|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β superfamily and participate in functional and morphogenetic regulation of various organs by, in part, controlling cell proliferation and apoptotic cell death . However, the mechanisms by which BMPs trigger growth inhibition and apoptosis remain to be elucidated.
We have previously found that BMP-2 induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we showed that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27^<KIP1>, p16^<INK4a>, p15^<INK4b>, but enhanced expression of p21^<CIP1/WAF1>. Accumulation of p21^<CIP1/WAF1> resulted in increased binding of p21^<CIP1/WAF1> to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21^<CIP1/WAF1> and Rb, rev
erted G1-arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21^<CIP1/WAF1> is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.
We also demonstrated that BMP-2 activated the mouse p21^<CIP1/WAF1> promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter, conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligoncleotide containing the 29-b region was found to be associated with Smad1 and Smad4 in the HS-72 nuclear extract. These results suggested that BMP-2 might activate p21^<CIP1/WAF1> transcription by inducing an indirect binding of Smad4 and Smad1 to the 29-b region in HS-72 cells. Less