The basal levels of mRNAs encoding two
metalloproteinases,
collagenase and
stromelysin, were increased as a function of in vitro serial subcultivation (cellular aging) of human fibroblasts.
Procollagenase and
prostromelysin synthesis and secretion were also greater in the old cultures (late passage). In contrast, the steady-state expression of
mRNA for an inhibitor of
metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1), in late-passage cultures was lower than that in young cell cultures (early passage). Each
mRNA was analyzed using total
RNA preparations isolated from normal fibroblast cultures at different phases of the in vitro life span and from cultures derived from donors with the premature senescence syndromes characterized as
Werner syndrome,
progeria (
Hutchinson-Gilford) syndrome, or
Cockayne syndrome. In normal cell cultures expression of
metalloproteinase mRNAs was increased after the culture had completed greater than 90% of the in vitro life span, and the reduction in
TIMP-1 mRNA expression occurred after the culture had completed greater than 74% of the in vitro lifespan. In
Werner syndrome cultures expression of
metalloproteinase and
TIMP-1 mRNAs was similar to the level of expression observed in late-passage cell cultures. Levels of
metalloproteinase and
TIMP-1 mRNA expression in
progeria and Cockayne syndromes were similar to those of early-passage cell cultures. To determine if young and old cells were each responsive to mediators of
metalloproteinase synthesis, cultures were treated with
phorbol ester or
cytokines. 12-O-tetradecanoylphorbol-13-acetate treatment increased the steady-state levels of all three mRNAs in young, old, and
Werner syndrome cultures and increased
procollagenase levels in all cultures. Early- and late-passage cell cultures also responded to
cytokines.
Interleukin-1 alpha treatment increased
collagenase and
stromelysin mRNA levels while
transforming growth factor-beta reduced the steady-state levels of both transcripts. Neither
cytokine affected the steady-state level of
TIMP-1 mRNA. The results indicate that in vitro cellular aging is associated with changes in expression of mRNAs encoding
proteins that mediate inflammatory responses and connective tissue remodeling.