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Elevation of PRH and inhibition of CK2 retards smooth muscle cell proliferation

      Vascular smooth muscle cell (VSMC) proliferation promotes intimal thickening and thereby restenosis. The transcription factor proline-rich homeodomain (PRH) inhibits haematopoietic and cancerous cell growth, but was previously proposed as an inducer of VSMC proliferation. We recently showed using siRNAs and ectopic overexpression that PRH retards VSMC proliferation by approximately 40%. In this study we aimed to determine the underlying mechanisms of the anti-proliferative effect to evaluate its potential as an anti-restenotic factor. We observed that PRH was elevated 3-fold (p<0.01, Student's t-test, n=3) late in the cell cycle in rat aortic VSMCs. Ectopic overexpression PRH mutants showed that PRH required DNA-binding activity, but not interaction with the co-repressor (TLE), to inhibit proliferation. Additionally, prolonging stability of PRH protein by mutation of phosphorylation sites extended the anti-proliferative effect (14±4 vs. 42±9%, ANOVA, n=3) for at least an additional 2 days compared to wild-type. Furthermore, we have established that knockdown of casein kinase-2 (CK2; which by phosphorylation promotes PRH inactivation and degradation) with CK2α/α’ siRNAs significantly inhibited proliferation (5±4% vs. 23±3%, p<0.05, Student's t-test, n=3); as did the CK2 inhibitor, TBB, in PDGF-BB+bFGF stimulated cells (1μM: 13±2% vs. 19±1%; 10μM: 20±2% vs. 43±5%; ANOVA, n=3). Importantly, we have also shown elevated expression of PRH in neointimal VSMCs after incorporation of bromodeoxyuridine (proliferation) in rats in vivo. This study has highlighted that PRH is elevated as a result of proliferation and retards cell growth. Consequently, elevation of PRH levels or inhibition of CK2 activity may be useful for retarding restenosis.
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