Endothelial-mesenchymal transition (EndMT) plays important roles in various physiological and pathological processes. While signals mediated by transforming growth factor (TGF)-β have been implicated in EndMT, the molecular mechanisms underlying it remain to be fully elucidated. Here, we examined the effects of TGF-β signals on the EndMT of mouse pancreatic microvascular endothelial cells (MS-1). By addition of TGF-β2, MS-1 cells underwent mesenchymal transition characterized by re-organization of actin stress fibre and increased expression of various mesenchymal markers such as α-smooth muscle actin (α-SMA) through activation of Rho signals. Whereas activation of Rho signals via TGF-β-induced non-Smad signals has been implicated in epithelial-mesenchymal transition (EMT), we found that Arhgef5, a guanine nucleotide exchange factor, is induced by Smad signals and contributes to the TGF-β2-induced α-SMA expression in MS-1 cells. We also found that TGF-β2 induces the expression of myocardin-related transcription factor-A (MRTF-A) in a Smad-dependent fashion and its nuclear accumulation in MS-1 cells and that MRTF-A is required and sufficient for TGF-β2-induced α-SMA expression. These results indicate that activation of Smad signals by TGF-β2 have dual effects on the activation of Rho signals and MRTF-A leading to the mesenchymal transition of MS-1 endothelial cells.

SummaryProx1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.

Transforming growth factor‐β (TGF‐β) plays central roles in endothelial–mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial–mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA‐31 (miR‐31) is a positive regulator of TGF‐β‐induced EndMT. Although the expression is not induced by TGF‐β, miR‐31 is required for induction of mesenchymal genes including α‐SMA, actin reorganization and MRTF‐A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF‐A activity, as a miR‐31 target. Global transcriptome analysis further showed that miR‐31 positively regulates EndMT‐associated unique secretory phenotype (EndMT‐SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL‐6 and Angptl2. As a mechanism for this phenomenon, TGF‐β and miR‐31 suppress Stk40, a negative regulator of NF‐κB pathway. Interestingly, TGF‐β induces alternative polyadenylation (APA)‐coupled miR‐31‐dependent Stk40 suppression without concomitant miR‐31 induction, and APA‐mediated exclusion of internal poly(A) sequence in Stk40 3′UTR enhances target efficiency of Stk40. Finally, miR‐31 functions as a molecular hub to integrate TGF‐β and TNF‐α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT.

Multiple microRNAs (miRNAs) regulate epithelial-mesenchymal transition and endothelial-mesenchymal transition (EndMT). Here we report that microRNA-27b (miR-27b) positively regulates transforming growth factor-β (TGF-β)-induced EndMT of MS-1 mouse pancreatic microvascular endothelial cells. TGF-β induced miR-23b/24-1/27b expression, and inhibition of miR-27 suppressed TGF-β-mediated induction of mesenchymal genes. Genome-wide miRNA target analysis revealed that miR-27 targets Elk1, which acts as a competitive inhibitor of myocardin-related transcription factor-serum response factor signalling and as a myogenic repressor. miR-27b was also found to regulate several semaphorin receptors including Neuropilin 2, Plexin A2 and Plexin D1. These results suggest important roles of miR-27 in TGF-β-driven EndMT.