Undermining the endothelium by ablation of MAPK-MEF2 signaling

Abstract

Numerous stimuli activate Big MAPK-1 (BMK1), an MAPK that activates the myocyte enhancer factor-2 (MEF2) transcription factor. Conditional gene deletion showed BMK1 to be required for survival of endothelial cells. An active form of MEF2C could partially bypass the requirement for BMK1 for endothelial cell survival in vitro. These findings reveal an essential role for BMK1-MEF2 signaling in an endothelial cell survival pathway and raise interesting questions about the molecular basis of this response.

A variety of extracellular stimuli transmit signals from the cell membrane to the nucleus via a cascade of MAPKs (1). MAPK pathways contain three distinct types of MAPKs that are activated sequentially (Figure 1). The most upstream kinase, MAPK kinase kinase (MEKK), is activated by G protein–coupled receptors and phosphorylates a subordinate MAPK kinase (MEK), which phosphorylates an MAPK. The final MAPK in the cascade phosphorylates various transcription factors, leading to activation of specific programs of gene expression.

Figure 1.

MAPK signaling pathways. Survival signals activate MEKK2/3, which activates MEK5, which activates BMK1. BMK1 stimulates the transcriptional activity of MEF2C by phosphorylating the transcription activation domain and by interacting directly with MEF2C and contributing its own transcriptional activation domain. MEF2C is required for cell survival and proliferation by activating downstream target genes that remain to be identified.

Big MAPK-1 (BMK1; also called ERK5) is the terminal MAPK that is activated by MEK5, which is activated by MEKK2/3 (Figure 1). BMK1 is unique among MAPKs because of its large size and bi-functionality. The N-terminal region of BMK1 contains the kinase domain, while the C-terminal region functions as a transcription activation domain (2). One of the best-characterized targets of BMK1 is the myocyte enhancer factor-2 (MEF2) family of transcription factors (3–6). There are four mammalian MEF2 genes, MEF2A, -B, -C, and -D, which are expressed in overlapping patterns in numerous cell types (7). MEF2 factors bind DNA as homo- and heterodimers and activate or repress transcription by recruiting positive or negative cofactors, many of which are cell type–specific and signal-responsive. First discovered as regulators of muscle development, MEF2 factors are now known to play diverse roles in the control of cell growth, survival, and apoptosis (7).

BMK1 acts in an endothelial cell survival pathway

Previous studies showed that KO mice lacking BMK1 die around embryonic day (E) 10 from severe abnormalities in cardiovascular development that are remarkably similar to defects seen in KO mice lacking MEKK3 or MEF2C, consistent with the sequential actions of these signaling molecules (8–13). However, the severity of the defects in these mutant embryos, and the associated abnormalities in yolk sac development, complicated the interpretation of the phenotypes and precluded the identification of the precise cell type responsible for embryonic lethality.

To further define the role of BMK1 in mouse development, Hayashi and coworkers generated mice harboring a conditional BMK1 allele (14). In this issue of the JCI, they report that excision of BMK1 after birth, using an inducible Cre recombinase transgene controlled by polyinosinic-polycytidylic (pIpC) acid, resulted in degeneration of the cardiovascular system, accompanied by multifocal hemorrhages, distended capillaries, and ruptures in the normally seamless endothelial lining of the vessels and the heart. Vascular demise following BMK1 deletion was attributable to apoptosis of endothelial cells (ECs). Subsequent analysis of the expression pattern of the pIpC-inducible Cre transgene using a floxed alkaline phosphatase reporter showed strong expression in endothelial and endocardial cells. In agreement with the conclusion that the lethal phenotype reflected an essential role of BMK1 in the endothelium, BMK1 deletion using a Cre transgene controlled by the endothelial-specific Tie2 promoter caused embryonic lethality at E9.5–10.5 with a phenotype indistinguishable from that of global BMK1-KO mice.

ECs normally proliferate and undergo a mesenchymal transformation in cardiac explant cultures. In contrast, explanted ECs from BMK1-KO mice failed to proliferate in vitro (14). The specific dependence of ECs on BMK1 signaling for proliferation and survival was further demonstrated by the finding that ECs isolated from conditional BMK1-KO mice stopped dividing and underwent apoptosis upon deletion of BMK1 with a Cre-expressing adenovirus in vitro, whereas fibroblasts were unaffected by BMK1 deletion.

VEGF and other growth factors that promote EC survival and proliferation activate BMK1 in cultured ECs. The possibility that MEF2C is a target of BMK1 in an EC survival pathway is supported by the finding that serum, a source of survival signals, stimulated activity of an MEF2C reporter in cultured ECs, and this stimulatory effect was lost upon deletion of BMK1 (14). Moreover, infection of BMK1-KO ECs with an adenovirus encoding a constitutively active form of MEF2C fused to the VP16 activation domain partially protected cells from apoptosis following BMK1 deletion.

A BMK1-MEF2 connection in endothelial survival

Together, these findings reveal a cell-autonomous requirement for BMK1-MEF2 signaling for EC proliferation and viability. The endothelial phenotype of BMK1-KO mice raises interesting questions about the cellular specificity of this response and the targets of BMK1 and MEF2 in the EC survival pathway. For example, it is unclear why ECs are so dependent on the BMK1-MEF2C signaling module for survival, whereas fibroblasts and hepatocytes have been shown to be unaffected by deletion of BMK1 (14). Previous studies have shown that BMK1 and MEF2C also promote survival of neurons in vitro (15–18). Perhaps other MAPK and MEF2 family members play redundant roles in other cell types, or perhaps ECs and neurons are more dependent on continual antiapoptotic signals for survival than other cell types. It will be of interest to identify the target genes of MEF2C in the EC survival pathway and to determine the extent to which BMK1 substrates other than MEF2C contribute to the EC phenotype of BMK1-KO mice. The fact that MEF2C-VP16 protected BMK1-KO ECs only partially from cell death suggests the existence of additional antiapoptotic BMK1 targets.

Other cardiovascular functions of BMK1-MEF2

The vascular endothelium is a seamless, yet dynamic, tissue required for multiple functions of the cardiovascular system, including maintenance of vascular tone, regulation of blood circulation, coagulation, inflammatory responses, and proper growth and development of vascular smooth muscle and cardiac myocytes. Perturbation of the vascular endothelium is responsible for a variety of cardiovascular disorders, including atherosclerosis, thrombosis, and hypertension. It will be of interest to determine whether BMK1 and MEF2 are involved in the pathogenic mechanisms associated with these endothelial disorders. In this regard, BMK1 is potently activated by fluid shear stress within the vessel wall, which is atheroprotective (19). Conversely, lack of shear stress has been shown to trigger EC apoptosis. Shear stress and activated MEK5 stimulate phosphorylation of the pro-apoptotic factor Bad, which prevents it from translocating to the mitochondria and activating caspase-3. BMK1 is also activated by and plays a protective role against oxidative stress. The apparent requirement of MEF2C for maintenance of endothelial integrity is also intriguing in light of the recent association of premature coronary artery disease and myocardial infarction with a mutation in the human MEF2A gene (20). Since MEF2A is highly expressed in the endothelium and is a substrate for BMK1, it is likely to act within the same EC survival pathway as MEF2C.

A remarkable number of processes within the cardiovascular system are dependent on signaling from MAPKs to MEF2 (7). In addition to its requirement for EC survival, this signaling pathway is important for differentiation and morphogenesis of cardiac and smooth muscle cells, and has been implicated in numerous cardiovascular disorders, including cardiac hypertrophy, dilated cardiomyopathy, coronary artery disease, and myocardial infarction. Further insights into the functions and mechanisms of action of this signaling module promise to provide new opportunities for its therapeutic manipulation in the settings of human disease.