Cytoplasmic Yes-associated Protein Research Articles

Nuclear Localization of Yes-Associated Protein Is Associated With Tumor Progression in Cutaneous Melanoma

Yes-associated protein (YAP), an effector molecule of the Hippo signaling pathway, is expressed at high levels in cutaneous melanoma. However, the role of YAP in melanoma progression according to cellular localization is poorly understood. Tissues from 140 patients with invasive melanoma were evaluated by immunohistochemistry. Flow cytometry, western blotting, viability assays, wound healing assays, verteporfin treatment, and xenograft assays were conducted using melanoma cell lines B16F1 and B16F10 subjected to YapS127A transfection and siYap knockdown. Nuclear YAP localization was identified in 63 tumors (45.0%) and was more frequent than cytoplasmic YAP in acral lentiginous and nodular subtypes (P = .007). Compared with cytoplasmic YAP melanomas, melanomas with nuclear YAP had higher mitotic activity (P = .016), deeper invasion (P < .001), and more frequently metastasized to lymph nodes (P < .001) and distant organs (P < .001). Patients with nuclear YAP melanomas had poorer disease-free survival (P < .001) and overall survival (P < .001). Nuclear YAP was an independent risk factor for distant metastasis (hazard ratio: 3.206; 95% CI, 1.032-9.961; P = .044). Proliferative ability was decreased in siYapB16F1 (P < .001) and siYapB16F10 (P = .001) cells and increased in YapS127AB16F1 (P = .003) and YapS127AB16F10 (P = .002) cells. Cell cycle analysis demonstrated relative G1 retention in siYapB16F1 (P < .001) and siYapB16F10 (P < .001) cells and S retention in YapS127AB16F1 cells (P = .008). Wound healing assays showed that Yap knockdown inhibited cell invasion (siYapB16F1, P = .001; siYapB16F10, P < .001), whereas nuclear YAP promoted it (YapS127AB16F, P < .001; YapS127AB16F1, P = .017). Verteporfin, a direct YAP inhibitor, reduced cellular proliferation in B16F1 (P = .003) and B16F10 (P < .001) cells. Proliferative effects of nuclear YAP were confirmed in xenograft mice (P < .001). In conclusion, nuclear YAP in human melanomas showed subtype specificity and correlated with proliferative activity and proinvasiveness. It is expected that YAP becomes a useful prognostic marker, and its inhibition may be a potential therapy for melanoma patients.

CaMKII may regulate renal tubular epithelial cell apoptosis through YAP/NFAT2 in acute kidney injury mice

Aim Renal tubular epithelial cell (RTEC) apoptosis is important in acute kidney injury (AKI). Calcium/calmodulin-dependent protein kinase II (CaMKII) plays an important role in cell apoptosis, but its potential role in AKI remains unknown. Methods Using co-immunoprecipitation, immunofluorescence, immunohistochemistry, western blotting, flow cytometry, and cell transfection, this study aimed to verify whether CaMKII is involved in RTEC apoptosis and to explore the underlying mechanism. Results We found that CaMKII was involved in RTEC apoptosis. In adriamycin-induced AKI mice, serum creatinine levels, cell apoptosis, CaMKII activity, and nuclear factor of activated T cells 2 (NFAT2) levels increased, whereas nuclear Yes-associated protein (YAP) expression decreased; inhibition of CaMKII activity reversed these changes. Phosphorylated CaMKII could bind to phosphorylated YAP in the cytoplasm and block it from entering the nucleus, thereby failing to inhibit NFAT2-mediated cell apoptosis. Sequestrated phosphorylated YAP in the RTEC cytoplasm was finally degraded by ubiquitination. Conclusion CaMKII may regulate RTEC apoptosis through YAP/NFAT2 in AKI mice. CaMKII may be a potent molecular target for AKI treatment.

Inhibition of YAP by lenvatinib in endothelial cells increases blood pressure through ferroptosis

Lenvatinib, a multitarget tyrosine kinase inhibitor (TKI), increases the incidence of severe hypertension and thus the incidence of cardiovascular complications. Inhibition of ferroptosis, a newly recognized type of cell death, alleviates endothelial dysfunction. Here, we report that lenvatinib-induced hypertension is associated with ferroptosis of endothelial cells. RNA sequencing (RNA-seq) showed that lenvatinib led to ferroptosis of endothelial cells and that administration of mouse with ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor, dramatically ameliorated lenvatinib-induced hypertension and reversed lenvatinib-induced impairment of endothelium-dependent relaxation (EDR). Furthermore, lenvatinib significantly reduced glutathione peroxidase 4 (GPX4) expressions in the mouse aorta and human umbilical vein endothelial cells (HUVECs) and increased lipid peroxidation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) levels in HUVECs. Immunofluorescence and Western blotting showed that lenvatinib significantly reduced Yes-associated protein (YAP) nuclear translocation but not cytoplasmic YAP expression in HUVECs. The data, generated from both in vivo and in vitro, showed that lenvatinib reduced total YAP (t-YAP) expression and increased the phosphorylation of YAP at both Ser127 and Ser397, without affecting YAP mRNA levels in HUVECs. XMU-MP-1 mediated YAP activation or YAP overexpression effectively attenuated the lenvatinib-induced decrease in GPX4 expression and increases in LDH release and MDA levels. In addition, overexpression of YAP in HUVECs ameliorated lenvatinib-induced decrease in the mRNA and protein levels of spermidine/spermine N (1)-acetyltransferase-1 (SAT1), heme oxygenase-1 (HO-1), and ferritin heavy chain 1 (FTH1). Taken together, our data suggest that lenvatinib-induced inhibition of YAP led to ferroptosis of endothelial cells and subsequently resulted in vascular dysfunction and hypertension.

Zinc Oxide Nanoparticles Promote YAP/TAZ Nuclear Localization in Alveolar Epithelial Type II Cells

We investigated roles of Hippo signaling pathway components in alveolar type II cells (AECII) after zinc oxide nanoparticle (ZnONP) exposure. ZnONPs physicochemistry was characterized using field emission-scanning electron microscopy (FE-SEM) and energy-dispersive X-ray (EDX) microanalysis. ZnONP deposition in human respiratory tract was estimated using multiple-path particle dosimetry (MPPD) model. MLE-12 AECII were cultured and exposed to 0, 1, and 5 μg/mL of ZnONPs for 24 h. Western blots were used to investigate signaling pathways associated with Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), cell adherens junctions, differentiation, and senescence. ZnONPs morphology was irregular, with Zn and O identified. Approximately 72% of inhaled ZnONPs were deposited in lungs, with 26% being deposited in alveolar regions. ZnONP exposure increased nuclear YAP expression and decreased cytoplasmic YAP expression by AECII. Adherens junction proteins, E-cadherin, α-catenin, and β-catenin, on AECII decreased after ZnONP exposure. ZnONP exposure of AECII increased alveolar type I (AECI) transition protein, LGALS3, and the AECI protein, T1α, while decreasing AECII SPC expression. ZnONP exposure induced Sirt1 and p53 senescence proteins by AECII. Our findings showed that inhalable ZnONPs can deposit in alveoli, which promotes YAP nuclear localization in AECII, resulting in decrease tight junctions, cell differentiation, and cell senescence.

Effects and mechanism of age on the stiffness and the fibrotic phenotype of fibroblasts of human hypertrophic scar

Objective: To explore the effects and potential molecular mechanism of age on the stiffness and the fibrotic phenotype of fibroblasts (Fbs) of human hypertrophic scar. Methods: The experimental research method was used. From January to June 2020, the surgically removed hypertrophic scar tissue of 10 scar patients (4 males and 6 females) and residual full-thickness normal skin tissue of 10 cases (5 males and 5 females, aged 7-41 years) were collected after operation in Department of Burns and Plastic Surgery of the Fourth Medical Center of the PLA General Hospital. The hypertrophic scar tissue of 6 patients aged (10.7±1.6) years was included into the young group and the hypertrophic scar tissue of 4 patients aged (40.0±2.2) years was included into the elderly group according to the age of patients. For the normal skin tissue and scar tissue in the two groups, hematoxylin eosin (HE) staining was performed to observe the tissue morphology, Masson staining was performed to observe the morphology and arrangement of collagen and quantify the content of collagen, and scanning electron microscope was used to observe the microscopic difference of dermal collagen fibers after the samples were freeze-dried and metal coated. The stiffness of scar tissue in the two groups was measured by atomic force microscope under the liquid phase. The scar tissue in the two groups was collected and the Fbs were isolated and cultured. The morphological differences of the Fbs were observed under the inverted phase contrast microscope, and the protein expression of paxillin was detected with cellular immunofluorescence to reflect the morphology of the Fbs. Cellular immunofluorescence was used to detect the expressions of pro-fibrosis protein α-smooth actin (α-SMA), transforming growth factor-β1 (TGF-β1), and type Ⅰ collagen, mechanotransduction-related protein Yes-associated protein (YAP), and the proliferation-related protein Ki67. Real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of pro-fibrosis genes of TGF-β1, α-SMA, and type Ⅰ collagen, fibrosis inhibiting gene of TGF-β3, and mechanotransduction-related genes of Rho-associated protein 1 (ROCK1) and YAP. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: HE staining showed that the epidermal layer of normal skin was uneven, and blood vessels and sweat glands could be seen in the dermal layer; the epidermal layer of the scar tissue in the two groups was relatively flat, and blood vessels and sweat glands were rare. Masson staining and scanning electron microscopy showed that the collagen fibers in normal skin arranged loosely and disorderly, while the collagen fibers in scar tissue of the two groups arranged densely and orderly, and the collagen fibers in scar tissue of the young group were denser than those of the elderly group. The collagen content in scar tissue of the young group and the elderly group was significantly higher than that of the normal skin tissue (t=8.02, 3.15, P<0.05 or P<0.01), and the collagen content in scar tissue of the elderly group was significantly lower than that of the young group (t=4.84, P<0.05). The dermal stiffness of scar tissue in the elderly group was (50.3±1.1) kPa, significantly higher than (35.2±0.8) kPa in the young group (t=11.43, P<0.05). There were no obvious differences in the morphology of scar Fbs in the two groups observed under inverted phase contrast microscope and by cellular immunofluorescence. The expressions of type Ⅰ collagen and TGF-β1 in scar Fbs cytoplasm of the elderly group were significantly higher than those in the young group, while the expressions of α-SMA in scar Fbs cytoplasm were close in the two groups. The expressions of YAP in cytoplasm and nucleus of scar Fbs in the elderly group were significantly higher than those in the young group, while the expressions of Ki67 in scar Fbs nucleus of the two groups were close. The mRNA expressions of TGF-β1 and type Ⅰ collagen in scar Fbs of the elderly group were significantly higher than those in the young group (t=2.87, 4.85, P<0.05 or P<0.01), the mRNA expression of TGF-β3 in scar Fbs of the elderly group was significantly lower than that in the young group (t=3.36, P<0.05), and the mRNA expressions of α-SMA in scar Fbs of the two groups were close (t=1.14, P>0.05). The mRNA expressions of ROCK1 and YAP in scar Fbs of the elderly group were significantly higher than those in the young group (t=2.98, 7.60, P<0.05 or P<0.01). Conclusions: The elderly are prone to scar healing after skin injury. The molecular mechanism may be attributed to the production of extracellular matrix components with higher stiffness, which increases tissue stiffness and thereby activates the expressions of ROCK and YAP/transcriptional co-activator with PDZ-binding motif genes, promoting pro-fibrosis gene and protein expression.

3D Confinement Regulates Cell Life and Death

AbstractBiophysical properties of the cellular microenvironment, including stiffness and geometry, have been shown to influence cell function. Recent findings have implicated 3D confinement as an important regulator of cell behavior. The understanding of how mechanical signals direct cell function is based primarily on 2D studies. To investigate how the extent of 3D confinement affects cell function, a single cell culture platform is fabricated with geometrically defined and fully enclosed microwells and it is applied to investigate how niche volume and stiffness affect human mesenchymal stem cells (hMSC) life and death. The viability and proliferation of hMSCs in confined 3D microniches are compared with unconfined cells in 2D. Confinement biases hMSC viability and proliferation, and this influence depends on the niche volume and stiffness. The rate of cell death increases and proliferation markedly decreases upon 3D confinement. The observed differences in hMSC behavior are correlated to changes in nuclear morphology and YES‐associated protein (YAP) localization. In smaller 3D microniches, hMSCs display smaller and more rounded nuclei and primarily cytoplasmic YAP localization, indicating reduced mechanical activation upon confinement. Interestingly, these effects scale with the extent of 3D confinement. These results demonstrate that the extent of confinement in 3D can be an important regulator of cell function.

Yes-Associated Protein Is Required for ZO-1-Mediated Tight-Junction Integrity and Cell Migration in E-Cadherin-Restored AGS Gastric Cancer Cells.

Yes-associated protein (YAP) regulates numerous cellular homeostasis processes and malignant transformation. We found that YAP influences ZO-1-mediated cell migration using E-cadherin-restored EC96 cells derived from gastric malignant AGS cells. Ectopic expression of E-cadherin enhanced straightforward migration of cells, in comparison to the meandering movement of parental AGS cells. In EC96 cells, YAP and ZO-1 expression increased but nuclear YAP levels and activity were reduced. Nuclear factor-κB (NF-κB) mediated the increase in ZO-1 expression, possibly stabilizing cytoplasmic YAP post-translationally. Downregulation of YAP expression using siYAP RNA or stable knock-down inhibited straightforward cell migration by fragmenting ZO-1 containing tight junctions (TJs) but not adherens junctions, implying involvement of YAP in ZO-1-mediated cell migration. The association of YAP with ZO-1 was mediated by angiomotin (AMOT) because downregulation of AMOT dissociated YAP from ZO-1 and reduced cell migration. E-cadherin restoration in malignant cancer cells induced NF-κB signaling to enhance ZO-1 expression and subsequently stabilize YAP. At high expression levels, YAP associates with ZO-1 via AMOT at TJs, influencing ZO-1-mediated cell migration and maintaining TJ integrity.

Delayed Senescence of Human Vascular Endothelial Cells by Molecular Mobility of Supramolecular Biointerfaces.

Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway, has been widely implicated in vascular aging and diseases. For preventing vascular endothelial cell senescence, the design and development of biomaterials to regulate YAP activity are required. This study prepares polyrotaxane-coated surfaces with molecular mobility and clarifies the role of the mobility on vascular endothelial cell senescence through Hippo-YAP signaling. The polyrotaxane surface with high mobility induces cytoplasmic YAP localization in endothelial cells, whereas the surface with low mobility induces nuclear YAP localization. After serial cultivation of endothelial cells using polyrotaxane surfaces with different mobilities for 35 d, the endothelial cells aged on the polyrotaxane surface with high mobility exhibit higher proliferative potential, smaller spreading size, and lower activity of senescence-associated β-galactosidase than those aged on the surface with low mobility. These findings suggest that cellular senescence can be delayed by modulating the molecular mobility on polyrotaxane surfaces.

Yes-associated protein regulates the hepatoprotective effect of vitamin D receptor activation through promoting adaptive bile duct remodeling in cholestatic mice.

Mounting clinical evidence has revealed that the vitamin D receptor (VDR) is associated with cholestatic liver injury, although the functions of VDR in this condition remain largely unexplored. Here, we investigated the effects of VDR activation on bile duct ligation (BDL) mice, and the underlying mechanisms were further investigated. A low-calcemic VDR agonist, paricalcitol (PAL, 200 ng/kg), was intraperitoneally injected into BDL mice every other day for 5 days or 28 days. Liver histology, liver function indicators, cholangiocyte proliferation, fibrosis scores, and inflammation were evaluated. Mice treated with PAL were rescued from the decreased survival rate induced by BDL and liver damage was reduced. Mechanistically, PAL promoted cholangiocyte proliferation, which was likely conducive to proliferating bile duct maturation and increased branching of bile ducts. PAL treatment also increased the expression of Yes-associated protein (YAP) and its target protein epithelial cell adhesion molecule (EpCam) and decreased the level of inactive cytoplasmic phosphorylated YAP. YAP knockdown abrogated PAL-induced primary bile duct epithelial cell proliferation, confirmed with YAP inhibitor administration. In addition, BDL-induced liver fibrosis and inflammatory cell infiltration were reduced by PAL treatment at both day 5 and day 28 post-BDL. In conclusion, VDR activation mitigates cholestatic liver injury by promoting adaptive bile duct remodeling through cholangiocytic YAP upregulation. Because PAL is an approved clinical drug, it may be useful for treatment of cholestatic liver disease. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The diverse roles of YAP in the regulation of human nasal epithelial remodeling

Yes-associated protein (YAP) is essential in maintaining tissue size. Aberrant epithelial remodeling is a key pathological alteration in both inflammation and benign tumors in nasal mucosa. We sought to investigate the expression and localization patterns of YAP in remodeled nasal epithelium of basal cell hyperplasia, goblet cell metaplasia and squamous metaplasia. YAP expression patterns were evaluated in tissues obtained from patients with NP (n = 45) and IP (n = 27), and control subjects with septal deviation (n = 17) and tissue-derived primary cell cultures. Compared to the normal epithelium, expressions of YAP were significantly higher in basal cell hyperplasia (NP, 11.4-fold; IP, 19.6-fold), followed by squamous metaplasia (8.2-fold) and mild to moderate goblet cell metaplasia (2.9-fold); while their expression was lower in severe goblet cell metaplasia (3.3-fold). Our resultsshowed that: 1) ectopic nuclear YAP expression associated with p63+ basal cell hyperplasia and the high proliferative potential epithelial cells; 2) increase of cytoplasmic YAP correlated with mild to moderate goblet cell metaplasia; 3) increase of cytoplasmic YAP correlated with squamous cell metaplasia. The in vitro cell model also demonstrated almost concordant changes of YAP with the mucosa findings. Different YAP expression and localization patterns should play critical but differential roles in the nasal epithelial remodeling processes under mucosal inflammation and benign tumor formation.

Improved epithelial cell-cell adhesion using molecular mobility of supramolecular surfaces.

Cells can sense the surrounding microenvironmental properties including contact with biomaterials. Although in vitro cell fates in response to the physical properties of cell-adhesive materials have been widely reported, their influence on cell-cell adhesion is unclear. Here, we investigated the role of molecular mobility on polyrotaxane surfaces in epithelial cell-cell adhesion. Polyrotaxane surfaces with high mobility induced cytoplasmic yes-associated protein (YAP) localization in epithelial cells, whereas those with low mobility induced nuclear YAP localization, suggesting that YAP localization is switched by the mobility of the polyrotaxane surface. The cytoplasmic YAP localization increased the expression of tight junction-associated genes. A scratch assay revealed that although the epithelial cells on the low mobile surface rapidly initiated their migration, the cells on the highly mobile surface delayed their migration. Thus, this finding suggests that polyrotaxane surfaces with higher mobility induce cytoplasmic YAP localization, leading to stronger cell-cell adhesion. The polyrotaxane biointerface is promising as a powerful tool to improve the physical immune system and repair biological tissues.

Increased Cytoplasmic Yes-associated Protein (YAP) Expression in Mismatch Repair Protein-Proficient Colorectal Cancer With High-grade Tumor Budding and Reduced Autophagy Activity.

Yes-associated protein (YAP) is a transcriptional coactivator regulated by autophagy that stimulates colorectal cancer (CRC) progression through activation of epithelial-mesenchymal transition (EMT), represented by tumor budding. The associations between these components in CRC are unknown. Archived surgically resected CRCs with known mismatch repair protein (MMR) status were retrieved (n=81; 2010 to 2016). Electronic medical records were reviewed for clinicopathologic variables including pathologic TNM stage and clinical stage. Tumor budding was graded according to consensus guidelines. Cytoplasmic and nuclear YAP and p62 (autophagy substrate) immunoreactivity were semiquantitatively scored within tumor samples. The Student t test, Fisher exact test, χ2 test, and Spearman correlation coefficient were performed with P<0.05 as a significance level. MMR proficiency (MMR-P) status correlated with high-grade tumor budding. The extent of cytoplasmic YAP staining and pathologic N stage was associated with tumor budding in multivariate analysis. Cytoplasmic YAP expression correlated with higher cytoplasmic p62 expression, suggesting an inverse correlation between autophagy activation and cytoplasmic YAP expression. Nuclear YAP expression correlated with pathologic N stage and clinical stage. A correlation between MMR-P status and tumor budding, combined with correlations between cytoplasmic YAP, tumor budding and p62 raise the possibility of 2 distinct neoplastic pathways concerning autophagy and YAP; one displaying relative activation of YAP and EMT, being commonly observed in MMR-P, and another with less active YAP and EMT, but active autophagy, being commonly seen in MMR-deficient CRC. Nuclear YAP staining could be useful in prognostication.

YAP‑mediated crosstalk between the Wnt and Hippo signaling pathways (Review).

Yes‑associated protein (YAP) acts as a transcriptional co‑activator in gene expression and cell proliferation control by binding to the transcriptional factor TEA domain(TEAD) of the Hippo signaling pathway in the nucleus, and also acts as a regulator by binding to another transcriptional co‑activator, β‑catenin of the Wnt signaling pathway. Whether YAP preferentially acts as a transcriptional co‑regulator of the activity of the Hippo signaling pathway or as a regulator in the Wnt signaling pathway depends on the cell type. Nuclear YAP upregulates the expression of β‑catenin, while cytoplasmic YAP has a negative effect on this expression. The present mini‑review focused on the important roles of YAP and further discussed the cross‑links between the Wnt and Hippo signaling pathways. The Wnt and Hippo signaling pathways are both related to the development of fibrosis or cancer. The current review discussed treatment approaches for these conditions based on the two pathways. YAP, the intersection of these two signaling pathways, has the potential to be developed as a novel treatment target, according to previous basic studies on fibroblasts and cancer cells.

&lt;p&gt;Rab14 Overexpression Promotes Proliferation and Invasion Through YAP Signaling in Non-Small Cell Lung Cancers&lt;/p&gt;

IntroductionSeveral reports have shown that Rab14 is dysregulated in human cancers suggesting that it is an oncogenic protein closely related to tumorigenesis. However, whether Rab14 plays a role in the development and progression of human non-small cell lung cancer (NSCLC) remains unclear.MethodsRab14 protein levels were examined in 115 cases of NSCLC tissues and 6 cancer cell lines. Rab14 knockdown was performed in H1299 and A549 cell lines. Rab14 plasmid transfection was performed in the LK2 cell line. The biological roles and mechanisms of Rab14 were examined using MTT, colony formation, Matrigel invasion assay, migration assay, cell cycle analysis, Western blotting, and RT-qPCR.ResultsWe found that Rab14 was upregulated in 65 of 115 lung cancer tissues. Rab14 high expression was significantly correlated with advanced TNM stage and nodal metastasis. Rab14 protein levels were higher in lung cancer cell lines than in normal bronchial cell line. Functionally, Rab14 overexpression increased growth rate, colony formation, invasion/migration ability and cell cycle progression, while Rab14 siRNA decreased the cell proliferation rate, colony numbers and inhibited invasion/migration ability and cell cycle progression. Rab14 upregulated cyclin D1, cyclin E, connective tissue growth factor (CTGF) and downregulated p27 protein and mRNA levels in both A549 and H1299 cell lines, while Rab14 siRNA produced the opposite effects. Further study showed that Rab14 overexpression increased luciferase reporter activity from transcriptional enhanced associate domain (TEAD) protein. Accordingly, Rab14 increased total Yes-associated protein (YAP) and nuclear YAP protein while decreased phosphorylated (p)-YAP and cytoplasmic YAP protein expression. Cycloheximide treatment showed that Rab14 downregulated the level of YAP degradation. Depletion of YAP using siRNA abolished the influence of Rab14 on cyclin D1, cyclin E, and CTGF. YAP knockdown also partly abolished the effects of Rab14 on cell proliferation and invasion.ConclusionIn summary, our data showed that Rab14 is overexpressed in human NSCLC. Rab14 facilitated proliferation and invasion, possibly through regulation of YAP signaling.

Abstract A17: The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma

Abstract Introduction: The effector protein of the Hippo pathway, Yes-associated protein (YAP), functions as a transcriptional coactivator and has been implicated in the biology of cholangiocarcinoma (CCA). Although the Hippo cascade consists of serine kinases, which act to sequester YAP in the cytosol, we have recently demonstrated the importance of tyrosine phosphorylation in regulating YAP localization and function in CCA. Furthermore, previous evaluation of clinical samples of CCA demonstrated a high frequency of mutations in tyrosine phosphatases. Evaluation of YAP-interacting proteins identified several tyrosine phosphatases, including SHP2. Herein, we describe the ability to modulate YAPY357 phosphorylation status, localization, and activity through manipulation of SHP2 levels and activity. Methods: The cholangiocarcinoma cell lines HuCCT-1 and KMCH were utilized. SHP2 activity was modulated utilizing the inhibitors NSC87877 and SHP099 (Selleckchem). SHP2 levels were modulated utilizing knockout via a doxycycline-inducible CRISPR/Cas9 system. Enforced expression of SHP2 was explored utilizing catalytically active and dead constructs. Tyrosine phosphorylation was assessed via immunoblot. Transcriptional activity was assessed by qRT-PCR for YAP target genes. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay utilizing SHP2 inhibitors and the Src-family kinase inhibitor dasatinib (Selleckchem). Results: The HuCCT-1 and KMCH cell lines were noted to have unique p-YAPY357 profiles (HuCCT-1 [p-YAPY357 high] KMCH [p-YAPY357 low]), and these profiles correlated with SHP2 levels and YAP subcellular localization with HuCCT1 cells demonstrating low levels of SHP2 and prominent nuclear YAP localization, while KMCH cells demonstrated high levels of SHP2 and predominately cytoplasmic YAP. In both cell lines, pharmacologic inhibition of SHP2 was accompanied by an increase in p-YAPY357 levels and YAP cotranscriptional activity (as noted by increased expression of cognate YAP target genes). In KMCH cells SHP2 inhibition shifted YAP from the cytoplasm to the nucleus, and these findings were confirmed by CRISPR/Cas9-mediated deletion of SHP2. In contrast, enforced expression of catalytically active SHP2 in HuCCT1 cells was associated with a decrease in p-YAPY357 levels and a shift of YAP from the nucleus to the cytoplasm. As predicted, based on an increase in YAP activity and nuclear localization, SHP2 inhibition was associated with an increase in cellular proliferation in both cell lines. Incubation of the cell lines with dasatinib was associated with decreased proliferation, which was partially rescued by pretreatment with an SHP2 inhibitor. Conclusions: The tyrosine phosphatase SHP2 can regulate YAPY357 phosphorylation, transcriptional coactivity, and subcellular localization in CCA. Inhibition of SHP2 activity protects cells from the effects of Src kinase inhibition on cellular proliferation and caspase activation. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Kabashima, Gregory Gores, Rory Smoot. The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A17.

Prostate-Derived ETS Factor (PDEF) Modulates Yes Associated Protein 1 (YAP1) in Prostate Cancer Cells: A Potential Cross-Talk between PDEF and Hippo Signaling.

PDEF (prostate-derived ETS factor, also known as SAM-pointed domain containing ETS transcription factor (SPDEF)) is expressed in luminal epithelial cells of the prostate gland and associates with luminal phenotype. The Hippo pathway regulates cell growth/proliferation, cellular homeostasis, and organ development by modulating phosphorylation of its downstream effectors. In previous studies, we observed decreased levels of PDEF during prostate cancer progression. In the present study, we evaluated the effects of the expression of PDEF on total/phosphoprotein levels of YAP1 (a downstream effector of the Hippo pathway). We observed that the PC3 and DU145 cells transfected with PDEF (PDEF-PC3 and PDEF-DU145) showed an increased phospho-YAP1 (Ser127) and total YAP1 levels as compared to the respective PC3 vector control (VC-PC3) and DU145 vector control cells (VC-DU145). We also observed an increased cytoplasmic YAP1 levels in PDEF-PC3 cells as compared to VC-PC3 cells. Moreover, our gene set enrichment analysis (GSEA) of mRNA expression in PDEF-PC3 and VC-PC3 cells revealed that PDEF resulted in inhibition of YAP1 target genes, directly demonstrating that PDEF plays a critical role in modulating YAP1 activity, and by extension in the regulation of the Hippo pathway. We also observed a decrease in YAP1 mRNA levels in prostate cancer tissues as compared to normal prostate tissues. Our analysis of multiple publicly available clinical cohorts revealed a gradual decrease in YAP1 mRNA expression during prostate cancer progression and metastasis. This decrease was similar to the decrease in PDEF levels, which we had reported earlier, and we observed a direct correlation between PDEF and YAP1 expression in CRPC data set. To the best of our knowledge, these results provide the first demonstration of inhibiting YAP1 activity by PDEF in any system and suggest a cross-talk between PDEF and the Hippo signaling pathway.

Nanotopography on titanium promotes osteogenesis via autophagy-mediated signaling between YAP and β-catenin

Nanostructured titanium implants are recognized for inducing osteogenesis, but the cell signal transductions related to topography are not fully understood. Implant topography is associated with the functionality of osteogenic transcription factors directed by β-catenin in the nucleus, and autophagic flux in the cytoplasm; YAP (Yes-associated protein) is implicated in the destruction of β-catenin in the cytoplasm and is susceptible to autophagic flux. This study investigated whether surface topography of the titanium implant modulates autophagy-lysosome degradation of cytoplasmic YAP. Titanium surfaces were modified with smooth, micro, or nanotopographies. Compared with the smooth and micro surfaces, nanotopography was associated with higher β-catenin nuclear translocation, osteogenic differentiation, and autophagy, and less cytoplasmic YAP. Blockade of the autophagy-lysosome pathway resulted in YAP retention in MC3T3-E1 cells. Cytoplasmic YAP restricted β-catenin nuclear translocation. In the nano surface group, β-catenin accumulation in the nucleus and expression of osteogenesis genes was improved. However, in the absence of cell-cell (confluent) contact, manipulation of YAP and β-catenin localization associated with topography-induced autophagy was lost. In summary, the osteogenesis observed in response to titanium implants with nanotopography involves a signaling link between YAP and β-catenin. Statement of SignificanceTitanium with rough topographical surfaces is extensively applied in orthopedic and dental clinics. However, the cellular response to topographies that promotes osteogenesis and underlying mechanisms are not fully understood. In this study, we modified titanium surfaces to produce smooth, micro, or nano topographies. Experiments indicated that the nanotopography induced a stronger autophagic response, leading to degraded cytoplasmic YAP. With the lower levels of YAP, β-catenin transported and accumulated in the nucleus to activate TCF/LEF transcription factors, resulting in stronger osteogenesis. Additionally, cell-cell contact was essential in the autophagy-mediated signaling link between YAP and β-catenin. Consequently, our investigation revealed a novel signal transduction in nanotopography-regulated osteogenesis, and supports the modification of biomaterial surfaces to maximize osseointegration.

Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Although the role of the Hippo signaling pathway in development and tumorigenesis has been extensively studied in multiple organs, its role in ovarian follicle development remains largely unknown. Here, we report that Yes-Associated Protein 1 (YAP1), the major effector of Hippo signaling, is spatiotemporally expressed in ovarian granulosa cells and plays a critical role in the regulation of follicle development. We found that the active form of YAP1 (nuclear YAP1) was predominantly expressed in proliferative granulosa cells, whereas the inactive form of YAP1 (cytoplasmic YAP1) was mainly detected in luteal cells (terminally differentiated granulosa cells). Pharmacological inhibition of YAP1 activity disrupted mouse ovarian follicle development in vitro and in vivo. Foxl2 promoter-driven knockout of Yap1 in ovarian granulosa cells resulted in increased apoptosis of granulosa cells, decreased number of corpora lutea, reduced ovarian size, and subfertility in transgenic mice. However, Cyp19a1 promoter-driven knockout of Yap1 in differentiated granulosa cells of preovulatory follicles and luteal cells of corpora lutea had no effect on ovarian morphology and fertility. Mechanistic studies demonstrated that YAP1 interacted with epidermal growth factor receptor and TGF-β signaling pathways to regulate granulosa cell proliferation, differentiation, and survival. Results from this study identify YAP1 as a critical regulator of granulosa cell proliferation and differentiation. Balanced expression and activation of YAP1 is essential for follicle development and successful reproduction. YAP1 is a promising target for treatment of subfertility associated with abnormal granulosa cell function.-Lv, X., He, C., Huang, C., Wang, H., Hua, G., Wang, Z., Zhou, J., Chen, X., Ma, B., Timm, B. K., Maclin, V., Dong, J., Rueda, B. R., Davis, J. S., Wang, C. Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Intricate role of yes-associated protein1 in human liver cirrhosis: TGF-β1 still is a giant player.

Numerous investigations have been performed on the role of the transforming growth factor-β1 (TGF-β1) pathway in the development of chronic liver diseases (CLDs); however, they failed to explain the underlying mechanism of its pathogenesis, suggesting that other alternative pathways might have been overlooked. The involvement of yes-associated protein1 (YAP1) has been attributed to liver fibrosis; yet, the precise function of this protein has not been fully understood. Therefore, this study aimed to investigate the activity of the YAP1 pathway in human liver cirrhosis (regardless of its causality) and its correlation with the TGF-β1 and sonic hedgehog (SHH) pathways. In this case-control study, the immunohistochemical and quantitative real-time polymerase chain reaction analyses were carried out to determine the activation of the YAP1 pathway in patients with liver cirrhosis (n = 38) and control 1 individuals (n = 10). The western blot analysis and ELISA method were also performed to assess the SHH and TGF-β1 pathways. Although significantly increased expression of cytoplasmic YAP1 was found in patients with liver cirrhosis (P < 0.045), the rate of the nuclear YAP1 expression was similar to that of the control 1 subjects. Moreover, the hepatic expression of amphiregulin (AREG), known as the YAP1 target, along with proteins involved in the TGF-β1 pathway was significantly elevated in all cirrhotic patients, compared with the control subjects. Our results showed that the increased activity of the TGF-β1 pathway is strongly associated with the expression of AREG, denoting a direct and positive relationship between the TGF-β1 and YAP1 pathways. It seems that, unlike the TGF-β1 and SHH pathways, the YAP1 pathway does not play a significant role in the development of liver cirrhosis.

Differential YAP nuclear signaling in healthy and dystrophic skeletal muscle.

Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin (mdx), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.