Abstract
A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle. Knock down experiments, immunoprecipitation and proximity ligation assays show that the ERK1/2 scaffold caveolin-1 mediates ERK1/2 activation in response to DPBA, but not FCS, and that ERK1/2 is released from caveolin-1 upon DPBA, but not FCS, stimulation. Conversely, ERK1/2 associated with the actin cytoskeleton is significantly reduced after FCS, but not DPBA stimulation, as determined by Triton X fractionation. Furthermore, cytochalasin treatment inhibits DPBA, but not FCS-induced ERK1/2 phosphorylation, indicating that the actin cytoskeleton is not only a target but also is required for ERK1/2 activation. Our results show that (1) at least two ERK1/2 fractions are regulated separately by specific stimuli, and that (2) the association of ERK1/2 with the actin cytoskeleton regulates the outcome of ERK1/2 signaling.
Highlights
Activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) leads to a proliferative response in many cell types, including smooth muscle, where it has been connected with pathologic hypertrophy and hyperplasia, prerequisites for the development of hypertension and atherosclerosis [1,2,3,4]
deoxyphorbol 13-isobutylate 20-acetate (DPBA) induced phosphorylation of caldesmon fully depends on ERK1/2 activation, since in the presence of the MEK inhibitor U0126, caldesmon phosphorylation is reduced to background levels
After fetal calf serum (FCS) stimulation, caldesmon phosphorylation is reduced by roughly 30% in the presence of U0126, indicating that caldesmon phosphorylation is only partly mediated by ERK1/2 after FCS stimulation (Figure 1B)
Summary
Activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) leads to a proliferative response in many cell types, including smooth muscle, where it has been connected with pathologic hypertrophy and hyperplasia, prerequisites for the development of hypertension and atherosclerosis [1,2,3,4]. It has been demonstrated that ERK1/2 activation can regulate contractility in fully differentiated smooth muscle [6,7,8], and ERK1/2 has been shown to contribute to elevated tone in hypertensive rats [9]. Subcellular targeting of ERK1/2 by scaffold proteins cannot fully explain the different outcomes of ERK1/2 activation, since even cytoskeletal ERK1/2 scaffolds have been associated with proliferative ERK1/2 signaling [12,13]. It is still not clear how ERK1/2 activation can be directed towards proliferation in some settings, and towards contraction in others
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