| Differentiated vascular smooth muscle cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors, SMCs retain the ability to de-differentiate into an "immature" proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanisms involved in maintaining SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-beta1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-beta1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs These characteristics are indicative of the differentiated phenotype. The effects of TGF-beta1 can be blocked by pretreatment of the cells with either HA 1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-beta1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM alpha-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF, GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-beta1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK1/2. Co-expression of dominant negative p38 MAPK is sufficient to abolish PKN-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-beta1 activates RhoA and PKN to promote differentiation of SMCs. |
