Tong-Cun Zhang

Vascular endothelial growth factor stimulates endothelial differentiation from mesenchymal stem cells via Rho/myocardin-related transcription factor--a signaling pathway.

Mesenchymal stem cells (MSCs) are pluripotent progenitors that can differentiate into a variety of cell types. Vascular endothelial growth factor (VEGF) is one of the major factors of initiating and regulating angiogenesis. It has been reported that VEGF can induce MSCs differentiated into endothelial cells (ECs). However, the mechanism that VEGF-induced MSC differentiation is not completely understood. Here, we showed that VEGF induced human and rat bone marrow-derived MSCs differentiation to ECs. Rho family plays an important role in VEGF-induced endothelial cell migration and angiogenesis. Our results indicated that in MSCs, VEGF activated Rho/ROCK signaling pathway and promoted nuclear translocation of myocardin-related transcription factor-A (MRTF-A), which is controlled by Rho/ROCK signaling. In addition, Rho inhibitor C3 transferase, ROCK inhibitor Y27632 or depletion of endogenous MRTF-A abolished the VEGF-induced differentiation of MSCs into ECs. Furthermore, VEGF also enhanced the expression levels of CYR61/CCN1, as a regulator of vascular development and angiogenesis, and knockdown of endogenous MRTF-A reduced VEGF-induced the upregulation of CYR61/CCN1. Report assays with site-direct mutation analysis of CYR61/CCN1 promoter demonstrated that MRTF-A transactivated CYR61/CCN1 promoter mainly depending on CArG box. In this study, we identify the Rho/MRTF-A signaling pathway as a main actor in controlling VEGF-induced differentiation of human and rat bone marrow-derived MSCs into endothelial cells.

Histone methyltransferase SMYD3 promotes MRTF-A-mediated transactivation of MYL9 and migration of MCF-7 breast cancer cells.

Myocardin-related transcription factor-A (MRTF-A) is a Rho signal-responsive transcriptional coactivator of serum response factor (SRF). Recent studies indicated that MRTF-A might be an important regulator of mammary gland and be involved in cancer metastasis. However, the roles of histone modification in the MRTF-A-dependent signal pathway and tumor migration are still not very clear. Here, we report that histone methylation is required for the MRTF-A-mediated upregulation of myosin regulatory light chain 9 (MYL9), an important cytoskeletal component which is implicated in cell migration. Furthermore, we demonstrate that SET and MYND domain containing protein 3 (SMYD3), a hitone methyltransferase (HMT) associated with carcinogenesis, might be the one which is responsible for the histone methylation occurred in the MRTF-A-mediated- transactivation of MYL9 and migration of breast cancer cells. Overexpression of SMYD3 promotes MRTF-A-mediated upregulation of MYL9 and migration of MCF-7 breast cancer cells, while contrary results were observed when the endogenous MRTF-A and SMYD3 were suppressed with specific siRNAs. In addition, the mutation analysis suggested that this cooperative transactivation is mainly mediated via the proximal binding element of MRTF-A in the promoter of MYL9, and the HMT activity of SMYD3 is required as well. Our findings reveal a new mechanism by which MRTF-A and SMYD3 functions in transcriptional regulation and cell migration, and provide a better understanding for metastasis of breast cancer.

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.

High-Level Expression of the Antimicrobial Peptide Plectasin in Escherichia coli

Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin could kill the sensitive strain as efficaciously as vancomycin and penicillin and without cytotoxic effects on mammalian cell viability. In order to establish a bacterium-based plectasin production system, in the present study, the coding sequence of plectasin was optimized, and then cloned into pET32a (+) vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lysate was separated by Ni2+-chelating affinity chromatography. The purified protein was then cleaved by Factor Xa protease to release mature plectasin. Final purification was achieved by Ni2+-chelating chromatography again. The recombinant plectasin exhibited the same antimicrobial activity as reported previously. This is the first study to describe the expression of plectasin in E. coli expression system, and these works might provide a significant foundation for the following production or study of plectasin, and contribute to the development and evolution of novel antimicrobial drugs in clinical applications.

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.

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.

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.

Myocardin-related transcription factor-A is a key regulator in retinoic acid-induced neural-like differentiation of adult bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can differentiate into neuron cells under appropriate environment in vitro and in vivo. Retinoic acid (RA), a vitamin A derivative, is known to facilitate the neuronal differentiation of MSCs. However, the mechanism by which RA induced MSC differentiation into neuron-like cells is not completely understood. Here, we show that RA can induce neural-like differentiation of bone marrow-derived MSCs, as evidenced by the increase of neuron-specific marker expression and the gradually decreased resting membrane potential. Of note, myocardin-related transcription factor-A (MRTF-A), a major co-activator of serum response factor (SRF), was significantly activated and its nuclear localization was observed during RA-induced neural-like differentiation. MRTF-A is recently reported to function in the development of the nervous system. Our results demonstrated that dominant-negative form of MRTF-A (DN-MRTF-A) or shRNA-MRTF-A strongly inhibited upregulation of neural markers in response to RA. Furthermore, reporter assays with NF-H promoter indicated that RA and MRTF-A can synergistically activate NF-H transcription and enhance the mRNA expression of NF-H. These findings reveal that MRTF-A is a key regulator in all-trans RA-induced neural-like differentiation of bone marrow-derived MSCs.