Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity.
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.
West
M D
MD
Roy M. and Phyllis Gough Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030.
Pereira-Smith
O M
OM
Smith
J R
JR
D015153:Blotting, Western; D002455:Cell Division; D002470:Cell Survival; D017364:Collagenases; D004791:Enzyme Inhibitors / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D003012:Microbial Collagenase / Q000378:metabolism*; D012867:Skin / Q000166:cytology* / Q000201:enzymology; D012995:Solubility; D019714:Tissue Inhibitor of Metalloproteinases; D014508:Urea
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D006133:Growth Substances; D019714:Tissue Inhibitor of Metalloproteinases; D014508:Urea; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase