Smad-mediated transcription is required for transforming growth factor-beta 1-induced p57(Kip2) proteolysis in osteoblastic cells

Cyclin-dependent kinase inhibitory proteins (CKIs) are negative regulators of the cell cycle. Of all CKls, only p57(Kip2) plays an essential role(s) t hat other CKIs cannot compensate for in embryonic development. Recently, we found that p57(Kip2) is degraded through the ubiquitin-proteasome pathway in osteoblastic cells stimulated to proliferation by transforming growth fa ctor (TGF)-beta1 (Urano, T., Yashiroda, H., Muraoka, RI., Tanaka, K., Hosoi , T.:, Inoue, S., Ouchi, Y., and Toyoshima, H. (1999) J. Biol, Chem. 274, 1 2197-12200). We report here that TGF-beta1-induced p57(Kip2) proteolysis is mediated through transcription by the Smad pathway. When the constitutivel y active form of the TGF-beta type I receptor ALK-5(TD) was ectopically exp ressed in osteoblastic cells, p57(Kip2) that had been accumulated by serum starvation causing the cell-cycle arrest was rapidly degraded in a manner a nalogous to TGF-beta1 stimulation. Moreover, Smad2 or Smad3 with Smad4 enha nced the proteolytic pathway of p57(Kip2). Th, degradation of p57(Kip2) evo ked by TGF-beta1 was blocked by forced expression of an inhibitory Smad cal led Smad7 or by the addition of actinomycin D or alpha -amanitin. These res ults indicate that accelerated degradation of p57(Kip2) by TGF-beta1/Smad s ignaling is mediated through a newly synthesized factor(s) that modifies p5 7(Kip2) or the ubiquitin-proteasome pathway.