PROTEIN-KINASE-C-ALPHA LEVELS ARE INVERSELY ASSOCIATED WITH GROWTH-RATE IN CULTURED HUMAN DERMAL FIBROBLASTS

Human dermal fibroblasts are known to express the alpha, delta, epsilo n, and zeta isoforms of protein kinase C (PKC). We asked whether the g rowth of human dermal fibroblasts correlates with expression of a part icular PKC isoform. Of total PKC activity measured in the presence of calcium, a condition permissive for activation of all PKC isoforms, 75 % was contributed by PKC-alpha, suggesting that PKC-a is the dominant isoform in human dermal fibroblasts. We then further studied PKC-alpha under different culture conditions and in cultures derived from diffe rent aged donors. In both subconfluent and confluent cultures, total P KC activity and the level of PKC-alpha protein were consistently highe r in slowly proliferating adult cells than in more rapidly proliferati ng newborn cells. Moreover, in newborn fibroblasts density strongly in fluenced these parameters. At subconfluent density, when cells were di viding exponentially, total PKC activity was 345 +/- 63 cpm/mu g prote in; whereas at confluent density, when cells were growth arrested, it was 6-7 fold higher, 2334 +/- 50 cpm/mu g protein. Immunoblot analysis using a specific monoclonal antibody against PKC-alpha exhibited a si milar 6-7 fold increase in the level of PKC-alpha protein at confluent density. However, in adult cells, density had no influence on the alr eady high total activity or level of PKC-alpha. To further determine w hether the increases in the levels of total PKC activity and the alpha isoform correlate with the decreased growth rate, a characteristic of both adult donor-derived and confluent cells, total PKC activity and the level of PKC-alpha in subconfluent quiescent cells was compared to that in paired exponentially growing cells at the same density. Total PKC activity was 8836 +/- 71 cpm/mu g protein in subconfluent quiesce nt cells versus 4415 +/- 175 cpm/mu g protein in dividing cells. The l evel of PKC-alpha protein was also 2-3 fold higher in quiescent than i n growing cultures. However, the amount of PKC-alpha mRNA in these two conditions was identical as determined by northern blot analysis. Tak en together, these results suggest an inverse relationship between the levels of total PKC activity and PKC-alpha protein and fibroblast gro wth rate that is regulated at the post-transcriptional level. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.