncouple the nuclear-cytoskeletal connection to investigate the role of this pathway on YAP transcription. Our results reveal that mechanical forces sensed at cell-cell junctions by the YAP target gene AmotL2 are directly involved in changes in global chromatin accessibility and activity of the histone methyltransferase EZH2, leading to modulation of YAP promotor activity. Functionally, shear stress-induced proliferation of endothelial cells in vivo was reliant on AmotL2 and YAP/TAZ (transcriptional coactivator with PDZ-binding motif) expression. Mechanistically, uncoupling of the nuclear-cytoskeletal connection from junctions and focal adhesions led to altered nuclear morphology, chromatin accessibility, and YAP promotor activity. Our findings reveal a role for AmotL2 and nuclear-cytoskeletal force transmission in modulating the epigenetic and transcriptional regulation of YAP to maintain a mechano-enforced positive feedback loop of vascular homeostasis. These findings may offer an explanation as to the proinflammatory phenotype that leads to aneurysm formation observed in AmotL2 endothelial deletion models.

Chromatin accessibility complex subunit 1 enhances tumor growth by regulating the oncogenic transcription of YAP in breast and cervical cancer.

As a component of chromatin remodeling complex, chromatin accessibility complex subunit 1 (CHRAC1) is critical in transcription and DNA replication. However, the significance of CHRAC1 in cancer progression has not been investigated extensively. This research aimed to determine the function of CHRAC1 in breast and cervical cancer and elucidate the molecular mechanism. The Bio-ID method was used to identify the interactome of transcriptional activator Yes-associated protein (YAP) and the binding between YAP and CHRAC1 was verified by immunofluorescence. CCK8, colony formation and subcutaneous xenograft assays were conducted to explore the function of CHRAC1 in cancer cell proliferation. RNA-seq analysis and RT-PCR were used to analyze the transcription program change after CHRAC1 ablation. The diagnostic value of CHRAC1 was analyzed by TCGA database and further validated by immunohistochemistry staining. In the current study, we found that the chromatin remodeler CHRAC1 was a potential YAP interactor. CHRAC1 depletion suppressed breast and cervical cancer cell proliferation and tumor growth. The potential mechanism may be that CHRAC1 interacts with YAP to facilitate oncogenic transcription of YAP target genes in Hippo pathway, thereby promoting tumorigenesis. CHRAC1 was elevated in cervical and breast cancer biopsies and the upregulation correlated with shorter survival, poor pathological stages and metastasis of cancer patients. Moreover, CHRAC1 expression was statistically associated with YAP in breast and cervical cancer biopsies. These findings highlight that CHRAC1 contributes to cancer progression through regulating the oncogenic transcription of YAP, which makes it a potential therapeutic target for cancer treatment.

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.

Mesenchymal stem cell-derived exosomal miR-27b-3p alleviates liver fibrosis via downregulating YAP/LOXL2 pathway

Lysyl oxidase-like 2 (LOXL2) is an extracellular copper-dependent enzyme that plays a central role in fibrosis by catalyzing the crosslinking and deposition of collagen. Therapeutic LOXL2 inhibition has been shown to suppress liver fibrosis progression and promote its reversal. This study investigates the efficacy and underlying mechanisms of human umbilical cord-derived exosomes (MSC-ex) in LOXL2 inhibition of liver fibrosis. MSC-ex, nonselective LOX inhibitor β-aminopropionitrile (BAPN), or PBS were administered into carbon tetrachloride (CCl4)-induced fibrotic livers. Serum LOXL2 and collagen crosslinking were assessed histologically and biochemically. MSC-ex’s mechanisms on LOXL2 regulation were investigated in human hepatic stellate cell line LX-2. We found that systemic administration of MSC-ex significantly reduced LOXL2 expression and collagen crosslinking, delaying the progression of CCl4-induced liver fibrosis. Mechanically, RNA-sequencing and fluorescence in situ hybridization (FISH) indicated that miR-27b-3p was enriched in MSC-ex and exosomal miR-27b-3p repressed Yes-associated protein (YAP) expression by targeting its 3ʹ untranslated region in LX-2. LOXL2 was identified as a novel downstream target gene of YAP, and YAP bound to the LOXL2 promoter to positively regulate transcription. Additionally, the miR-27b-3p inhibitor abrogated the anti-LOXL2 abilities of MSC-ex and diminished the antifibrotic efficacy. miR-27b-3p overexpression promoted MSC-ex mediated YAP/LOXL2 inhibition. Thus, MSC-ex may suppress LOXL2 expression through exosomal miR-27b-3p mediated YAP down-regulation. The findings here may improve our understanding of MSC-ex in liver fibrosis alleviation and provide new opportunities for clinical treatment.

Cyclic tensile strain-induced yes-associated protein activity modulates the response of human periodontal ligament mesenchymal stromal cells to tumor necrosis factor-α

ObjectivesThis study aimed to evaluate the role of yes-associated protein (YAP) in the inflammatory processes induced in human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) by cyclic tensile strain (CTS). DesignhPDL-MSCs from five periodontally healthy individuals were stimulated with 12% CTS and/or TNF-α for 24 h. YAP activity was determined by analyzing the YAP nuclear localization and the target genes expression, using immunofluorescence and qPCR, respectively. Verteporfin was used to inhibit the activation of YAP. The gene expression of interleukin (IL)-6, IL-8, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 was analyzed by qPCR. ResultsIn the absence of TNF-α, application of CTS resulted in the nuclear YAP translocation and upregulation of YAP target genes. Verteporfin inhibited the activation of YAP pathway and upregulated the basal expression of IL-6 and IL-8. TNF-α induced the activation of YAP pathway, which was inhibited by verteporfin. However, application of CTS under these conditions diminished TNF-α-induced YAP activation. TNF-α-induced expression of IL-6, VCAM-1, and ICAM-1 was inhibited after the application of CTS. Inhibition of YAP activation by verteporfin diminished TNF-α-induced gene expression of IL-6, VCAM-1, and ICAM-1, and under these conditions no inhibitory effect of CTS on these parameters was observed. ConclusionsYAP is at least partially involved in the CTS-activated mechanotransduction pathway. The effects of CTS and YAP on the inflammatory responses depend on the inflammatory environment. A better understanding of the inflammatory modulation by mechanical stress may help improve the orthodontic strategies, especially in the patient with periodontitis.

Cmarr/miR-540-3p axis promotes cardiomyocyte maturation transition by orchestrating Dtna expression

The immature phenotype of embryonic stem cell-derived cardiomyocytes (ESC-CMs) limits their application. However, the molecular mechanisms of cardiomyocyte maturation remain largely unexplored. This study found that overexpression of long noncoding RNA (lncRNA)-Cmarr, which was highly expressed in cardiomyocytes, promoted the maturation change and physiological maturation of mouse ESC-CMs (mESC-CMs). Moreover, transplantation of cardiac patch overexpressing Cmarr exhibited better retention of mESC-CMs, reduced infarct area by enhancing vascular density in the host heart, and improved cardiac function in mice after myocardial infarction. Mechanism studies identified that Cmarr acted as a competitive endogenous RNA to impede the repression of miR-540-3p on Dtna expression and promoted the binding of the dystrophin-glycoprotein complex (DGC) and yes-associated protein (YAP), which in turn reduced the proportion of nuclear YAP and the expression of YAP target genes. Therefore, this study revealed the function and mechanism of Cmarr in promoting cardiomyocyte maturation and provided a lncRNA that can be used as a functional factor in the construction of cardiac patches for the treatment of myocardial infarction.

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.

SIRT6 Protects Against Liver Fibrosis by Negatively Regulating YAP/TAZ

Hepatic fibrosis often occurs after chronic liver injury, and it is largely attributable to the activation of hepatic stellate cells (HSCs). It is a determining step in the pathophysiology of non‐alcoholic steatohepatitis (NASH), an advanced form of fatty liver disease. The Hippo pathway and its effectors Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), have been implicated in hepatic fibrogenesis. However, how YAP and TAZ are regulated in HSCs remains incompletely understood. Here in this work, we have identified Sirtuin 6 (SIRT6), a NAD+ dependent deacetylase, as a regulator of YAP and TAZ in HSCs. Using primary HSCs deficient in SIRT6 from mice fed a moderately‐high‐fat‐cholesterol‐cholate diet (HFCC) for one week as well as in vitro models using transforming growth factor‐β (TGF‐β) treated LX‐2 cells, we have revealed that YAP and TAZ were acetylated during the HSC activation. Moreover, expression of YAP and TAZ target genes including connective tissue growth factor (Ctgf), cysteine‐rich angiogenic inducer 61 (Cyr61) and ankyrin repeat domain 1 (Ankrd1) was increased in those HSCs. Immunofluorescence microscopy analysis of liver sections obtained from Sirt6 HSC‐specific knockout mice fed HFCC diet for six weeks showed elevated YAP nuclear localization in the HSCs, suggesting an activation of YAP during fibrosis. Further in vitroanalysis of YAP/TAZ activity upon gain‐ and loss‐of‐function of SIRT6 in TGF‐β treated LX‐2 cells showed a decrease or increase of Ctgf, Cyr61 and Col3a1gene expression,respectively. To understand how SIRT6 regulates YAP and TAZ, we performed immunoprecipitation and lysine acetylation analysis, and our data showed that SIRT6 indeed interacted with YAP and TAZ and deacetylated them. Taken together, our study has identified a key regulatory axis in which SIRT6 interacts with YAP/TAZ and deacetylates them at several lysine residues, thereby suppressing their transcriptional co‐activator activity during fibrosis.

Twinfilin-1 is an essential regulator of myogenic differentiation through the modulation of YAP in C2C12 myoblasts

Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.

Adriamycin-Induced Podocyte Injury Disrupts the YAP-TEAD1 Axis and Downregulates Cyr61 and CTGF Expression.

The most severe forms of kidney diseases are often associated with irreversible damage to the glomerular podocytes, the highly specialized epithelial cells that encase glomerular capillaries and regulate the removal of toxins and waste from the blood. Several studies revealed significant changes to podocyte cytoskeletal structure during disease onset, suggesting possible roles of cellular mechanosensing in podocyte responses to injury. Still, this topic remains underexplored partly due to the lack of appropriate in vitro models that closely recapitulate human podocyte biology. Here, we leveraged our previously established method for the derivation of mature podocytes from human induced pluripotent stem cells (hiPSCs) to help uncover the roles of yes-associated protein (YAP), a transcriptional coactivator and mechanosensor, in podocyte injury response. We found that while the total expression levels of YAP remain relatively unchanged during Adriamycin (ADR)-induced podocyte injury, the YAP target genes connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61) are significantly downregulated. Intriguingly, TEAD1 is significantly downregulated in podocytes injured with ADR. By examining multiple independent modes of cellular injury, we found that CTGF and Cyr61 expression are downregulated only when podocytes were exposed to molecules known to disrupt the cell's mechanical integrity or cytoskeletal structure. To our knowledge, this is the first report that the YAP-TEAD1 signaling axis is disrupted when stem cell-derived human podocytes experience biomechanical injury. Together, these results could help improve the understanding of kidney disease mechanisms and highlight CTGF and Cyr61 as potential therapeutic targets or biomarkers for patient stratification.

NGF Signaling Interacts With the Hippo/YAP Pathway to Regulate Cervical Cancer Progression.

Nerve growth factor (NGF) is increasingly implicated in cervical cancer progression, but its mechanism in cervical cancer is unclear. Here, studies demonstrate that NGF inhibits the Hippo signaling pathway and activates Yes-associated protein (YAP) to induce cervical cancer cell proliferation and migration. Our results suggested that stimulation of NGF promoted cell growth and migration and activated YAP in HeLa and C-33A cell lines. The expression of YAP target genes (CTGF and ANKRD1) was upregulated after NGF treatment. The NGF inhibitor Ro 08-2750 and siRNA-mediated NGF receptor gene silencing suppressed HeLa and C-33A cells proliferation and migration, activated large suppressor kinase 1 (LATS1) kinase activity, and suppressed YAP function. In addition, the expression of YAP target genes (CTGF and ANKRD1) was suppressed by Ro 08-2750 treatment in HeLa and C-33A cells. Interestingly, proliferation was significantly higher in NGF-treated cells than in control cells, and this effect was completely reversed by the YAP small molecule inhibitor-verteporfin. Furthermore, the mouse xenograft model shows that NGF regulates YAP oncogenic activity in vivo. Mechanistically, NGF stimulation inactivates LATS1 and activates YAP, and NGF inhibition was found to induce large suppressor kinase 1 (LATS1) phosphorylation. Taken together, these data provide the first direct evidence of crosstalk between the NGF signaling and Hippo cancer pathways, an interaction that affects cervical cancer progression. Our study indicates that combined targeting of the NGF signaling and the Hippo pathway represents a novel therapeutic strategy for treatment of cervical cancer.

Periodontal ligament fibroblast-derived exosomes induced by compressive force promote macrophage M1 polarization via Yes-associated protein

ObjectivesThis study aimed to investigate the biological roles and mechanisms of compressive force-stimulated periodontal ligament fibroblasts (PDLFs) on polarization of macrophages DesignPDLFs were stimulated with or without static compressive force, and then conditioned medium, high-molecular weight proteins and low-molecular weight proteins were collected to treat THP-1 macrophages. RT-qPCR and flow cytometric analysis were used to evaluate the polarization of macrophages. Exosomes were isolated by ultracentrifugation method and identified via transmission electron microscopy, western-blot and nano-tracking analysis. The protein level of Yes-Associated Protein (YAP) contained in exosomes was detected by western blot. GW4869 and Verteporfin were used to inhibit exosome secretion and YAP- TEA domain transcription factor (TEAD) interaction respectively. ResultsExosomes were successfully purified from PDLFs and could be efficiently incorporated into THP-1 macrophages. conditioned medium, HMW proteins and exosomes derived from compressive force-treated PDLFs significantly induce M1 polarization of macrophages. While inhibiting exosomes secretion by GW4869 treatment eliminated the inductive effect. YAP target genes, connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) were upregulated in macrophages when treated with exosomes derived from compressive force-treated PDLFs (F-Exo). YAP level was elevated in the F-Exo. When macrophages were treated with Verteporfin, expression of YAP target genes and M1 polarization were significantly downregulated. ConclusionThese results suggested that exosomes derived from compressive force-treated PDLFs promoted the M1 polarization of the THP-1 macrophages. The elevated level of YAP in the exosomes may be a critical factor for this response.

Transcriptional regulation of miR-30a by YAP impacts PTPN13 and KLF9 levels and Schwann cell proliferation

The Hippo pathway is a key regulatory pathway that is tightly regulated by mechanical cues such as tension, pressure, and contact with the extracellular matrix and other cells. At the distal end of the pathway is the yes-associated protein (YAP), a well-characterized transcriptional regulator. Through binding to transcription factors such as the TEA Domain TFs (TEADs) YAP regulates expression of several genes involved in cell fate, proliferation and death decisions. While the function of YAP as direct transcriptional regulator has been extensively characterized, only a small number of studies examined YAP function as a regulator of gene expression via microRNAs. We utilized bioinformatic approaches, including chromatin immunoprecipitation sequencing and RNA-Seq, to identify potential new targets of YAP regulation and identified miR-30a as a YAP target gene in Schwann cells. We find that YAP binds to the promoter and regulates the expression of miR-30a. Moreover, we identify several YAP-regulated genes that are putative miR-30a targets and focus on two of these, protein tyrosine pohosphatase non-receptor type 13 (PTPN13) and Kruppel like factor 9. We find that YAP regulation of Schwann cell proliferation and death is mediated, to a significant extent, through miR-30a regulation of PTPN13 in Schwann cells. These findings identify a new regulatory function by YAP, mediated by miR-30a, to downregulate expression of PTPN13 and Kruppel like factor 9. These studies expand our understanding of YAP function as a regulator of miRNAs and illustrate the complexity of YAP transcriptional functions.

Abstract 2294: NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output

Abstract Background: Despite recent development in prostate cancer (PCa) therapeutics, castration-resistant prostate cancer still accounts for high morbidity and mortality in patients, partly because of incomplete knowledge of the major players that contribute to the androgen independent growth of prostate tumor. NIMA related kinase 1 (NEK1), a dual specificity kinase, mainly functions in maintaining genomic stability by regulating DNA damage response (DDR) and G2/M transition of the cells. We previously reported that tousled-like kinase 1 (TLK1) mediated phosphorylation of NEK1 on T141 can lead to rapid androgen independent progression of PCa cells upon anti-androgen treatment by activating DDR. However, activating DDR alone may not be sufficient to trigger androgen insensitive growth of PCa cells. We hypothesize that TLK1>NEK1 axis may also regulate hippo pathway oncoprotein YAP as a previous study reported that NEK1 can phosphorylate YAP homolog TAZ and upregulate YAP/TAZ transcriptional targets. Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of hippo kinases that binds to transcription factor TEAD and promotes many oncogenic activities in the cells. Methods and Results: To determine if NEK1 can regulate Hippo pathway, our western blotting (WB) analysis revealed higher cleaved YAP (cl-YAP) and lower total YAP level in the NEK1-T141A hypoactive variant overexpressing LNCaP than wt-NEK1 overexpressing cells treated with bicalutamide (anti-androgen) and/or THD (a specific inhibitor of TLK1) which suggest that NEK1 may play a role in stabilizing YAP. TLK1 inhibition also resulted in the reduced expression of some YAP target genes such as CTGF, CDH2, Twist1, and P53AIP1. Co-IP assay revealed that NEK1 can interact with YAP regardless of its kinase activity. We generated a CRISPR-Cas9 mediated NEK1 knockout NT1 (a mouse PCa cell line) cells which showed reduced level of YAP in NEK1 KO clones as well as downregulation of YAP target genes such as CTGF, Zeb1, Twist1, and Cyr61. TLK1 knockdown and inhibition also resulted in reduced pNEK1 (T141) and total YAP level which suggested TLK1 mediated NEK1 activation is necessary for YAP stabilization. An IVK assay using [γ-32P] ATP demonstrated that catalytically active purified NEK1 can phosphorylate purified YAP1. MS analysis determined eight NEK1 mediated phospho-acceptor sites of YAP1: T83, T361, S366, S388, Y407, and T493. Y407 and T493 are the first ever reported phosphorylation sites on YAP1 especially by NEK1 which lies in the transactivation and PDZ binding domain. Our bioinformatics analysis revealed mRNA downregulation of YAP1, while upregulation of YAP protein level was observed in high-grade PCa, which is consistent with our model of post-transcriptional protein stabilization. Conclusion: Our data support that TLK1>NEK1 signaling promotes YAP stabilization and may contribute to castration resistant growth of PCa during AR blockade. Citation Format: Md Imtiaz Khalil, Ishita Ghosh, Vibha Singh, Jing Chen, Haining Zhu, Arrigo De Benedetti. NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2294.

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biophysical Forces of Blood Flow

Hematopoietic stem cells (HSCs) are first specified during embryonic development from hemogenic endothelial cells (HEC) in the ventral dorsal aorta (VDA). Prior studies from our lab and others demonstrated that HSC formation is coordinated with the onset of rigorous circulatory flow, which exposes the vascular endothelium to both wall shear stress (WSS) and circumferential stretch (CS). To better understand how these biomechanical forces drive HEC specification and HSC production, we engineered a microfluidics-based dorsal aorta-on-a-chip that recapitulates the biophysical features present in the vasculature. Using this biomimetic microsystem, human iPS-derived HEC exposed to WSS and CS through continuous media flow and cyclic stretching of the adherent cell layer showed an upregulation of RUNX1 (WSS, p<0.01; CS, p<0.05), the main transcription factor involved in HSC specification, by qPCR compared to cells in the static control. Interestingly, the YAP (Yes-associated protein) target genes CYR61, ANKRD1 and CTGF were also upregulated in human iPS-derived HEC after exposure to CS, suggesting a possible downstream mechanism for the effects of blood flow on HEC. YAP is the most downstream effector protein in the Hippo signaling pathway and is known to regulate stem cell dynamics and organ size. Prior work identified YAP as a mechanosensor, where it is activated in response to Rho GTPase activity and actomyosin filament rearrangement independent of the core Hippo pathway. To test whether YAP functions in response to mechanical force-activated Rho, iPS-derived HEC were exposed to circumferential stretch in the presence of a Rho inhibitor, which abolished CS-induced activation of YAP targets and RUNX1, indicating that Rho proteins are involved in mediating the effects of blood flow on HECs. Furthermore, treatment of iPS-derived HEC cultured under static conditions with either direct or indirect activators of the Rho GTPase family increased YAP activity and RUNX1 levels, demonstrating that biophysical forces from blood flow can be mimicked in vitro with Rho modulation. To investigate the implication of these findings in vivo, we utilized the zebrafish model: as in mammalian systems, HSCs are born in the VDA (24-36 hours post fertilization (hpf)) and migrate to the secondary sites of hematopoiesis to expand and differentiate before colonizing adult tissues. Heat-shock mediated overexpression of Yap at 12 hpf significantly increased runx1 + HEC specification at 36 hpf by both in situ hybridization and qPCR (p<0.01). This impact on HSC number was sustained throughout development, as Yap overexpression also significantly increased the number of Cd41+ HSCs present in the caudal hematopoietic tissue (CHT) at 72 hpf (p<0.05). In contrast, Yap knockout mutants exhibited a significant reduction in runx1 at 36 hpf (p<0.0001) despite normal Flk1+ vascular structure, indicative of a role for Yap in HSC specification. In line with the human in vitro data, activation of Rho GTPase increased expression of Yap target genes and runx1 at 36 hpf (p<0.05), suggesting a pathway in which Yap induces Runx1 downstream of Rho stimulation. Prior analysis of the silent heart mutant line as well as morpholino knockdown of cardiac troponin T type A (tnnt2a) revealed a significant deficiency in HEC specification and HSC production in the absence of heartbeat and blood flow. Importantly, compound-mediated activation of Rho GTPase (12-36 hpf) significantly increased runx1 expression in tnnt2a morphant embryos by in situ hybridization and qPCR (p<0.01), demonstrating that Rho activation can mimic the effects of blood flow on HSC production in vivo . Taken together, our findings point to a functional intersection between blood flow, Rho GTPase function and YAP activation in RUNX1-dependent HSC production; this mechanochemical mechanism may be exploited to improve in vitro human HSC differentiation protocols for the treatment for blood disorders. DisclosuresIngber:Emulate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Radiation-induced YAP activation confers glioma radioresistance via promoting FGF2 transcription and DNA damage repair

Although radiotherapy is a well-known effective non-surgical treatment for malignant gliomas, the therapeutic efficacy is severely limited due to the radioresistance of tumor cells. Previously, we demonstrated that Yes-associated protein (YAP) promotes glioma malignant progression. However, whether YAP plays a role in radioresistance and its potential value in cancer treatment are still unclear. In this study, we found that high YAP expression is associated with poor prognosis in malignant glioma patients undergoing radiotherapy. Research in immortalized cell lines and primary cells from GBM patients revealed that YAP exhibited a radioresistant effect on gliomas via promoting DNA damage repair. Mechanistically, after radiation, YAP was translocated into the nucleus, where it promoted the expression and secretion of FGF2, leading to MAPK–ERK pathway activation. FGF2 is a novel target gene of YAP. Inhibition of YAP–FGF2–MAPK signaling sensitizes gliomas to radiotherapy and prolongs the survival of intracranial cell-derived and patient-derived xenograft models. These results suggest that YAP–FGF2–MAPK is a key mechanism of radioresistance and is an actionable target for improving radiotherapy efficacy.

TRPV4 regulates matrix stiffness‐dependent activation of YAP/VEGFR2 signaling via Rho/Rho kinase/LATS1/2 pathway

TRPV4 is a mechanosensitive ion channel in endothelial cells and shown to regulate physiological and pathological angiogenesis. We have recently shown that TRPV4 deletion and/or down regulation activates vascular endothelial growth factor receptor-2 (VEGFR2) via yes-associated protein (YAP) in human EC. However, the molecular mechanisms of TRPV4-dependant YAP/VEGFR2 activation are unknown. We have employed human microvascular endothelial cells (HMEC-1; EC) on ECM gels of varying stiffness that mimic normal and failing hearts (8 and 50 kPa) and examined nuclear translocation of YAP and disappearance of perinuclear VEGFR2, after siRNA knockdown of TRPV4. Next, we investigated the role of upstream and downstream regulators of YAP translocation by focusing on LATS-1, CYR61 and Rho/Rho Kinase, using phospho-specific antibodies (pYAP and pLATS) and pharmacological inhibitor of Rho kinase (Y-27632). siRNA knockdown of TRPV4 significantly increased nuclear translocation of YAP with concomitant disappearance of perinuclear-VEGFR2 in matrix stiffness-dependent manner. Further, we found increased Rho-GTP and decreased LATS-1 phosphorylation in TRPV4 knockdown EC compared to control siRNA treated EC. Furthermore, pharmacological inhibition of Rho kinase with Y-27632, increased phosphorylation of LATS1/2 kinase, inhibited nuclear translocation of YAP and VEGFR2 redistribution. Consistent with above results, YAP target genes CYR61 and CTGF mRNA expression levels were increased in a matrix stiffness-dependent manner in TRPV4 knockdown EC than the control siRNA treated EC. Our results suggest that TRPV4 regulates YAP/VEGFR2 activation in a matrix stiffness dependent manner during angiogenesis via Rho/Rho kinase/LATS1/2 pathway.

Geranylgeranyl pyrophosphate-mediated protein geranylgeranylation regulates endothelial cell proliferation and apoptosis during vasculogenesis in mouse embryo

Vascular development is essential for the establishment of the circulatory system during embryonic development and requires the proliferation of endothelial cells. However, the underpinning regulatory mechanisms are not well understood. Here, we report that geranylgeranyl pyrophosphate (GGPP), a metabolite involved in protein geranylgeranylation, plays an indispensable role in embryonic vascular development. GGPP is synthesized by geranylgeranyl pyrophosphate synthase (GGPPS) in the mevalonate pathway. The selective knockout of Ggpps in endothelial cells led to aberrant vascular development and embryonic lethality, resulting from the decreased proliferation and enhanced apoptosis of endothelial cells during vasculogenesis. The defect in protein geranylgeranylation induced by GGPP depletion inhibited the membrane localization of RhoA and enhanced yes-associated protein (YAP) phosphorylation, thereby prohibiting the entry of YAP into the nucleus and the expression of YAP target genes related to cell proliferation and the antiapoptosis process. Moreover, inhibition of the mevalonate pathway by simvastatin induced endothelial cell proliferation defects and apoptosis, which were ameliorated by GGPP. Geranylgeraniol (GGOH), a precursor of GGPP, ameliorated the harmful effects of simvastatin on vascular development of developing fetuses in pregnant mice. These results indicate that GGPP-mediated protein geranylgeranylation is essential for endothelial cell proliferation and the antiapoptosis process during embryonic vascular development.

YAP Activation and Implications in Patients and a Mouse Model of Biliary Atresia.

Background and Aim: Biliary atresia (BA), an inflammatory destruction of the bile ducts, leads to liver fibrosis in infants and accounts for half of cases undergoing pediatric liver transplantation. Yes-associated protein (YAP), an effector of the Hippo signaling pathway, is critical in maintaining identities of bile ductal cells. Here, we evaluated the expression of YAP and YAP target genes in BA livers and a rhesus rotavirus (RRV)-induced BA mice model.Methods: Liver specimens collected from 200 BA patients were compared with those of 30 non-BA patients. Model mice liver tissues were also collected. The expression of YAP and YAP target genes were measured by transfection, RNA-seq, immunohistochemistry, immunoblot, and quantitative PCR. Masson's trichrome staining and the Biliary Atresia Research Consortium (BARC) system were utilized to score liver fibrosis status.Results: The expression of YAP is elevated and positively correlated with fibrosis in BA livers. Moreover, ANKRD1, which is identified as the target gene of YAP, is also highly expressed in BA livers. Consistent with clinical data, YAP and ANKRD1 are significantly upregulated in RRV-induced BA mouse model.Conclusions: YAP expression is closely correlated with the bile duct hyperplasia and liver fibrosis, and may serve as an indicator for liver fibrosis and BA progression. This study indicates an involvement of the Hippo signaling pathway in the development of BA, and the YAP induced ANKRD1 expression may also be related to bile duct hyperplasia in BA. This provides a new direction for more in-depth exploration of the etiology and pathogenesis of biliary atresia.