Choi S W, Park H Y, Rubeiz N G, Sachs D, Gilchrest B A
Boston University School of Medicine, Department of Dermatology, MA 02118-2394, USA.
J Dermatol Sci. 1998 Sep;18(1):54-63. doi: 10.1016/s0923-1811(98)00025-5.
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.
已知人皮肤成纤维细胞可表达蛋白激酶C（PKC）的α、δ、ε和ζ亚型。我们探究了人皮肤成纤维细胞的生长是否与特定PKC亚型的表达相关。在存在钙的条件下（此条件允许所有PKC亚型被激活）所测得的总PKC活性中，75%由PKC-α贡献，这表明PKC-α是人皮肤成纤维细胞中的主要亚型。然后我们在不同培养条件下以及来自不同年龄供体的培养物中进一步研究了PKC-α。在亚汇合和汇合培养物中，缓慢增殖的成年细胞中的总PKC活性和PKC-α蛋白水平始终高于增殖较快的新生细胞。此外，在新生成纤维细胞中，密度对这些参数有很大影响。在亚汇合密度下，当细胞呈指数分裂时，总PKC活性为345±63 cpm/μg蛋白；而在汇合密度下，当细胞生长停滞时，其活性高6 - 7倍，为2334±50 cpm/μg蛋白。使用针对PKC-α的特异性单克隆抗体进行的免疫印迹分析显示，在汇合密度下PKC-α蛋白水平也有类似的6 - 7倍增加。然而，在成年细胞中，密度对已经很高的总活性或PKC-α水平没有影响。为了进一步确定总PKC活性水平和α亚型的增加是否与生长速率降低相关（这是成年供体来源细胞和汇合细胞的共同特征），我们将亚汇合静止细胞中的总PKC活性和PKC-α水平与相同密度下成对的指数生长细胞进行了比较。亚汇合静止细胞中的总PKC活性为8836±71 cpm/μg蛋白，而分裂细胞中的为4415±175 cpm/μg蛋白。静止培养物中PKC-α蛋白水平也比生长培养物中的高2 - 3倍。然而，通过Northern印迹分析确定，这两种条件下PKC-α mRNA的量是相同的。综上所述，这些结果表明总PKC活性水平和PKC-α蛋白与成纤维细胞生长速率之间存在负相关关系，且这种关系在转录后水平受到调控。
