Clark S D, Wilhelm S M, Stricklin G P, Welgus H G
Arch Biochem Biophys. 1985 Aug 15;241(1):36-44. doi: 10.1016/0003-9861(85)90358-3.
Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.
佛波醇肉豆蔻酸酯乙酸酯（PMA）是一种已知能刺激成纤维细胞和内皮细胞产生胶原酶的肿瘤促进剂，我们研究了它调节人皮肤成纤维细胞分泌的胶原酶抑制剂表达的能力。将汇合的人皮肤成纤维细胞与浓度范围为10⁻¹¹至10⁻⁷M的PMA一起孵育，并用酶联免疫吸附测定法分析条件培养基中的免疫反应性胶原酶和胶原酶抑制剂。PMA刺激几种细胞系中胶原酶和胶原酶抑制剂的产生至相似的最大速率，分别为300至350与230至330 pmol 10μg DNA⁻¹ 48 h⁻¹。由于这些蛋白质基础表达水平的差异，这样的速率反映出胶原酶产生有2至7倍的刺激，相比之下抑制剂合成有更一致的2至3倍增强。抑制剂的产生在7×10⁻⁹M时达到最大值的50%，在佛波醇浓度为10⁻⁷M时达到最大值。这种浓度依赖性效应与观察到的胶原酶表达非常相似。通过将[³H]亮氨酸掺入新合成的蛋白质来研究佛波醇处理的细胞的总蛋白质合成，并未显著增加，细胞DNA含量也未增加。PMA对抑制剂产生作用的起始发生在4至8小时之间，8小时时达到最大值，并至少持续64小时不减弱。在最初的8小时后，抑制剂的产生以大约10 pmol 10μg DNA⁻¹ h⁻¹的大致恒定速率继续。有趣的是，从培养基中去除佛波醇后，这种成纤维细胞继续产生增加量的抑制剂蛋白至少72小时。代谢标记研究中，成纤维细胞暴露于[³H]亮氨酸，然后用抑制剂特异性抗体进行免疫沉淀，结果表明PMA对抑制剂产生的刺激是通过新抑制剂蛋白合成的增加介导的。因此，响应肿瘤促进剂PMA，人皮肤成纤维细胞中胶原酶和胶原酶抑制剂的表达似乎是共同调节的。
