Characterization of the Role of PKN in TGF-Beta 1-Mediated Cell Cycle Regulation of Vascular Smooth Muscle Cells

Chang Su, Characterization of the role of PKN in TGF-beta-1 induced cell cycle inhibition in vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), November 2005, 173 pp, 2 tables, 34 illustrations, 225 references. Mature vascular smooth muscle cells (VSMCs) are unique in that they can switch between proliferative and differentiated phenotypes. Aberrant proliferation of VSMC is regarded as a central feature in vascular diseases such as atherosclerosis and restenosis following balloon angioplasty. Transforming growth factor-β1 (TGF-β1) is known to inhibit smooth muscle cell progression; however, the signaling pathway(s) through which this is accomplished is poorly understood. Entry into mitosis in dividing VSMCs is triggered by Cdc2/cyclin B1 complex, which is tightly controlled by phosphatase Cdc25C that dephosphorylates tyrosine-15 and threonine-14 on Cdc2 at onset of mitosis. A serine/threonine protein kinase, PKN, was recently reported to inhibit Cdc25C activity. PKN has been identified as a downstream target for TGF-β1 signaling in VSMCs. Therefore we hypothesize that PKN mediates TGF-β1-delayed cell cycle progression by inhibiting Cdc25C. In this study, TGF-β1 is shown to delay G2/M phase progression timing in PAC-1 VSMCs. This effect is blocked by pretreatment of cells with either HA1077 of Y-27632, two pharmacological inhibitors of PKN, as well as by reduced expression of PKN by RNA interference (RNAi). Oscillation of PKN activity temporally correlates with G2/M phase progression. Co-immunoprecipitation suggests that Cdc25C and PKN physically associate with each other. Immunocytochemistry demonstrate that PKN and Cdc25C co-localize in the nuclei and peri-nuclear region of only dividing (M phase) cells but not in the interphase cells. Additionally, PKN phosphorylates Cdc25C in PAC-1 cell cultures. Finally, TGF-β1-induced delay of Cdc2 activation is abolished by pretreating the cells with Y-27632. These data suggest that PKN inhibits G2/M progression by directly binding to Cdc25C and inhibiting its activity by phosphorylation. In addition to the PKN-Cdc25C signaling pathway, TGF-β1 strongly induces the transcriptional activity of the Smad-dependent enhancer in PAC-1 cells. This effect is attenuated by blocking PKN function by either chemical inhibitors or RNAi. Active forms of MKK3/6 alone are sufficient to increase the Smad enhancer activity, and co-expression of dominant negative MKK3/6 decreases TGF-β1-induced activation of the Smad enhancer. Lastly, the Smad reporter activity induced by TGF-β1 is also significantly attenuated by SB203580, a highly specific pharmacological inhibitor for p38 MAPK. These data demonstrate a novel mechanism of PKN-MKK3/6-p38 MAPK cascade to cross talk with the Smad pathway in PAC-1 VSMCs. Taken together, findings presented in this dissertation identify components of important intracellular signaling pathways through which TGF-β1 activates PKN to inhibit proliferation and promote differentiation of SMCs. Augmenting PKN-Cdc25C-Cdc2 signaling may provide a potential therapeutic approach to counter abnormal VSMC proliferation, prevent the clinical consequences of atherosclerosis and improve outcomes after angioplasty.