STAT1 requirement for PKR-induced cell cycle arrest in vascular smooth muscle cells in response to heparin

- Protein kinase PKR inhibits VSMC proliferation in response to heparin.
- PKR regulates p27kip1 protein levels in VSMC by stabilizing the protein.
- PKR activation leads to phosphorylation of transcription factor STAT1.
- STAT1 is essential for PKR-mediated p27kip1 stabilization.
- PKR and STAT1 null cells are insensitive to heparin.

Interferons (IFNs) are a family of cytokines that exhibit antiviral, antiproliferative, and immunomodulatory properties. PKR (protein kinase, RNA activated) is of central importance in mediating the antiproliferative actions of IFNs. Our research has established that PKR inhibits vascular smooth muscle cell (VSMC) proliferation by regulating G1 to S transition. Many cardiovascular diseases result from complications of atherosclerosis, a chronic and progressive inflammatory condition often characterized by excessive proliferation of VSMC. Thus, an effective method for inhibiting VSMC proliferation is likely to arrest atherosclerosis and restenosis at early stages. Our research establishes that PKR activation in VSMC leads to a G1 arrest brought about by an inhibition of cyclin-dependent kinase 2 (Cdk2) activity by p27kip1. In quiescent VSMC, p27kip1 levels are high and when stimulated by serum/growth factors, p27kip1 levels drop by destabilization of the protein. Under conditions that lead to activation of PKR, there is a marked inhibition of p27kip1 down-regulation due to increased stability of p27kip1 protein. In order to understand the mechanism of heparin-induced stabilization of p27kip1 in VSMC, we examined the involvement of the Signal Transducer and Activator of Transcription-1 (STAT1), which is an important player in mediating antiproliferative effects of IFNs. Our results demonstrate that PKR overexpression in VSMC leads to an increase in p27kip1 protein levels and this increase requires the catalytic activity of PKR. PKR activation induced by antiproliferative agent heparin leads to phosphorylation of STAT1 on serine 727, which is essential for the cell cycle block. STAT1 null VSMCs are largely defective in heparin-induced cell cycle arrest and in PKR null cells the STAT1 phosphorylation in response to heparin was absent. These results establish that heparin causes STAT1 phosphorylation on serine 727 via activation of PKR and that this event is required for the G1 arrest in VSMC.