Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by aging dermal fibroblasts from normal and Down's syndrome patients.
Previous evidence has shown a deficiency in microfilament stress fiber formation upon short-term cycloheximide treatment of cultured human dermal fibroblasts while cytoplasmic spreading appeared completely normal and other cytoskeletal networks organized normally. This deficiency applied to collagen substrata (not fibronectin substrata) and was specific for in vitro-aged normal fibroblasts and for fibroblasts from three different Down's syndrome patients at any passage level. To identify the mechanism(s) for matrix receptor deficiency in aging cells, cells were evaluated for amounts and distributions of several integrin subunits using specific monoclonal antibodies and two complementary experimental approaches. Flow cytometric analyses have shown that all these cells at all passage levels have large amounts of alpha 3 and beta 1 integrin subunits and smaller amounts of the alpha 5 subunit, directed to fibronectin, which are minimally affected in their cell surface availability by cycloheximide treatment. In contrast, cycloheximide treatment leads to the loss from surface availability of most of the alpha 2 subunit, directed to collagen, in late-passage papillary and reticular normal fibroblasts and in all three Down's patient cells at all passages. Prior growth of cells in ascorbate-supplemented medium, which overcomes the deficiency in stress fiber formation, conserves the large amounts of cell surface-available alpha 2 subunit detectable by flow cytometry. When amounts of integrin subunits were evaluated by immunoprecipitation of [35S]methionine-radiolabeled cells, there was no diminution of the alpha 2 subunit or any other subunit for any cells upon cycloheximide treatment; however, there was much less alpha 2 subunit complexed with beta 1 in aging normal and Down's cells. Therefore, cycloheximide treatment does not lead to loss in the amounts of the alpha 2 subunit but rather to its masking at the cell surface and inability to transmit signals across the plasma membrane to effect stress fiber formation. This aging-related deficiency in integrin-mediated signaling can now be studied mechanistically with a variety of approaches to determine the nature of cell-surface molecules interacting with integrins (cis- and/or trans-acting molecules) that discriminate functional from nonfunctional receptors.
Flickinger
K S
KS
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
Carter
W G
WG
Culp
L A
LA
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000328:Adult; D000368:Aged; D000375:Aging / Q000378:metabolism*; D002478:Cells, Cultured; D016922:Cellular Senescence; D003094:Collagen / Q000378:metabolism; D004314:Down Syndrome / Q000378:metabolism* / Q000473:pathology; D005347:Fibroblasts; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016023:Integrins / Q000378:metabolism*; D008297:Male; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism* / Q000648:ultrastructure
D005353:Fibronectins; D016023:Integrins; D003094:Collagen