Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells

The transcript (mRNA), protein levels, enzyme activity, and cellular locali zation of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR-108)were studied at confluent density and during mechan ical stress (cyclic stretch). Western blot analysis indicated that growth t o confluent density significantly increased the protein levels of cPKC-alph a (11.6-fold), nPKC-delta (5.3-fold), and nPKC-epsilon (22.0-fold) but not aPKC-xi. Northern blot analysis indicated a significant (2.3-fold) increase in the 10 kb transcript of cPKC-alpha, a slight (1.3-fold) increase in tha t of nPKC-epsilon but no detectable change in that of the remaining isozyme s. Enzyme activity assays of the individually immunoprecipitated isozymes y ielded detectable kinase activity only for PKC-alpha, PKC-delta, and PKC-ep silon and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR-108 cells showed a dramatic or ientation response to mechanical stress with cell reshaping and alignment o f the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which s tained: for actin, vinculin, and PKC in separate experiments. Longer term m echanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC i sozymes investigated. The results indicate that there are isozyme-selective increases in the protein levels of PKC isozymes of osteoblastic UMR-108 ce lls upon growth to confluence which may be regulated at the transcriptional or the post-transcriptional level. The results from UMR-108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999-1011) that PKC could be involved in the early phase of m echanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton. (C) 2001 Wiley-Liss, Inc.