Figure 1: MRTF activation and nuclear translocation induced angiogenesis via CCN1 and CCN2 activation.
From: MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2
(a,b) MRTF-A transfection enhanced endothelial cell migration in a wound-scratch assay in vitro (red area=uncovered area, scale bar: 200 μm), and (c,d) tube formation of human microvascular endothelial cells (HMECs) in vitro (lpf=low-power field). Overexpression of Tß4, a G-actin sequestering peptide activating MRTFs, displayed a similar effect unless an MRTF-shRNA was co-applied or a Tß4 mutant (Tß4 m), which lacked the G-actin-binding motif KLKKTET, were used (scale bar: 200 μm). (e) Tß4 transfection of myocytic HL-1 cells enabled nuclear (blue fluorescence) translocation of MRTF-A (green fluorescence), an effect absent when the Tß4 m construct was used lacking the G-actin-binding site (scale bar: 20 μm). (f) Tß4 transfection of HL-1 cells induced an MRTF-SRF-sensitive luciferase reporter (containing three copies of the c-fos SRF-binding site=p3DA.Luc, cf.44), unlike Tß4 mutant transfection. (g) Tß4-induced tube formation was abolished in case of shRNA-co-transfection of the MRTF/SRF target geneCCN1 (Cyr61, scale bar: 200 μm) (h,i). Tube maturation, assessed as pericyte recruitment (PC, green fluorescence) to endothelial rings (EC rings, red fluorescence, scale bar: 200 μm) was induced by MRTF-A and Tß4. Co-transfection of shRNA versus the MRTF-target gene CCN2 (CTGF) abolished the Tß4 effect. (All error bars: mean±s.e.m., n=5, *P<0.05, **P<0.001, using analysis of variance (ANOVA) with the Student–Newman–Keul’s procedure)
