Xing-Hua Liao

STAT3 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin

Background: The Hippo-Yap signaling pathway is one of the critical pathways regulating cell proliferation, differentiation, and apoptosis. Results: Knockdown of endogenous Yap1 impairs VSMC proliferation and enhances VSMC contractile phenotype by promoting the association of the myocardin-SRF-CArG complex. Conclusion: The Yap1 signaling pathway is a central regulator of the phenotypic switch of VSMCs. Significance: The phenotypic switch of VSMCs and vessel injury response can be controlled by modulation of Hippo-Yap signaling. The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.

MRTF-A and STAT3 synergistically promote breast cancer cell migration

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism on metastasis is still not fully understood; we now report that both MRTF-A and STAT3 play important role in breast cancer migration of MDA-MB-231 cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers Myl-9 and Cyr-61. Importantly, we identified a detailed molecular mechanism of MDA-MB-231 cell migration controlled via physical interaction between MRTF-A and STAT3, which synergistically promote the transactivity of the migration marker Myl-9 and Cyr-61 by CArG box binding. Interestingly, the two signaling pathways RhoA-MRTF-A and JAK-STAT3 across talk to regulate MDA-MB-231 cell migration. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.

STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells.

Skin cancer is a major cause of morbidity and mortality worldwide. Mounting evidence shows that exposure of the skin to solar UV radiation results in inflammation, oxidative stress, DNA damage, dysregulation of cellular signaling pathways and immunosuppression thereby resulting in skin cancer. Signal transducer and activator of transcription 3 (STAT3) is well known to function as an anti-apoptotic factor, especially in numerous malignancies, but the relationship between STAT3 activation and DNA damage response in skin cancer is still not fully understood. We now report that STAT3 inhibited DNA damage induced by UV and STAT3 mediated upregulation of GADD45γ and MDC-1 and the phosphorylation of H2AX in UV induced DNA damage. Notably, STAT3 can increase the expression of ATR in A431 cells. Luciferase assay shows that STAT3 activates the transcription of ATR promoter. More importantly, microRNA-383 suppressed ATR expression by targeting 3 (untranslated regions)UTR of ATR in A431 cells, and STAT3 down-regulates the transcription of miR-383 promoter. Thus, these results reveal the new insight that ATR is down-regulated by STAT3-regulated microRNA-383 in A431 cells. Moreover, overexpression of STAT3 enhanced expression of antiapoptosis genes BCL-1 and MCL-1, and depletion of STAT3 sensitized A431 cells to apoptotic cell death following UV. Collectively, these studies suggest that STAT3 may be a potential target for both the prevention and treatment of human skin cancer.

Re-expression and epigenetic modification of maspin induced apoptosis in MCF-7 cells mediated by myocardin

Breast cancer is the leading cause of cancer death in women worldwide. It is well known that oncogene activation and anti-oncogene inactivation affect the development and progression of breast cancer, but the role of oncogene activation and anti-oncogene inactivation in breast cancer is still not fully understood. We now report that maspin acts as a tumor suppressor gene to induce MCF-7 cell apoptosis. In addition, maspin promoter hypermethylation and histone hypoacetylation lead to silencing of maspin gene expression in MCF-7 cells. Moreover, DNA methyltransferase (DNMT) inhibitor 5-aza-2-deoxycytidine (5-aza-dc) and/or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) strongly up-regulated the expression of maspin in MCF-7 cells. Notably, myocardin can promote the re-expression of maspin in MCF-7 cells. Luciferase assay shows that myocardin activates the transcription of maspin promoter by CArG box. More importantly, 5-aza-dc/TSA and myocardin synergetically enhance re-expression of maspin and augment maspin-mediated apoptosis in MCF-7 cells. Thus, these data reveal the new insight that myocardin meditates apoptosis in breast cancer through affecting maspin re-expression and epigenetic modification to regulate the development of breast cancer, thereby raising the possibility of its use in breast cancer therapy.

STAT3 is required for MiR-17-5p-mediated sensitization to chemotherapy-induced apoptosis in breast cancer cells

Signal transducer and activator of transcription 3 (STAT3) controls cell survival, growth, migration, and invasion. Here, we observed that STAT3 exerted anti-apoptotic effects in breast cancer cells. On the other hand, miR-17-5p induced apoptosis in breast cancer cells, and overexpression of miR-17-5p sensitized MCF-7 cells to paclitaxel-induced apoptosis via STAT3. Overexpression of STAT3 in MCF-7 cells decreased paclitaxel-induced apoptosis, but STAT3 knockout abolished the miR-17-5p-induced increases in apoptosis. Finally, miR-17-5p promoted apoptosis by increasing p53 expression, which was inhibited by STAT3. These results demonstrate a novel pathway via which miR-17-5p inhibits STAT3 and increases p53 expression to promote apoptosis in breast cancer cells.

MiR-93-5p inhibits the EMT of breast cancer cells via targeting MKL-1 and STAT3

ABSTRACT Epithelial‐mesenchymal transition (EMT) plays an important role in breast cancer cell metastasis. Both (megakaryoblastic leukemia)/myocardin‐like 1 (MKL‐1) and Signal transducer and activator of transcription 3 (STAT3) have been implicated in the control of cellular metabolism, survival and growth. Our previous study has shown that cooperativity of MKL‐1 and STAT3 promoted breast cancer cell migration. Herein, we demonstrate a requirement for MKL‐1 and STAT3 in miRNA‐mediated cellular EMT to affect breast cancer cell migration. Here we show that cooperativity of MKL‐1 and STAT3 promoted the EMT of MCF‐7 cells. Importantly, MKL‐1 and STAT3 promoted the expression of Vimentin via its promoter CArG box. Interestingly, miR‐93‐5p inhibits the EMT of breast cancer cells through suppressing the expression of MKL‐1 and STAT3 via targeted their 3′UTR. These results demonstrated a novel pathway through which miR‐93‐5p regulates MKL‐1 and STAT3 to affect EMT controlling breast cancer cell migration. HighlightsCooperativity of MKL‐1 and STAT3 promoted the EMT of MCF‐7 cells.Cooperativity of MKL‐1 and STAT3 promoted the expression of Vimentin via its promoter CArG box.MiR‐93‐5p inhibits the EMT of MCF‐7 cells through suppressing MKL‐1 and STAT3 via targeted their 3′UTR.

NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms

Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1.

MRTF-A and STAT3 promote MDA-MB-231 cell migration via hypermethylating BRSM1

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism of metastasis is still not fully understood. We now report that both MRTF‐A and STAT3 play important roles in migration of MDA‐MB‐231 breast cancer cells. Moreover, MRTF‐A and STAT3 synergistically increased MDA‐MB‐231 cell migration by promoting the expression of migration markers urokinase‐type plasminogen activator (uPA) and osteopontin (OPN) and inhibiting the expression of breast cancer metastasis suppressor 1 (BRMS1). Luciferase reporter assays demonstrated that MRTF‐A and STAT3 do not affect transcription of the BRMS1 promoter. Instead, we identified a newly molecular mechanism by which MRTF‐A and STAT3 synergistically controlled MDA‐MB‐231 cell migration by recruiting DNMT1 to hypermethylate the promoter of BRMS1 and thus affect the expression of BRMS1. Interestingly, physical interaction between MRTF‐A and STAT3 synergistically promotes the transactivity of DNMT1 by binding to the GAS element within the DNMT1 promoter. Our data thus provide important and novel insights into the roles of MRTF‐A and STAT3 in regulating MDA‐MB‐231 cell migration.

Ca2 + signal-induced cardiomyocyte hypertrophy through activation of myocardin

Hypertrophic growth of cardiomyocytes in response to pressure overload is an important stage during the development of many cardiac diseases. Ca(2+) overload as well as subsequent activation of Ca(2+) signaling pathways has been reported to induce cardiac hypertrophy. Myocardin, a transcription cofactor of serum response factor (SRF), is a key transducer of hypertrophic signals. However, the direct role of myocardin in Ca(2+) signal-induced cardiomyocyte hypertrophy has not been explained clearly. In the present study, we discovered that embryonic rat heart-derived H9c2 cells responded to the stimulation of calcium ionophore A23187 with a cell surface area enlargement and an increased expression of cardiac hypertrophy marker genes. Increased Ca(2+) also induces an organization of sarcomeres in neonatal rat cardiomyocytes, as revealed by α-actinin staining. Increased Ca(2+) could upregulate the expression of myocardin. Knockdown of myocardin by shRNA attenuates hypertrophic responses triggered by increased intracellular Ca(2+), suggesting that Ca(2+) signals induce cardiomyocyte hypertrophy partly through activation of myocardin. Furthermore, A23187 treatment directly activates myocardin promoter, chelation of Ca(2+) by EGTA inhibits this activation and knockdown of myocardin expression using shRNA also abrogates A23187-induced ANF and SK-α-actin promoter activity. CSA (calcineurin inhibitor) and KN93 (CaMKII inhibitor) inhibit A23187-induced the increase in myocardin expression. These results suggest that myocardin plays a critical role in Ca(2+) signal-induced cardiomyocyte hypertrophy, which may serve as a novel mechanism that is important for cardiac hypertrophy.

Myocardin inhibited the gap protein connexin 43 via promoted miR-206 to regulate vascular smooth muscle cell phenotypic switch

Myocardin is regarded as a key mediator for the change of smooth muscle phenotype. The gap junction protein connexin 43 (Cx43) has been shown to be involved in vascular smooth muscle cells (VSMCs) proliferation and the development of atherosclerosis. However, the role of myocardin on gap junction of cell communication and the relation between myocardin and Cx43 in VSMC phenotypic switch has not been investigated. The goal of the present study is to investigate the molecular mechanism by which myocardin affects Cx43-regulated VSMC proliferation. Data presented in this study demonstrated that inhibition of the Cx43 activation process impaired VSMC proliferation. On the other hand, overexpression miR-206 inhibited VSMC proliferation. In additon, miR-206 silences the expression of Cx43 via targeting Cx43 3 Untranslated Regions. Importantly, myocardin can significantly promote the expression of miR-206. Cx43 regulates VSMCs proliferation and metastasis through miR-206, which could be promoted by myocardin and used as a marker for diagnosis and a target for therapeutic intervention. Thus myocardin affected the gap junction by inhibited Cx43 and myocardin-miR-206-Cx43 formed a loop to regulate VSMC phenotypic switch.