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-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition 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 associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27(kip1) abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared 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 occurs secondary to a suppression in cyclin D1 and induction of p27(kip1) expression, which in turn inhibits Rb phosphorylation.
Stuart
W D
WD
Departments of Medicine and Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267, USA.
Maeda
S
S
Khera
P
P
Fagin
J A
JA
Clemens
T L
TL
D003994:Bucladesine / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003994:Bucladesine; D000242:Cyclic AMP; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases