West M D, Pereira-Smith O M, Smith J R
Roy M. and Phyllis Gough Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030.
Exp Cell Res. 1989 Sep;184(1):138-47. doi: 10.1016/0014-4827(89)90372-8.
The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.
细胞外基质萎缩是结缔组织衰老过程中的常见现象。在本研究中，我们验证了这样一个假设：衰老细胞受血清生长因子调节的能力改变与胶原酶合成调控的丧失相关。我们检测了体外培养的年轻和衰老成纤维细胞中免疫反应性前胶原酶和胶原酶抑制剂（金属蛋白酶组织抑制剂，TIMP）的水平。在低浓度（0.5%）胎牛血清中培养的年轻成纤维细胞，在血清刺激4.0小时后，随着血清浓度升高（至10%），前胶原酶和TIMP水平会升高。相比之下，衰老的成纤维细胞即使在低血清水平培养时也会持续产生相对高水平的前胶原酶，并且不会因血清刺激而增加前胶原酶的合成。此外，衰老的成纤维细胞持续表达相对少量的TIMP，血清刺激后也不会被诱导。胶原酶和TIMP的这种表达改变似乎是衰老表型所特有的，而不仅仅是生长抑制的结果，因为因密度依赖性生长抑制而生长停滞的年轻细胞在血清刺激后，前胶原酶和TIMP的表达时间模式与未汇合的年轻细胞培养物相似。对净胶原酶活性的检测显示，在低浓度胎牛血清中培养时，衰老成纤维细胞经胰蛋白酶激活的提取物中的活性比年轻成纤维细胞高出20倍以上。这些结果首次证明了调节结缔组织稳态的分子途径改变与复制性衰老的分子途径改变之间存在直接关联。与在低血清浓度下培养的年轻成纤维细胞相比，衰老成纤维细胞的胶原olytic活性增加表明，衰老的成纤维细胞可能变得越来越具有纤维分解性，从而导致体内衰老过程中观察到的许多与年龄相关的真皮胶原改变。
