Smad-mediated Transcription is Required for Transforming Growth Factor-β1-induced p57Kip2 Proteolysis in Osteoblastic Cells

Abstract

Cyclin-dependent kinase inhibitory proteins (CKIs) are negative regulators of the cell cycle. Of all CKIs, only p57Kip2 plays an essential role(s) that other CKIs cannot compensate for in embryonic development. Recently, we found that p57Kip2 is degraded through the ubiquitin-proteasome pathway in osteoblastic cells stimulated to proliferation by transforming growth factor (TGF)-β1 (Urano, T., Yashiroda, H., Muraoka, M., Tanaka, K., Hosoi, T., Inoue, S., Ouchi, Y., and Toyoshima, H. (1999) J. Biol. Chem. 274, 12197-12200). We report here that TGF-β1-induced p57 Kip2 proteolysis is mediated through transcription by the Smad pathway. When the constitutively active form of the TGF-β type I receptor ALK-5(TD) was ectopically expressed in osteoblastic cells, p57Kip2 that had been accumulated by serum starvation causing the cell-cycle arrest was rapidly degraded in a manner analogous to TGF-β1 stimulation. Moreover, Smad2 or Smad3 with Smad4 enhanced the proteolytic pathway of p57 Kip2. The degradation of p57Kip2 evoked by TGF-β1 was blocked by forced expression of an inhibitory Smad called Smad7 or by the addition of actinomycin D or α-amanitin. These results indicate that accelerated degradation of p57Kip2 by TGF-β1/Smad signaling is mediated through a newly synthesized factor(s) that modifies p57Kip2 or the ubiquitin-proteasome pathway.