Cyclin D-1 is required for S phase traversal in bovine tracheal myocytes

We examined the role of cyclin D-1 in cultured bovine tracheal myocyte mitogenesis. Immunoblots using a polyclonal antibody against cyclin D-1 showed the appearance of this protein 4 h after treatment with platelet-derived growth factor (PDGF), a potent mitogen for these cells. Immunoblots utilizing an antibody against the 110-kDa retinoblastoma protein (Rb), a downstream phosphorylation target of the cyclin D-1/cyclin-dependent kinase 4 (cdk4) complex, showed reduced electrophoretic mobility of this protein as early as 8 h after PDGF treatment, suggesting phosphorylation of Rb by the cyclin D-1/cdk4 dimer in vivo. Epidermal growth factor (EGF), a nonmitogen, failed to induce either cyclin DI synthesis or Rb phosphorylation. PDGF treatment of cells transiently transfected with the full-length cyclin D-1 promoter subcloned into a luciferase reporter increased luciferase activity almost threefold, demonstrating transcriptional activation of the cyclin D-1 promoter with mitogenic stimulation. Finally, microinjection of individual myocytes with an affinity-purified antibody against cyclin D-1 reduced the percentage of cells traversing S phase after serum stimulation, as assessed by fractional bromodeoxyuridine labeling (isotype control antibody, 0.74 +/- 0.10; anti-cyclin D-1, 0.22 +/- 0.04; P = 0.0001). We conclude that 1) mitogenic stimulation of cultured bovine tracheal myocytes by PDGF induces cyclin D-1 transcriptional activation and protein expression, 2) cyclin D-1 expression is accompanied by Rb phosphorylation, which is evidence of increased cyclin D-1-associated kinase activity in vivo, and 3) microinjection of anti-cyclin D-1 antibodies inhibits cellular DNA synthesis, which is evidence that cyclin D-1 is required for airway smooth muscle S phase traversal.