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, epsilon, and zeta isoforms of protein kinase C (PKC). We asked whether the growth of human dermal fibroblasts correlates with expression of a particular 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-alpha is the dominant isoform in human dermal fibroblasts. We then further studied PKC-alpha under different culture conditions and in cultures derived from different aged donors. In both subconfluent and confluent cultures, total PKC activity and the level of PKC-alpha protein were consistently higher in slowly proliferating adult cells than in more rapidly proliferating newborn cells. Moreover, in newborn fibroblasts density strongly influenced these parameters. At subconfluent density, when cells were dividing exponentially, total PKC activity was 345+/-63 cpm/,ug protein; whereas at confluent density, when cells were growth arrested, it was 6-7 fold higher, 2334+/-50 cpm/ug protein. Immunoblot analysis using a specific monoclonal antibody against PKC-alpha exhibited a similar 6-7 fold increase in the level of PKC-alpha protein at confluent density. However, in adult cells, density had no influence on the already high total activity or level of PKC-alpha. To further determine whether 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/microg protein in subconfluent quiescent cells versus 4415+/-175 cpm/microg protein in dividing cells. The level of PKC-alpha protein was also 2-3 fold higher in quiescent than in growing cultures. However, the amount of PKC-alpha mRNA in these two conditions was identical as determined by northern blot analysis. Taken together, these results suggest an inverse relationship between the levels of total PKC activity and PKC-alpha protein and fibroblast growth rate that is regulated at the post-transcriptional level.
Choi
S W
SW
Boston University School of Medicine, Department of Dermatology, MA 02118-2394, USA.
Park
H Y
HY
Rubeiz
N G
NG
Sachs
D
D
Gilchrest
B A
BA
D000328:Adult; D000367:Age Factors; D002455:Cell Division; D002478:Cells, Cultured; D016922:Cellular Senescence; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007231:Infant, Newborn; D007527:Isoenzymes / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000096:biosynthesis*; D051571:Protein Kinase C-alpha; D012867:Skin / Q000166:cytology* / Q000201:enzymology*
D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha