Hongpeng He

Knockdown of DNMT1 and DNMT3a Promotes the Angiogenesis of Human Mesenchymal Stem Cells Leading to Arterial Specific Differentiation

Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5‐aza‐dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube‐like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss‐of‐function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273–1283

Histone acetyltransferase p300 promotes MRTF-A-mediates transactivation of VE-cadherin gene in human umbilical vein endothelial cells

Vascular endothelial cadherin (VE-cadherin) is the major determinant of endothelial cell contact integrity and is required in vascular development and angiogenesis. Serum response factor (SRF) plays essential roles in postnatal retinal angiogenesis and adult neovascularization. It is unclear whether transcription of VE-cadherin is mediated by a SRF co-activator, myocardin-related transcription factor-A (MRTF-A). Here we have demonstrated that MRTF-A is a key regulatory factor to activate the transcription of VE-cadherin in human umbilical vein endothelial cells (HUVECs). siRNA-mediated knockdown of MRTF-A decreased the level of VE-cadherin in HUVECs. Vascular endothelial growth factor (VEGF) induced MRTF-A binding to the SRF-binding site (CArG box) within VE-cadherin promoter. Histone acetyltransferase p300 and MRTF-A could synergistically augment the expression of VE-cadherin by enhancing acetylation of histone3K9 (H3K9Ac), histone3K14 (H3K14Ac) and histone4 at the SRF-binding site within VE-cadherin promoter. Taken together, these data identified a detailed regulatory mechanism of VE-cadherin gene expression.

Transcriptional factors p300 and MRTF-A synergistically enhance the expression of migration-related genes in MCF-7 breast cancer cells

The transcriptional coactivator p300 is highly expressed in breast cancer tissues. MRTF-A is a transcription factor governed by the Rho-GTPase-actin signaling pathway. The purpose of this study was to explore the role of p300 in breast cancer metastasis. Here we showed that the motility of breast cancer cells was enhanced by the overexpression of p300, meanwhile, the transcription of migration-related genes was upregulated. Depletion of p300 downregulated the migration-related genes and slowed down the migration of breast cancer cells. p300 worked synergistically with MRTF-A to activate the transcription of MYH9, MYL9 and CYR61. As identified by co-IP, p300 interacted with the C-terminal TAD domain of MRTF-A. And together with MRTF-A, p300 was associated with the target gene promoters. Furthermore, MRTF-A was found to be acetylated in MCF-7 breast cancer cells. These results demonstrated the involvement of p300 in the MRTF-A mediated gene regulation and breast cancer cell migration.

Rho/MRTF-A-Induced Integrin Expression Regulates Angiogenesis in Differentiated Multipotent Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are known to undergo endothelial differentiation in response to treatment with vascular endothelial growth factor (VEGF), but their angiogenic ability is poorly characterized. In the present study, we aimed to further investigate the role of Rho/MRTF-A in angiogenesis by MSCs and the effect of the Rho/MRTF-A pathway on the expression of integrins α1β1 and α5β1, which are known to mediate physiological and pathological angiogenesis. Our results showed that increased expression of α1, α5, and β1 was observed during angiogenesis of differentiated MSCs, and the Rho/MRTF-A signaling pathway was demonstrated to be involved in regulating the expression of integrins α1, α5, and β1. Luciferase reporter assay and ChIP assay determined that MRTF-A could bind to and transactivate the integrin α1 and α5 promoters. Treatment with the Rho inhibitor C3 transferase, the Rho-associated protein kinase (ROCK) inhibitor Y27632 or with shMRTF-A inhibited both the upregulation of α1, α5, and β1 as well as angiogenesis. Furthermore, in human umbilical vein endothelial cells (HUVECs), MRTF-A deletion led to marked reductions in cell migration and vessel network formation compared with the control. These data demonstrate that Rho/MRTF-A signaling is an important mediator that controls integrin gene expression during MSC-mediated angiogenic processes.

Transcription of HOTAIR is regulated by RhoC-MRTF-A-SRF signaling pathway in human breast cancer cells

HOTAIR is a long non-coding RNA highly expressed in cancer tissues and is a negative prognostic factor, whereas the mechanism by which HOTAIR expression is upregulated in cancers remains elusive. In the present study, the regulation of HOTAIR transcription was investigated in breast cancer cells MCF7 and T47D. We found that, when the RhoC-ROCK signaling was disturbed by specific siRNAs or chemical inhibitors, the expression of HOTAIR would be down-regulated. Further, MRTF-A and SRF were found to affect HOTAIR expression. HOTAIR promoter activity was demonstrated to be regulated by the RhoC-MRTF-A-SRF signaling in a CArG-box-dependent manner. Moreover, MRTF-A was identified to physically interact with HOTAIR promoter, and RNA polymerase II association on HOTAIR promoter was enhanced by MRTF-A overexpression. Taken together, our results suggest that HOTAIR is regulated by the RhoC-MRTF-A-SRF signaling pathway in breast cancer cells.

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.

Generation of insulin-producing cells from C3H10T1/2 mesenchymal progenitor cells.

Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate β cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes.

NFATc4 and myocardin synergistically up-regulate the expression of LTCC α1C in ET-1-induced cardiomyocyte hypertrophy.

AIMS Dysregulation of Ca(2+) is a central cause of cardiac hypertrophy. The α1C subunit of L-type Ca(2+) channel (LTCC) is a pore-forming protein which is responsible for the voltage-dependent channel gating and channel selectivity for Ca(2+). Myocardin and nuclear factor of activated T-cells c4 (NFATc4) are two key transcription factors in cardiac hypertrophy. We aimed to investigate the underlying mechanism of the transcriptional regulation of LTCC α1C by myocardin and NFATc4 in hypertrophic cardiomyocytes. MAIN METHODS Endothelin-1 (ET-1) was used to induce cardiomyocyte hypertrophy. Cyclosporin A (CSA) was used to block the activation of calcineurin/NFATc4 pathway in ET-1-treated cardiomyocytes and the expression of LTCC α1C were examined. Overexpression or RNAi interfering experiments were performed to investigate the effects of NFATc4 or myocardin on the transcriptional regulation of LTCC α1C. Interactions between NFATc4 and myocardin or the association of NFATc4 with myocardin promoter were assessed via Co-IP or ChIP assays respectively. KEY FINDINGS In the present study, we found that ET-1 stimulated LTCC α1C transcription in neonatal rat cardiomyocytes partially via the activation of calcineurin/NFATc4 pathway. Overexpression of NFATc4 or myocardin promoted LTCC α1C expression in cardiomyocytes. Ca(2+) channel blocker verapamil or knockdown of α1C inhibited myocardin-induced cardiomyocyte hypertrophy. Further studies showed that NFATc4 interacted with myocardin to synergistically activate the expression of LTCC α1C, moreover, NFATc4 activated myocardin expression by binding to its promoter. SIGNIFICANCE Our results suggest a novel mechanism of the transcriptional regulation of LTCC α1C by synergistic activities of NFATc4 and myocardin in ET-1-induced cardiomyocyte hypertrophy.

SAHA inhibits the transcription initiation of HPV18 E6/E7 genes in HeLa cervical cancer cells

High risk human papillomavirus (HPV) is a well recognized causative agent of cervical cancer. Suberoylanilide hydroxamic acid (SAHA) is a potential anti-cervical cancer drug; however, its effect on the expression of HPV E6 and E7 genes remains unclear. Here, we show that, in SAHA treated HeLa cells, HPV18 E6 and E7 mRNA and protein levels were reduced, HPV18 promoter activity was decreased, and the association of RNP II with HPV18 promoter was diminished, suggesting that SAHA inhibited the transcription initiation of HPV18 E6 and E7 genes. In SAHA-treated HeLa, although the level of lysine 9-acetylated histone H3 in the whole cell extracts increased obviously, its enrichment on HPV18 promoter was significantly reduced which is correlated with the down-regulation of HPV E6 and E7.

Histone acetyltransferase p300 promotes MKL1-mediated transactivation of catechol-O-methyltransferase gene

Previous studies have revealed that histone acetyltransferase p300 is recruited to the promoters of certain cardiac and smooth muscle specific genes to enhance the transactivation activity of myocardin, which is a master regulator in cardiovascular differentiation and development. Here, we found that the gene encoding catechol-O-methyltransferase (COMT), an important metabolic enzyme catalyzing the conversion of estrogen, is also a target gene of myocardin-related transcription factors (MRTFs). Megakaryoblastic leukemia 1 (MKL1, also named MRTF-A) and p300 could synergistically augment the expression of COMT gene, increase the metabolic rate of estrogen, and thus reduce the proliferation of MCF-7 breast cancer cells stimulated by estrogen.