Parathyroid hormone-related protein induces G1 phase growth arrest of vascular smooth muscle cells

In this study, we investigated the mechanisms responsible for the growth-in hibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascu lar smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysi s of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) de monstrated an enrichment of cells in G1 and a reduction in the S phase. Mea surement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibi tion by 12-16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its ass ociated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a redu ction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting o f extracts from cAMP-treated cells with antibodies to cdk inhibitors reveal ed a striking increase in p27(kip1) abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepar ed from cAMP-treated cells followed by immunoblotting with antisera to p27( kip1) disclosed a threefold increase in p27(kip1) associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This o ccurs secondary to a suppression in cyclin D1 and induction of p27(kip1) ex pression, which in turn inhibits Rb phosphorylation.