Although the major upstream signaling pathways mediating VEGF proangiogenic effects on endothelial cells have been well characterized, relatively less is known regarding the transcription factors that mediate these effects. MEF2C has been demonstrated to play a critical role in vasculogenesis and angiogenesis during vascular development. Mice with targeted deletion of the MEF2C gene exhibit severe vascular abnormalities
7 10 and a phenotype similar to that of mice lacking VEGF or Flt-1.
7 Importantly, transcripts for VEGF and its receptors, Flt-1 and Flk-1, were detected at similar levels in wild-type and MEF2C mutant embryos, suggesting that the vascular defects in MEF2C mutant embryos did not arise from downregulation of these molecules.
7
We were, therefore, interested in the concept that MEF2C might mediate, at least in part, the angiogenic effects of VEGF on endothelial cells. We initially looked for evidence that VEGF might regulate the expression of MEF2C. We found that VEGF strongly induces the expression of MEF2C RNA and protein, and this induction is completely abrogated by the inhibition of PKC. We were interested in testing the possible effects of specific PKC isoforms, particularly PKC-β given its known effects in mediating VEGF action in retinal endothelial cells and its effects on retinal neovascularization.
44 We found that VEGF induction of MEF2C expression was significantly reduced by the inhibition of PKC-β and PKC-δ. To our knowledge, this is the first example of a cell-extrinsic signal or growth factor that induces MEF2C expression in any cell type. In contrast, the cell-intrinsic regulation of MEF2C expression has received greater attention, especially in the context of skeletal muscle development. The basic helix-loop-helix transcription factor myogenin has been shown to upregulate MEF2C expression in vitro,
45 46 and myogenic basic helix-loop-helix proteins have been shown to activate MEF2C expression during skeletal muscle development.
47 48 MEF2C, in turn, participates in the developmental program for myogenesis. It is conceivable that VEGF induction of MEF2C expression in endothelial cells plays an analogous role in angiogenesis.
MEF2C is known to be regulated at the protein level in multiple cell types by several signaling pathways. One important mechanism of regulation is activation of its transcriptional activation domain, notably by p38 MAPK and BMK1/ERK5 signaling. p38 MAPK is thought to activate MEF2C through phosphorylation of the Thr293, Thr300, and Ser387 sites within the MEF2C transactivation domain,
30 whereas BMK1/ERK5 enhances the transactivating activity of MEF2C by phosphorylating Ser 387.
31 Because VEGF is known to activate the phosphorylation of p38 MAPK
34 and BMK1/ERK5,
35 we investigated whether these pathways play a role in VEGF stimulation of MEF2-driven transcription. For our experiments investigating MEF2-driven transcription, we used a 3×-MEF2 reporter approach that is widely used as a readout of cellular MEF2 activity both in vitro
14 and in vivo.
13 This reporter construct allows the investigation of transcription specifically driven by MEF2, in isolation from other transcription factors that might also be induced by VEGF. We found that the modulation of p38 either by pharmacologic inhibition or by transfection of dominant-negative p38 reduced VEGF stimulation of MEF2-driven transcription, whereas the inhibition of BMK1/ERK5 using a dominant-negative mutant did not.
In addition to p38 MAPK, we found that VEGF activation of MEF2-driven transcription is strongly dependent on the calcium-dependent protein phosphatase calcineurin because this activation was strongly inhibited by cyclosporin and by the calcineurin regulator, RCAN1/DSCR1. With regard to calcineurin regulation of transcription factors, the NFAT family is a well-known target of calcineurin, and NFAT transcription factors translocate to the nucleus in response to dephosphorylation by calcineurin.
49 Of note, VEGF induces NFAT dephosphorylation and nuclear translocation in endothelial cells, and this induction is blocked by the calcineurin inhibitor cyclosporin.
50 Furthermore, VEGF activation of NFAT in endothelial cells results in the induction of gene expression of DSCR1 (recently renamed RCAN1
38 ), which acts as a feedback inhibitor of calcineurin.
21 42 43 In nonendothelial cells, calcineurin activates MEF2 using several potential mechanisms.
28 Calcineurin dephosphorylates MEF2 in neurons
51 and skeletal muscle.
52 Calcineurin-mediated dephosphorylation of MEF2 transcription factors (specifically, Ser396 in MEF2C) inhibits sumoylation of a nearby lysine residue,
53 54 thereby inhibiting repression of MEF2C.
29 An additional mechanism by which calcineurin can activate MEF2 is the recruitment of NFAT, which associates with MEF2 and recruits the p300 coactivator.
32 It will be of great interest to determine the specific mechanism(s) by which calcineurin activates MEF2C in endothelial cells.
The role of calcineurin in mediating VEGF activation of MEF2 prompted us to investigate the calcium-dependent protein kinase, calmodulin-dependent protein kinase (CaMK). CaMK has been demonstrated to activate MEF2 in nonendothelial cells by releasing it from class 2 histone deacetylases (HDACs), well-known inhibitors of MEF2.
55 Although VEGF has not been reported to regulate CaMK in endothelial cells, CaMKII expression has been detected in endothelial cells,
56 and CaMKII has been found to play a regulatory role in eNOS expression
57 and nitric oxide synthesis
58 in endothelial cells. Our results demonstrated that VEGF activation of MEF2-driven transcription is indeed partially dependent on CaMK because this activation was inhibited by the CaMK inhibitor KN93 and by a dominant-negative mutant of CaMKII. It will be interesting to determine whether CaMKII activates MEF2C in endothelial cells through the regulation of HDACs or through an alternative mechanism.
Because our studies demonstrate strong evidence that VEGF induces MEF2C expression and MEF2-dependent transcription, we sought to determine the potential functional effect on endothelial cells. Endothelial cell migration is an important facet of the angiogenic process. Our experiments showed that a dominant-negative mutant of MEF2C significantly inhibited VEGF-stimulated retinal endothelial cell migration
(Fig. 7) . We consider this to be particularly significant given the relatively short period (4 hours) of VEGF treatment in this assay. In this experiment, there was also a trend toward a reduction in basal migration (i.e., in the absence of VEGF) in endothelial cells transfected with dominant-negative MEF2C compared with control plasmid, though this did not reach statistical significance. In any event, these results indicate that MEF2 had a significant effect in modulating endothelial cell migration in this assay system. It will be of great interest to investigate other functional end points, including endothelial cell proliferation, survival, and tube formation.
VEGF has pleiotropic effects on endothelial cells resulting from its ability to activate a complex network of signaling pathways.
4 In the context of this study, VEGF activation of MEF2C appears to be dependent on several different signaling pathways, suggesting a role for MEF2C as an important downstream target of these pathways. p38 MAPK and calcineurin have been proposed as important mediators of VEGF-induced migration,
34 59 and our results suggest that MEF2C might serve as one of the downstream effectors of these two signaling molecules. Clearly, further studies will be necessary to fully dissect the role and importance of MEF2C in modulating the effects of these pathways.
Based on the current understanding of MEF2C and our results in this study, we propose a schematic profile of VEGF regulation of MEF2C in endothelial cells
(Fig. 8) . We hypothesize that VEGF induces MEF2C in endothelial cells at two levels. First, VEGF induces the expression of MEF2C RNA (and, therefore, of protein) in a PKC-dependent fashion and possibly by stimulation of transcription factors including Ets-1. In addition, VEGF increases the activity of MEF2C protein by activating signaling molecules, including p38 MAPK, calcineurin, and calmodulin-dependent protein kinase II. Activated MEF2C then plays a role in modulating the effects of VEGF on endothelial cells (including such activities as migration).
Although our results suggest that MEF2C in particular may be important for the effects of VEGF, we cannot rule out the possible involvement of other MEF2 family members. In addition, it is highly likely that other transcription factors also play critical roles in the angiogenic actions of VEGF. VEGF has been shown to induce Egr-1 expression in endothelial cells,
60 and this transcription factor plays a major role in VEGF induction of tissue factor.
61 VEGF induction of Ets-1 expression is important for retinal angiogenesis.
12 VEGF activates NFAT through dephosphorylation and nuclear translocation,
50 and NFAT appears to have a role in VEGF-induced angiogenesis through Cox-2 induction.
62
Nevertheless, VEGF regulation of MEF2C expression and MEF2-dependent transcription suggest an important role for this transcription factor in VEGF action. The stimulation of MEF2C expression and MEF2-driven transcription was specific to VEGF because we did not find evidence of stimulation by other proangiogenic growth factors. It will be important to delineate the spectrum of MEF2C functional roles in VEGF-induced angiogenesis and to elucidate its transcriptional program and critical target genes in endothelial cells.
The authors thank Eric Olson, Jiahuai Han, Leslie Griffith, Susan Birren, and J. Silvio Gutkind for providing plasmids, and Chaitali Sarkar for technical assistance.