PKN activation via transforming growth factor-beta 1 (TGF-beta 1) receptor signaling delays G2/M phase transition in vascular smooth muscle cells.

The transition of vascular smooth muscle cells (VSMCs) from G2 phase into the M (mitosis) phase of the cell cycle is a tightly controlled process. As an arterial SMC prepares for a G2/M transition, the cell has primed the Cdc2/cyclinB1 complex for activation by the phosphorylation of threonine-161 residue on Cdc2. This phosphorylation is necessary but not sufficient for the VSMC to enter into the M phase. In order to enter into mitosis, a phosphatase, Cdc25C, must first dephosphorylate two other critical residues: tyrosine-15 and threonine-14. If Cdc25C phosphatase activity is blocked, VSMC entry into mitosis is delayed. However, how the activity of Cdc25C is regulated has not been fully illustrated. In an earlier published study we have demonstrated that exposure of the VSMC line, PAC-1, to Transforming growth factor-beta1 (TGF-beta1), activated PKN (a RhoA-dependent kinase). Here we show that exposure to TGF-beta1 delays the G2/M transition by 2 hrs in G1/S synchronized and released PAC-1 culture. This delay is abolished by the RhoA kinase inhibitors, HA1077 or Y-27632. More importantly, RNAi knockdown of PKN expression prevents the G2/M transition delay induced by TGF-beta1. Changes in PKN activity temporally correlates to the G2/M transition timing. Moreover, Cdc25C is phosphorylated by the TGF-beta1-activated PKN. PKN and Cdc25C coimmunoprecipitate with each other. Finally, PKN and Cdc25C colocalize to the nuclear region only during the critical period of time prior to entry into the M phase. Our data demonstrate that Cdc25C activity is negatively regulated by TGF-beta1-stimulated PKN. Once activated through TGF-beta1 signaling, PKN binds to and phosphorylates Cdc25C. The physical interaction and phosphorylation result in an inactivation of Cdc25C and delay the VSMC entry into the M stage of the cell cycle.

Transforming growth factor-beta1-induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK.

Differentiated vascular smooth muscle cells (SMCs) exhibit a work phenotype characterized by expression of several well documented contractile apparatus-associated proteins. However, SMCs retain the ability to de-differentiate into a proliferative phenotype, which is involved in the progression of vascular diseases such as atherosclerosis and restenosis. Understanding the mechanisms involved in maintaining SMC differentiation is critical for preventing proliferation associated with vascular disease. In this study, the molecular mechanisms through which transforming growth factor-beta1 (TGF-beta1) induces differentiation of SMCs were examined. TGF-beta1 stimulated actin re-organization, inhibited cell proliferation, and up-regulated SMC marker gene expression in PAC-1 SMCs. These effects were blocked by pretreatment of cells with either HA1077 or Y-27632, which inhibit the kinases downstream of RhoA. Moreover, TGF-beta1 activated RhoA and its downstream target PKN. Overexpression of active PKN alone was sufficient to increase the transcriptional activity of the promoters that control expression of smooth muscle (SM) alpha-actin, SM-myosin heavy chain, and SM22alpha. In addition, PKN increased the activities of serum-response factor (SRF), GATA, and MEF2-dependent enhancer-reporters. RNA interference-mediated inhibition of PKN abolished TGF-beta1-induced activation of SMC marker gene promoters. Finally, examination of MAPK signaling demonstrated that TGF-beta1 increased the activity of p38 MAPK, which was required for activation of the SMC marker gene promoters. Co-expression of dominant negative p38 MAPK was sufficient to block PKN-mediated activation of the SMC marker gene promoters as well as the serum-response factor, GATA, and MEF2 enhancers. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-beta1 activates PKN to promote differentiation of SMCs.

CaM kinase IIdeltaC phosphorylation of 14-3-3beta in vascular smooth muscle cells: activation of class II HDAC repression.

The myocyte enhancer factor-2 (MEF2) family of transcription factors regulates transcription of muscle-dependent genes in cardiac, skeletal and smooth muscle. They are activated by calcium/calmodulin (CaM)-dependent protein kinases I and IV and silenced by CaM KIIdeltaC. MEF2 is held in an inactive form by the class II histone deacetylases (HDAC) until phosphorylated by either CaM kinase I or IV. Upon phosphorylation, HDAC is transported out of the nucleus via a 14-3-3 dependent mechanism freeing MEF2 to drive transcription. The 14-3-3 chaperone protein exists as a homodimer. In the region of homodimerization, there are two canonical CaM kinase II phosphorylation sites (ser60 and ser65). In vitro phosphorylation assay results indicate that 14-3-3beta is indeed a substrate for CaM kinase II. We hypothesize that CaM kinase IIdeltaC phosphorylation of 14-3-3beta will disrupt homodimer formation resulting in the return of HDAC to the nucleus and their reassociation with MEF2. To test this, we mutated serines 60 and 65 of 14-3-3beta to aspartates to mimic the phosphorylated state. In MEF2 enhancer-reporter assays in smooth muscle cells, expression of the 14-3-3beta double mutant attenuated MEF2-enhancer activity driven by CaM kinase I or IV. The intracellular fate of HDAC4 was followed by transfection of smooth muscle cells with an HDAC4-Green Fluorescent Protein fusion hybrid. The 14-3-3beta double mutant prevented HDAC4 cytoplasmic localization in the presence of active CaM kinase I or IV. These data suggest that the mechanism of CaM kinase IIdeltaC silencing of MEF-2-dependent genes is by phosphorylation of 14-3-3beta, which allows HDAC to return to the nucleus to reform a complex with MEF2, thereby silencing MADS box-dependent gene induction in smooth muscle.