Yes-associated Protein Activation Research Articles

YAP Activates STAT3 Signalling to Promote Colonic Epithelial Cell Proliferation in DSS-Induced Colitis and Colitis Associated Cancer.

Background and AimsYes-associated protein (YAP) is a key transcriptional coactivator of cell proliferation and differentiation. In this study, we sought to identify the roles of YAP in colonic epithelial regeneration and tumourigenesis.MethodsMurine DSS-induced colitis and YAP overexpression models were constructed via lentiviral intraperitoneal injection. Stable YAP-overexpressing cells, protein immunoprecipitation, and ChIP were used to deeply explore the molecular mechanism.ResultsWe found that the expression of YAP was dramatically diminished in the colonic crypts during the acute colitis phase, while YAP was strikingly enhanced to initiate tissue repair after DSS withdrawal. Overexpressing YAP in mice drastically accelerated epithelial regeneration, presenting with more intact structural integrity and reduced inflammatory cell infiltration in the mucosa. Further mechanistic studies showed that the expression of YAP in the nucleus was significantly increased by 2 h post-DSS removal, accompanied by upregulated protein levels of activated STAT3. Overexpression of YAP (YAPWT) elevated the expression of activated STAT3 and its transcriptional targets and strengthened the proliferation and “wound healing” ability of colonic cells. However, these effects were reversed when STAT3 was silenced in YAPWT cells. Moreover, YAP could directly interact with STAT3 in the nucleus, and c-Myc and CyclinD1 were the transcriptional targets. Finally, during colitis-associated cancer (CAC), YAPWT promoted the progression of CAC, while the phosphomimetic YAP downregulated the expression of STAT3 and inhibited the development and progression of CAC.ConclusionYAP activates STAT3 signalling to facilitate mucosal regeneration after DSS-induced colitis. However, excessive YAP activation in the colonic epithelium promotes CAC development.

Exosomes derived from mesenchymal stem cells attenuate diabetic kidney disease by inhibiting cell apoptosis and epithelial-to-mesenchymal transition via miR-424-5p.

Diabetic kidney disease (DKD) is well-acknowledged as one of the most common complications in diabetes mellitus. Recent studies have demonstrated the promising role of mesenchymal stem cell-derived exosomes (MSC-exos) as a cell-free treatment strategy for DKD. The present study sought to investigate the therapeutic potential and the underlying mechanisms of MSC-exos in DKD. The authentication of MSC-exos was validated by western blot, transmission electron microscope (TEM), and nanosight tracking analysis (NTA). Apoptosis was detected by western blot, TUNEL staining, and flow cytometry. Epithelial-to-mesenchymal transition (EMT) was evaluated by western blot and immunofluorescence. The relationship between miR-424-5p and Yes-associated protein 1 (YAP1) was revealed by dual luciferase reporter assay. We observed that MSC-exos could attenuate DKD by decreasing cell apoptosis and inhibiting epithelial-to-mesenchymal transition (EMT) in diabetic kidneys in db/db mice. Besides, we documented that MSC-exos could reverse high glucose-induced apoptosis and EMT in HK2 cells. Interestingly, miR-424-5p derived from MSC-exos could inhibit YAP1 activation in HK2 cells, resulting in alleviation of high glucose-induced cell apoptosis and EMT. Our study provides novel insights into MSC-exos-mediated protective effect in DKD. MSC-exos could inhibit high glucose-induced apoptosis and EMT through miR-424-5p targeting of YAP1.

Inhibition of YAP/TAZ-TEAD activity induces cytotrophoblast differentiation into syncytiotrophoblast in human trophoblast.

During placentation, placental cytotrophoblast (CT) cells differentiate into syncytiotrophoblast (ST) cells and extravillous trophoblast (EVT) cells. In the placenta, the expression of various genes is regulated by the Hippo pathway through a transcription complex, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-TEA domain transcription factor (TEAD) (YAP/TAZ-TEAD) activity. YAP/TAZ-TEAD activity is controlled by multiple factors and signaling, such as cAMP signaling. cAMP signaling is believed to be involved in the regulation of trophoblast function but is not yet fully understood. Here we showed that YAP/TAZ-TEAD expressions and their activities were altered by cAMP stimulation in BeWo cells, a human choriocarcinoma cell line. The repression of YAP/TAZ-TEAD activity induced the expression of ST-specific genes without cAMP stimulation, and transduction of constitutively active YAP, i.e. YAP-5SA, resulted in the repression of 8Br-cAMP-induced expressions of ST-specific genes in a TEAD-dependent manner. We also investigated the role of YAP/TAZ-TEAD in maintaining CT cells and their differentiation into ST and EVT cells using human trophoblast stem (TS) cells. YAP/TAZ-TEAD activity was involved in maintaining the stemness of TS cells. Induction or repression of YAP/TAZ-TEAD activity resulted in marked changes in the expression of ST-specific genes. Using primary CT cells, which spontaneously differentiate into ST-like cells, the effects of YAP-5SA transduction were investigated, and the expression of ST-specific genes was found to be repressed. These results indicate that the inhibition of YAP/TAZ-TEAD activity, with or without cAMP stimulation, is essential for the differentiation of CT cells into ST cells.

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.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

SHP2 inhibition enhances Yes-associated protein-mediated liver regeneration in murine partial hepatectomy models.

Disrupted liver regeneration following hepatectomy represents an “undruggable” clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.

Abstract P3065: Myeloid YAP Inhibition Improves Cardiac Phenotype During Pressure Overload Stress

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcription co-factor, in modulating cardiac inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7 days PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) were subjected to PO stress. After 4 weeks cardiac hypertrophy was similar between myeloid YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, apoptosis, “fetal gene” induction, and additional indicators of pathological remodeling were attenuated in myeloid YAP KO mice compared to controls. Additionally, inflammatory gene expression and macrophage infiltration in the myocardium were significantly attenuated in myeloid YAP KO mice after PO indicating reduced inflammation compared to controls. Conversely, angiogenic indicators and pro-resolving genes were enhanced in myeloid YAP KO hearts. Experiments using primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression attenuated “M1-like” inflammatory gene expression, while “M2-like” resolving genes were augmented following stimulation. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the injured heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin to test the translational potential of targeting YAP to prevent heart failure. An alternate-day dosing regimen of drug-loaded nanoparticles attenuated inflammation and fibrosis, and improved myocardial capillary density after PO compared to DMSO nanoparticle control treatment. Together these data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and pathology during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Akt1-dependent expression of angiopoietin 1 and 2 in vascular smooth muscle cells leads to vascular stabilization

Retinal angiogenesis was delayed in VSMC-specific Akt1-deficient mice (Akt1∆SMC) but not in Akt2∆SMC mice. The proliferation of ECs, recruitment of pericytes, and coverage of VSMCs to the endothelium were defective in Akt1∆SMC. The silencing of Akt1 in VSMCs led to the downregulation of angiopoietin 1 (Ang1) and the upregulation of Ang2. The activation of Notch3 in VSMCs was significantly reduced in the retinas of Akt1∆SMC mice. Silencing Akt1 suppressed the activation of Notch3. Moreover, the silencing of Notch3 downregulated Ang1, whereas the overexpression of Notch3 intracellular domain (NICD3) enhanced Ang1 expression. The nuclear localization and transcriptional activity of yes-associated protein (YAP) were affected by the expression level of Akt1. Silencing YAP downregulated Ang2 expression, whereas overexpression of YAP showed the opposite results. Ang1 antibody and Ang2 suppressed endothelial sprouting of wild-type aortic tissues, whereas the Ang2 antibody and Ang1 facilitated the endothelial sprouting of aortic tissues from Akt1∆SMC mice. Finally, severe hemorrhage was observed in Akt1∆SMC mice, which was further facilitated under streptozotocin (STZ)-induced diabetic conditions. Therefore, the Akt1-Notch3/YAP-Ang1/2 signaling cascade in VSMCs might play an essential role in the paracrine regulation of endothelial function.

EGF Receptor Signaling Modulates YAP Activation and Promotes Experimental Proliferative Vitreoretinopathy.

PurposeBoth epidermal growth factor receptor (EGFR) and the Yes-associated protein (YAP) signaling pathway are implicated in cell proliferation and differentiation. In this study, we explored whether the formation of proliferative vitreoretinopathy (PVR) depends on the interaction of the EGFR receptor and YAP pathway.MethodsWe studied the effects of EGFR and YAP activation on retinal fibrosis in a PVR mouse model as well as in knockout mice (conditional deletion of EGFR or YAP specifically in RPE cells). Reversal and knockdown experiments were performed to induce a model of ARPE-19 cells treated with TGF-β2 in vitro. The effect of EGFR/YAP signaling blockade on the PVR-induced cell cycle and TGF-β2–induced ARPE-19 cell activation was determined.ResultsThe EGFR inhibitor erlotinib or conditional deletion of EGFR attenuated YAP activation and decreased the expression of YAP and its downstream target Cyr61 and of connective tissue growth factor in vivo and in vitro. EGFR-PI3K-PDK1 signaling induced by PVR promoted YAP activation and cell cycle progression. Furthermore, activated EGFR signaling bypassed RhoA to increase the protein levels of YAP, C-Myc, CyclinD1, and Bcl-xl.ConclusionsOur work highlights that EGFR-PI3K-PDK1–dependent YAP activation plays a crucial role in the formation of PVR. Targeting EGFR and the YAP pathway provides promising therapeutic treatments for PVR.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

KAT7 promoted gastric cancer progression through promoting YAP1 activation

Lysine acetyltransferase 7 (KAT7) was upregulated in gastric cancer (GC) patient tissues, and associated with poor prognosis and metastasis. However, its specific role in GC remains unclear. This study aimed to annotate the role of KAT7 in GC cells. The results showed that the overexpression of KAT7 promoted cell growth, migration, and invasion, while KAT7 inhibition has the opposite effect. Besides, KAT7 participated in cell cycle phase distribution and epithelial-mesenchymal transition (EMT) process of GC cells. In addition, KAT7 promoted the transcription and nuclear translocation of Yes-associated protein 1 (YAP1) in MKN45 cells. Silence of YAP1 partly reversed the promoting effect of KAT7 on GC cells progression. In summary, this study indicates that KAT7 promoted GC cells progression through promoting YAP1 activation, contributes to understand the specific role of KAT7 in GC.

YAP1 controls degeneration of human cartilage chondrocytes in response to mechanical tension

Disc herniation is a kind of disease caused by degenerative discs, which is common in the elderly, bringing substantial financial burden to families and society. Mechanical tension has a vital effect on the maintenance of cartilage function, however, the molecular mechanism by which mechanical tension causes degenerative discs to remain unclear. This study was the first to reveal Yes-associated protein 1(YAP1) is a key regulator in mechanical tension-mediated degenerative discs. Activation of YAP1 may be a valuable strategy to delays the degeneration of human cartilage chondrocytes. We found that YAP1 expression was significantly decreased in degenerative human endplate cartilage and tissue with the strength and time of mechanical stimulation, but the cell cycle distribution was significantly changed under the 10% cyclic mechanical tension(CMT). Besides, the degeneration of endplate cartilage can be delayed by activating the expression level of YAP1 in vitro and it has also been verified in the cartilage endplate tissue in vitro. Furthermore, We found that YAP1 and TEAD1 overexpression increased the activity of the ACAN or COL2A1 promoter to enhance the transcriptional activity of human chondrocyte collagen. The CMT activates the classic Hippo signaling pathway of YAP1, and piezo1 may regulate YAP1 expression through the Hippo signaling pathway. In conclusion, these results suggest the novel mechanism of YAP contributes to delaying the degeneration of endplate cartilage and targeting YAP in combination with Piezo1 is a potential therapeutic approach for the treatment of endplate cartilage degeneration.

The effect of AKT in extracellular matrix stiffness induced osteogenic differentiation of hBMSCs

Extracellular matrix (ECM) stiffness is an important biophysical factor in human bone marrow mesenchymal stem cells (hBMSCs) differentiation. Although there is evidence that Yes-associated protein (YAP) plays an important role in ECM elasticity induced osteogenesis, but the regulatory mechanism and signaling pathways have not been distinctly uncovered. In this study, hBMSCs were cultured on collagen-coated polydimethylsiloxane hydrogels with stiffness corresponding to Young's moduli of 0.5 kPa and 32 kPa, and gene chip analyses revealed the phosphoinositide 3-kinase (PI3K)-AKT pathway was highly correlated with ECM stiffness. Following western blots indicated that AKT phosphorylation was evidently affected in 5th–7th days after ECM stiffness stimulation, while PI3K showed little difference. The AKT activator SC79 and inhibitor MK2206 were utilized to modulate AKT phosphorylation. SC79 and MK2206 caused alteration in the mRNA expression and protein level of alkaline phosphatase (ALP), collagen type I alpha 1 (COL1A1) and runt related transcription factor 2 (RUNX2). On 32 kPa substrates, YAP enrichment in nucleus were significantly promoted by SC79 and remarkably decreased by MK2206. Besides, the ratio of YAP/p-YAP is upregulated by SC79 on both 32 kPa and 0.5 kPa substrates. In conclusion, these findings suggest that AKT is involved in the modulation of ECM stiffness induced osteogenesis, and AKT phosphorylation also influences the subcellular localization and activation of YAP.

Pin1/YAP pathway mediates matrix stiffness-induced epithelial-mesenchymal transition driving cervical cancer metastasis via a non-Hippo mechanism.

Cervical cancer metastasis is an important cause of death in cervical cancer. Previous studies have shown that epithelial-mesenchymal transition (EMT) of tumors promotes its invasive and metastatic capacity. Alterations in the extracellular matrix (ECM) and mechanical signaling are closely associated with cancer cell metastasis. However, it is unclear how matrix stiffness as an independent cue triggers EMT and promotes cervical cancer metastasis. Using collagen-coated polyacrylamide hydrogel models and animal models, we investigated the effect of matrix stiffness on EMT and metastasis in cervical cancer. Our data showed that high matrix stiffness promotes EMT and migration of cervical cancer hela cell lines in vitro and in vivo. Notably, we found that matrix stiffness regulates yes-associated protein (YAP) activity via PPIase non-mitotic a-interaction 1 (Pin1) with a non-Hippo mechanism. These data indicate that matrix stiffness of the tumor microenvironment positively regulates EMT in cervical cancer through the Pin1/YAP pathway, and this study deepens our understanding of cervical cancer biomechanics and may provide new ideas for the treatment of cervical cancer.

C12orf59 Promotes Esophageal Squamous Cell Carcinoma Progression via YAP-Mediated Epithelial-Mesenchymal Transition.

C12orf59 is a novel gene widely expressed in diverse normal human tissues. Aberrant expression of C12orf59, which is involved in tumor progression, has been reported in a few types of cancer. However, its expression and biological function in esophageal squamous cell carcinoma (ESCC) remain largely unclear. Here, we found that the mRNA and protein levels of C12orf59 were prominently higher in both tumor tissues and most ESCC cell lines. Functionally, C12orf59 overexpression promoted ESCC cell proliferation, migration and invasion, whereas C12orf59 depletion worked oppositely. Mechanistically, C12orf59 exerted its oncogenic function through the induction of epithelial-mesenchymal transition (EMT) of ESCC cells, which relied on Yes-associated protein (YAP) dephosphorylation and nuclear translocation. Constitutively active YAP further facilitated cell migration, invasion and EMT induced by enforced C12orf59 overexpression. On the contrary, increased cell motility and EMT caused by enforced C12orf59 overexpression were dramatically repressed upon YAP inactivation by verteporfin. Thus, we conclude that YAP activation driven by C12orf59 contributes to the malignancy of ESCC through EMT and that targeting drugs for C12orf59 combined with YAP inhibitor may be a potential therapeutic strategy for ESCC.

Kank1 Is Essential for Myogenic Differentiation by Regulating Actin Remodeling and Cell Proliferation in C2C12 Progenitor Cells.

Actin cytoskeleton dynamics are essential regulatory processes in muscle development, growth, and regeneration due to their modulation of mechanotransduction, cell proliferation, differentiation, and morphological changes. Although the KN motif and ankyrin repeat domain-containing protein 1 (Kank1) plays a significant role in cell adhesion dynamics, actin polymerization, and cell proliferation in various cells, the functional significance of Kank1 during the myogenic differentiation of progenitor cells has not been explored. Here, we report that Kank1 acts as a critical regulator of the proliferation and differentiation of muscle progenitor cells. Kank1 was found to be expressed at a relatively high level in C2C12 myoblasts, and its expression was modulated during the differentiation. Depletion of Kank1 by siRNA (siKank1) increased the accumulation of filamentous actin (F-actin). Furthermore, it facilitated the nuclear localization of Yes-associated protein 1 (YAP1) by diminishing YAP1 phosphorylation in the cytoplasm, which activated the transcriptions of YAP1 target genes and promoted proliferation and cell cycle progression in myoblasts. Notably, depletion of Kank1 suppressed the protein expression of myogenic regulatory factors (i.e., MyoD and MyoG) and dramatically inhibited myoblast differentiation and myotube formation. Our results show that Kank1 is an essential regulator of actin dynamics, YAP1 activation, and cell proliferation and that its depletion impairs the myogenic differentiation of progenitor cells by promoting myoblast proliferation triggered by the F-actin-induced nuclear translocation of YAP1.

FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1.

We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities. Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms. The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK. We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.

Functional characteristics of YAP-positive hepatocytes expression in an early stage of NASH with transcriptome sequence analysis

Objective: To analyze and compare the differentially expressed genes (DEGs) of Yes-associated protein (YAP)-positive and negative hepatocytes and further understand the preliminary functional characteristics of YAP-positive hepatocytes in an early mouse model of nonalcoholic steatohepatitis (NASH) with transcriptome sequence (RNA-Seq). Methods: C57BL/6 mice were fed with methionine-choline deficiency (MCD) diet for 2 weeks to establish an early NASH model, and the control group was fed with normal diet. Liver tissue was stained with hematoxylin-eosin (HE) and Sirius red, and the pathological score was recorded. The expression of YAP and P-YAP were determined by immunohistochemistry (IHC) in liver tissues. Primary hepatocytes with viability greater than 90% were isolated and purified by collagenase perfusion combined with Percoll density gradient centrifugation. YAP-positive and negative hepatocytes were assessed by YAP antibody, flow cytometry and RNA-Seq analyses. Sequencing results were screened by GO, KEGG and interaction network analysis methods. RT-PCR was used to verify the expression levels of YAP and some DEGs in liver tissue model group. Two samples mean was compared by independent samples t-test. Results: Compared with the control group, the HE-stained liver tissue of MCD-induced mice at 2 weeks showed steatosis (pathological score 1.07±0.21), accompanied by lobular inflammation (pathological score 1.13±0.32) and ballooned hepatocyte (pathological score 0.80) ±0.20). Sirius red staining showed non-significant liver fibrosis (pathological score 0.40±0.40). IHC showed partial YAP-positive hepatocytes expression in an early stage of NASH. RNA-Seq analysis showed that clean reads of YAP-positive and negative hepatocytes were 49 310 604 and 5 4820 036, respectively. Compared with YAP-negative hepatocytes, YAP-positive hepatocytes had differential expression of 5 565 genes, including 1 662 up-regulated genes and 3 903 down-regulated genes. GO analysis of up-regulated genes showed that the metabolic processes related to mitochondrial functions, such as purine nucleoside triphosphate and nucleoside triphosphate were significantly enriched in biological processes (BP), while down-regulated gene analysis showed that olfactory-related receptor were significantly enriched in BP. KEGG analysis showed that DEGs were enriched in 292 pathways, and oxidative phosphorylation (OXPHOS) pathway was significantly enriched in signaling pathway. RT-PCR validated that inflammatory factors (interleukin-1β, interleukin-6), YAP and its target genes (Cyr61, Ankrd1), and Cox5b and Sdhc genes were significantly up-regulated in the OXPHOS pathway, which was consistent with the sequencing results. In addition, eight key genes with interaction network analysis were predicted. Conclusion: Changes in hepatocyte metabolic levels may be associated with increased YAP activity in an early stage of NASH.

Senescent hepatic stellate cells promote liver regeneration through IL-6 and ligands of CXCR2.

Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts, such as wound healing. We have found that hepatic stellate cells (HSCs) underwent senescence within 2 days after 2/3 partial hepatectomy (PHx) in young (2–3 months old) mice, and the elimination of these senescent cells by using the senolytic drug ABT263 or by using a genetic mouse model impaired liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype, which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces Yes-associated protein (YAP) activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restored liver regeneration in mice with senescent cell elimination, and the combination of both fully restored liver weight recovery. Furthermore, the matricellular protein central communication network factor 1 (CCN1, previously called CYR61) was rapidly elevated in response to PHx and induced HSC senescence. Knockin mice expressing a mutant CCN1 unable to bind integrin α6β1 were deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.

SUFU suppresses ferroptosis sensitivity in breast cancer cells via Hippo/YAP pathway

SummaryFerroptosis is a new kind of regulated cell death that is characterized by highly iron-dependent lipid peroxidation. Cancer cells differ in their sensitivity to ferroptosis. Here we showed that the Suppressor of fused homolog (SUFU), a critical component in Hedgehog signaling, regulates ferroptosis sensitivity of breast cancer cells. Ectopic SUFU expression suppressed, whereas depletion of SUFU enhanced the sensitivity of breast cancer cells to RSL3-triggered ferroptosis through deregulation of ACSL4. Moreover, SUFU depletion promoted the activation of Yes-associated protein (YAP), thereby increasing the expression of ACSL4. Mechanistically, SUFU is associated with LATS1. Deletion of a region comprising residues 174–385 in SUFU disrupted SUFU binding to LATS1, thus abrogating SUFU-mediated downregulation of the YAP-ACSL4 axis and sensitivity to ferroptosis. Noteworthy, we showed that vincristine downregulated SUFU, thus increasing breast cancer cell sensitivity to RSL3 in vitro and in vivo. Together, our findings uncover SUFU as a novel regulator in ferroptosis sensitivity.