Role Of Yes-associated Protein Research Articles

Role of Yes-associated protein 1 in angiotensinⅡ-induced pulmonary fibrosis in rats

Objective: To explore the mechanism of Yes-associated protein 1 (Yap1) in angiotensinⅡ(AngⅡ)-induced pulmonary fibrosis. Methods:In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group (BLM), and BLM+ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical (IHC) staining for Yap1. In vitro, the isolated fibroblasts were treated with 10(-7) mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif (TAZ), and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups: the empty plasmid (vector) group, the vector+ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA+ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity. Results:In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM+ AngⅡ group than the BLM group (both P<0.05). In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group (all P<0.05). Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group (0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015). However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015). Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ+ irbesartan group were higher in cytoplasm (0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059), while lower in the nucleus (0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015) (all P<0.05). Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA+ AngⅡ group were distinctly lower than the vector+ AngⅡ group (all P<0.05). In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector+ AngⅡ group exceeded that of vector group (both P<0.05). However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA+ AngⅡgroup were less than the vector+ AngⅡ group (both P<0.05). Conclusion: AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.

Interference of Yes associated protein 1 expression inhibits the migration of U87MG glioma cells

Objective To evaluate the effect of Yes associated protein 1 (YAP1) on the migration of human glioma cell line U87MG in the downstream of Hippo signaling pathway. Methods YAP specific small interfering RNA (siRNA) was used to knock down the YAP expression of U87MG cells, which was identified by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). By comparison among the Blank group, NC group and YAP1 interference group, the cell scratch assay was used to verify the role of YAP1 in migration of glioma cells. Results The siRNA YAP1 sequence selected could significantly interfered with the YAP1 expression with the interference efficiency being over 90%. The migration of glioma cells was significantly decreased after siRNA interference of YAP1 expression. The quantitative analysis showed that there was significant difference in the migration rate of U87MG cells between YAP1 interference group and control group. Conclusion YAP1 regulates U87MG glioma cells, and interference of the YAP1 expression can inhibit the migration of U87MG glioma cells. Key words: Glioma; Yes associated protein 1; Small interfering RNA; Cell migration

Elevation of YAP promotes the epithelial-mesenchymal transition and tumor aggressiveness in colorectal cancer

Tumor metastasis is the leading cause of death in cancer patients. Identifying metastatic biomarkers in tumor cells would help cancer diagnoses and the development of therapeutic targets. Yes-associated protein (YAP) plays an important role in organ development and has gained much attention in tumorigenesis. However, the role of YAP and the underlying mechanism in tumor metastasis of colorectal cancer (CRC) is still unclear. In this study, we generated metastatic 116-LM cells from the HCT116 CRC cell line. We found that the capacity for tumor aggressiveness was elevated in 116-LM cells and identified that YAP and its mRNA level were upregulated in 116-LM cells. Moreover, expression of YAP was found to correlate with epithelial-mesenchymal transition (EMT) marker expressions, whereas suppression of YAP decreased EMT marker expressions and impeded tumor migration and invasion. Additionally, upregulation of YAP was identified in colon cancer patients, and it was correlated with EMT gene expressions. Furthermore, we identified LBH589, a histone deacetylase inhibitor, that was capable of inhibiting tumor growth and aggressiveness in both HCT116 and 116-LM cells. LBH589 potentially inhibited YAP and its mRNA expression, accompanied by diminished expressions of YAP downstream genes and EMT markers. Together, YAP plays a crucial role in aggressiveness and metastasis of CRC, and YAP may be an attractive therapeutic target.

YAP1 enhances cell proliferation, migration, and invasion of gastric cancer in vitro and in vivo.

Yes-associated protein 1 (YAP1) plays an important role in the development of carcinomas such as breast, colorectal, and gastric (GC) cancers, but the role of YAP1 in GC has not been investigated comprehensively. The present study strongly suggests that YAP1 and P62 were significantly up-regulated in GC specimens, compared with normal gastric mucosa. In addition, the YAP1high P62high expression was independently associated with poor prognosis in GC (hazard ratio: 1.334, 95% confidence interval: 1.045–1.704, P = 0.021). Stable YAP1 silencing inhibited the proliferation, migration, and invasion of BGC-823 GC cells in vitro and inhibited the growth of xenograft tumor and hematogenous metastasis of BGC-823 GC cells in vivo. The mechanism was associated with inhibited extracellular signal-regulated kinases (ERK)1/2 phosphorylation, elevated E-cadherin protein expression and decreased vimentin protein expression, down-regulated β-catenin protein expression and elevated α-catenin protein expression, and down-regulated long non-coding RNA (lncRNA) expressions including HOX transcript antisense RNA (HOTAIR), H19, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), human large tumor suppressor-2 (LATS2)-AS1-001, and LATS2. YAP1 over-expression promoted the proliferation, migration, and invasion of human immortalized normal gastric mucosa GES-1 cells in vitro by reversing the above signal molecules. Subcutaneous inoculation of GES-1 cells and YAP1-over-expressing GES-1 cells into nude mice did not generate tumors. We successfully established the xenograft tumor models using MKN-45 GC cells, but immunochemistry showed that there was no YAP1 expression in MKN-45 cells. These results suggest that YAP1 is not a direct factor affecting tumor formation, but could accelerate tumor growth and metastasis. Collectively, this study highlights an important role for YAP1 as a promoter of GC growth and metastasis, and suggests that YAP1 could possibly be a potential treatment target for GC.

Unraveling the expression of the oncogene YAP1, a Wnt/beta-catenin target, in adrenocortical tumors and its association with poor outcome in pediatric patients.

BackgroundOverexpression of the oncogene yes-associated-protein-1 (YAP1) is associated with increased cell proliferation in human cancers. YAP1 is a potential target of the Wnt/beta-catenin pathway, which plays an important role in adrenocortical tumors (ACT). The role of YAP1 in adrenocortical tumorigenesis has not been assessed.Aims: To evaluate YAP1 expression in normal adrenals and pediatric ACT and its association with disease outcome. To investigate the interaction between YAP1 and the Wnt/beta-catenin pathway in adrenocortical cells.ResultsStrong YAP1 staining was present in fetal adrenals and pediatric ACT but weak in postnatal adrenals. In pediatric ACT, YAP1 mRNA overexpression was associated with death, recurrent/metastatic disease and lower overall survival. The inhibition of the Wnt/beta-catenin pathway increased YAP1 mRNA expression. siYAP1 increased CTNNB1/beta-catenin expression and nuclear staining regardless of DLV2, moreover, it decreased cell growth and impaired cell migration.Materials and MethodsWe assessed in 42 pediatric ACT samples the YAP1 protein expression by immunohistochemistry and mRNA expression by RT-qPCR and analyzed their association with outcome. As controls, we resort 32 fetal and postnatal normal adrenals for IHC and 10 normal adrenal cortices for RT-qPCR. The interaction between YAP1 and the Wnt/beta-catenin pathway was assessed in NCI-H295 adrenocortical cells by inhibiting the TCF/beta-catenin complex and by knocking down YAP1.ConclusionYAP1 overexpression is a marker of poor prognosis for pediatric patients with ACT. In adrenocortical cells, there is a close crosstalk between YAP1 and Wnt/beta-catenin. These data open the possibility of future molecular therapies targeting Hippo/YAP1 signaling to treat advanced ACT.

Aurora A kinase activates YAP signaling in triple-negative breast cancer.

The Yes-associated protein (YAP) is an effector that transduces the output of the Hippo pathway to transcriptional modulation. Considering the role of YAP in cancers, this protein has emerged as a key node in malignancy development. In this study, we determined that Aurora A kinase acts as a positive regulator for YAP-mediated transcriptional machinery. Specifically, YAP associates with Aurora A predominantly in the nucleus. Activation of Aurora A can impinge on YAP activity through direct phosphorylation. Moreover, aberrant expression of YAP and Aurora A signaling is highly correlated with triple-negative breast cancer (TNBC). We herein provide evidence to establish the functional relevance of this newly discovered regulatory axis in TNBC.

Role of Yes-associated protein in cancer: An update.

Yes-associated protein (YAP) is an oncoprotein located in the cytoplasm in an inactive form, and when activated, it translocates to the nucleus and activates the transcription of genes responsible for cell division and apoptosis. YAP is one of the downstream regulatory proteins in the Hippo signaling pathway, which is important in cell proliferation and regeneration. Due to its great importance, YAP is regulated very strictly by different regulatory systems. The present review will focus on the canonical pathways of YAP, and will provide details on the most recent findings regarding its regulation and role in tumorigenesis, specifically in prostate tumor progression.

Abstract 73: Yap Plays a Crucial Role in the Development of Cardiomyopathy in Lysosomal Storage Diseases

Rag proteins play a critical role in regulating lysosomal functions. Muscle-specific deletion of RagA and RagB (Rag A/B) results in lysosomal dysfunction, suppression of autophagy, and development of cardiomyopathy, reminiscent of lysosomal storage diseases (LSDs). We investigated the molecular mechanism by which cardiomyopathy is developed in RagA/B knockout (KO) mice. We generated cardiac-specific Rag A/BKO (RagA/BcKO) mice using αMHC-Cre. Similar to the muscle-specific KO mice, RagA/BcKO mice showed prominent cardiac hypertrophy (heart weight (HW)/ tibial length (TL): 12.35 ± 0.41 vs. 7.12 ± 0.15, p&lt; 0.01) at 3 months of age. RagA/BcKO mice exhibited significantly greater phospho-histone H3-positive cardiomyocytes than control mice, suggesting that cardiomyocyte proliferation is stimulated in RagA/BcKO mice. However, RagA/BcKO mice exhibited a significantly smaller percentage of fractional shortening (%FS: 23.0 ± 0.82 vs. 45.8 ± 1.06, p&lt; 0.01) and significantly greater mortality than control mice (50% vs. 0%, p&lt;0.01). RagA/BcKO mouse hearts showed upregulation of β-MHC and α-smooth muscle actin, indicating de-differentiation. Yes-associated protein (YAP) is a transcription co-factor that controls growth and survival and is a key downstream target of the Hippo pathway. YAP associates with p62/SQSTM1 and is degraded through autophagy. YAP is significantly more accumulated (5.9-fold, p&lt;0.05) in RagA/BcKO mouse hearts, where autophagy is inhibited and p62/SQSTM1 is accumulated, than in control hearts. In order to elucidate the role of YAP in mediating cardiomyopathy, we crossed RagA/BcKO mice with cardiac-specific heterozygous YAPKO mice. Heterozygous deletion of YAP in RagA/BcKO mice reduced the heart size (HW/ TL: 9.12 ± 0.10 vs. 11.9 ± 0.27, p&lt; 0.05) and improved cardiac function (%FS: 35.0 ± 2.89 vs. 24.0 ± 0.58, p&lt; 0.05) in RagA/BcKO mice. Pharmacological inhibition of YAP by verteporfin treatment also suppressed cardiac hypertrophy (HW/ TL: 8.92 ± 0.11 vs. 12.09 ± 0.30, p&lt; 0.05) and heart failure (%FS: 33.3 ± 2.29 vs. 23.3 ± 0.63, p&lt; 0.05) in RagA/BcKO mice. These results indicate that YAP plays a crucial role in the development of cardiomyopathy in RagA/BcKO mice, a mouse model of LSDs.

Abstract 2146: YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas

Abstract Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. Using lung adenocarcinoma cell lines, we detected enhanced YAP expression and activity mediated by EGFR signaling due to enhanced protein stability. The Src family protein YES was involved in EGFR-regulated YAP expression. The YAP/YES pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP levels by mechanisms bypassing EGFR were effective in killing EGFR-dependent cells including EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP in EGFR mutant lung adenocarcinomas and identified YAP as an promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance. Citation Format: Ting-Fang Lee, Yu-Chi Tseng, Ying-Pu Liu, Yu-Rung Kao, Cheng-Wen Wu. YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2146.

MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status.

Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the “loser” and actively eliminated, while the cell with the relatively higher fitness level is the “winner” and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells.

Abstract 168: YAP and TAZ Mediate Mechanical Control of Vasculogenesis

Physical cues such as the mechanical properties of the extracellular matrix are known to modulate blood vessel formation, but the underlying mechanisms remain poorly understood. Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) were recently identified as key mediators of cellular mechanotransduction [1], but the roles of YAP and TAZ in neovascular mechanobiology have not been investigated. We hypothesized that YAP and TAZ mediate neovascular mechanotransduction of extracellular matrix rigidity by vasculogenic endothelial colony forming cells (ECFCs) cultured on/in matrices of controllable rigidity. YAP and TAZ depletion abrogated ECFC migration (Fig. 1A), and YAP/TAZ expression and nuclear localization (i.e., activation) was significantly upregulated in migrating cells (Fig. 1B). Combinatorial depletion also abrogated tubular network formation and 3D vasculogenesis in collagen matrices of variable rigidity but constant matrix density (data not shown). In 2D, PDMS rigidity regulated YAP/TAZ subcellular localization (data not shown). In 3D, vasculogenesis was enhanced in stiff (G’= 1049 Pa) collagen oligomer matrices compared to soft (G’ = 305 Pa) monomer matrices formulated at constant collagen matrix density. YAP and TAZ were nuclear localized (activated) in the stiff oligomer matrices, but cytosolic in soft monomer matrices (Fig. 1C). Mechanistically, YAP and TAZ depletion reduced cytoskeletal stress fiber formation (data not shown), implicating YAP and TAZ in a feedback mechanism to control cytoskeletal remodeling. Delivered in oligomer matrices in vivo , ECFCs formed a functional human neovascular plexus and exhibited strong expression and nuclear localization of YAP and TAZ and expression of downstream genes, Cyr61 and CTGF (data not shown). In summary, YAP and TAZ are essential for mechanical control of ECFC migration and vasculogenesis. [1] Dupont et al. 2011 Nature. 474:179-185.

Role of Hippo-YAP/TAZ signaling pathway in mechanotransduction.

Transcriptional coactivators YAP(yes associated protein)and TAZ(transcriptional coactivator with PDZ-binding motif)regulate gene expression through binding to transcription factors. Recently, some studies showed that YAP/TAZ activity responded to mechanical inputs such as stiffness of the extracellular matrix and the mechanical regulation of YAP/TAZ controlled cell proliferation or differentiation. Additionally, we revealed important roles of YAP in tissue formation and homeostasis through cellular tension and pressure. These reports indicate that YAP/TAZ are major factors in mechanotransduction connecting between the mechanical environment and cell responses.

YAP and ERK mediated mechanical strain-induced cell cycle progression through RhoA and cytoskeletal dynamics in rat growth plate chondrocytes.

Yes-associated protein (YAP) and extracellular signal-regulated kinase (ERK) have been considered as key regulators in tissue homeostasis, organ development, and tumor formation. However, the roles of YAP and ERK in the mediating strain mechanosensing in the growth plate cartilage have not been determined. In this study, chondrocytes obtained from the growth plate cartilage of 2-week-old Sprague-Dawley rats were subjected to the mechanical strain with different magnitudes and durations at a frequency of 0.5 Hz. We found that YAP and ERK activation in response to mechanical strain was time and magnitude dependent. Pretreatment with a RhoA inhibitor (C3 toxin) or a microfilament cytoskeleton disrupting reagent (cytochalasin D) could suppress their activation. In addition, activated YAP and ERK were able to induce cell cycle progression by up-regulating the expression of cell cycle-related genes. These results shed new light on the function of YAP and ERK in mechanical strain-promoted growth plate development. Our results also provided evidence that RhoA and cytoskeletal dynamics are required for this mechanotransduction. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1121-1129, 2016.

Abstract B157: YAP is a promising target in cutaneous melanoma

Abstract The transcriptional regulator YAP (Yes-associated protein) has been implicated in tissue development and homeostasis and in cancer growth (Harvey et al. 2013). In uveal melanoma, YAP mediates oncogenic signaling of mutated G-proteins, driving tumor growth in a therapeutically exploitable manner (Yu et al. &amp; Feng et al. 2014). The role of YAP in cutaneous melanoma (CM) is less well defined, although previous studies implicating YAP in metastasis and disease maintenance/progression (Nallet-Staub et al. 2014; Menzel et al. 2014), and encouraging data suggesting synergism between combination targeting of YAP and the Mitogen-Activated Protein Kinases (MAPK) pathway (Lin et al. 2015). Here we show that YAP is a critical mediator of disease maintenance and progression in some but not all CMs. In a primary CM we identified novel YAP mutations, which are rare in human cancer but are predicted to relieve YAP from inhibition by the Hippo pathway. By quantifying YAP expression by immunofluorescence, we found YAP was increased early in human melanocytic transformation, being elevated in nevi and in over 50% of primary CMs. Gene targeting of YAP with inducible shRNAs induced profound proliferative arrest and death in cultured CM cells (measured by MTT assay, EdU incorporation and propidium iodide staining respectively). The dependency on YAP of melanoma maintenance was validated in vivo using xenograft models of cell lines and of early passage patient-derived melanomas. Further, efficacy of YAP targeting synergized with MAPK inhibition in BRAF mutant CMs. However, some CMs were resistant to YAP targeting independent of BRAF/NRAS genotype and of levels of YAP protein expression or activation. These data compel the development of anti-YAP therapies in CM and the identification of biomarkers that predict response to YAP targeting. Citation Format: Jian zhong Tang, Xiaomeng Zhang, Ismael Vergara, Anthony Papenfuss, Kieran Harvey, Mark Shackleton. YAP is a promising target in cutaneous melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B157.

Role of YAP and TAZ in pancreatic ductal adenocarcinoma and in stellate cells associated with cancer and chronic pancreatitis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic and inflammatory microenvironment that is formed primarily by activated, myofibroblast-like, stellate cells. Although the stellate cells are thought to contribute to tumorigenesis, metastasis and drug resistance of PDAC, the signaling events involved in activation of the stellate cells are not well defined. Functioning as transcription co-factors, Yes-associated protein (YAP) and its homolog transcriptional co-activator with PDZ-binding motif (TAZ) modulate the expression of genes involved in various aspects of cellular functions, such as proliferation and mobility. Using human tissues we show that YAP and TAZ expression is restricted to the centroacinar and ductal cells of normal pancreas, but is elevated in cancer cells. In particular, YAP and TAZ are expressed at high levels in the activated stellate cells of both chronic pancreatitis and PDAC patients as well as in the islets of Langerhans in chronic pancreatitis tissues. Of note, YAP is up regulated in both acinar and ductal cells following induction of acute and chronic pancreatitis in mice. These findings indicate that YAP and TAZ may play a critical role in modulating pancreatic tissue regeneration, neoplastic transformation, and stellate cell functions in both PDAC and pancreatitis.

YAP is a critical oncogene in human cholangiocarcinoma.

Yes-associated protein (YAP), a transcriptional co-activator, has important regulatory roles in cell signaling and is dysregulated in a number of cancers. However, the role of YAP in cholangiocarcinoma (CCA) progression remains unclear. Here, we demonstrated that YAP was overexpressed in CCA cells and human specimens. High levels of nuclear YAP (nYAP) correlated with histological differentiation, TNM stage, metastasis and poor prognosis in CCA. Silencing YAP increased tumor sensitivity to chemotherapy and inhibited CCA tumorigenesis and metastasis both in vivo and in vitro. YAP overexpression in vivo and in vitro promoted CCA tumorigenesis and metastasis. Additionally, we found that YAP induced epithelial-mesenchymal transition (EMT) and formed a regulatory circuit with miR-29c, IGF1, AKT and gankyrin to promote the progression of CCA. Results of CCA tissue microarray showed positive correlations between nYAP and gankyrin or p-AKT expression. Combination of nYAP and gankyrin or p-AKT exhibited improved prognostic accuracy for CCA patients. In conclusion, YAP promotes carcinogenesis and metastasis by up-regulating gankyrin through activation of the AKT pathway.

Abstract 427: Flow Regulation of YAP/TAZ in Endothelial Inflammation and Atherosclerosis

The focal nature of atherosclerotic lesions in regions such as arterial branch points and curvatures suggests a regulatory mechanism by local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechano-transduction and vascular homeostasis. However, the mechanism by which YAP/TAZ modulates endothelial phenotypes in response to atheroprone vs. atheroprotective hemodynamic flows remains unknown. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of endothelial inflammation and the consequential development of atherosclerotic lesions. Experiments were conducted on cultured vascular endothelial cells (ECs) and atherosclerotic mouse model. We found that exposure of ECs to the atheroprone oscillatory shear stress (OS) results in YAP/TAZ activation and translocation into EC nucleus to upregulate the target genes, including connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61). In contrast, the atheroprotective laminar shear stress suppresses YAP/TAZ. Consistent with these in vitro results, ex vivo analysis of mouse arteries demonstrated a more nuclear localization of YAP/TAZ and elevated levels of CTGF and Cyr61 in the endothelium in atheroprone areas than in atheroprotective areas. Functionally, YAP/TAZ knockdown significantly attenuated the OS- and cytokine-inductions of pro-inflammatory adhesion molecules in ECs and monocytes, as well as the consequential monocyte adhesion to ECs. Overexpression of constitutively active YAP increases endothelial inflammation and monocyte adhesion to ECs. Furthermore, in vivo blockade of YAP/TAZ translation by Morpholino oligos in apolipoprotein E-deficient mice significantly reduced endothelial inflammation, mononuclear cell infiltration, and the size of atherosclerotic plaques. Our results demonstrate a critical role of YAP/TAZ activation by atheroprone flow in promoting endothelial inflammation and the development of atherosclerotic lesions. Therefore, inhibition of YAP/TAZ activation may hold promise as a novel atheroprotective therapeutic strategy.

Mo1885 Role of the Hippo-YAP Pathway in the Regulation of DNA Synthesis by cAMP in Intestinal Epithelial Cells and Swiss 3T3 Fibroblasts

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.

Mo1884 Regulation of YAP Localization and Phosphorylation in Intestinal Epithelial Cells

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.

Mo1907 A Cohort Study of Risk Factors for Developing Erosive Esophagitis Following Negative Index Endoscopy in a Large Health Check-Up Population

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.