Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: Aging in vivo or in vitro

Abstract

Attachment, spreading, and microfilament reorganization have been evaluated in human dermal reticular fibroblasts isolated from the inner, upper aspect of the arm of a newborn male (RET 5 cells) and a 78-year-old male (RET 8 cells). Substrata were tested using a set of complementary fragments from individual polypeptide chains of human plasma fibronectin (pFN) or cellular FNs (cFN). With both cell classes, fragments containing the C-terminal heparin-binding (Hep II) domain only elicited linear bundles of microfilaments in spreading cells but no stress fibers; fragments containing the RGDS-dependent cell-binding (Cell I) domain elicited only partial spreading with condensations of F-actin at ruffling membranes and at other regions along the plasma membrane. The minimum sequence required to obtain responses identical to those on intact pFN (broad spreading with extensive stress fiber formation) was found in fragment 155 (F155) from the β chain of pFN; F155 contains both Hep II and Cell I domains. In contrast, the analogous fragment from the α chain of pFN (F145) was notably less effective for generating stress fibers. This evidence along with the better attachment, spreading, and microfilament bundle formation on the Hep II fragment from the β chain than the analogous fragment from the α chain indicates that the extra type III homology unit permits more effective interaction of β chain fragments with cell-surface heparan sulfate proteoglycan and possibly integrin (binding efficiency to the substratum was similar for fragments from both chains). Therefore, alternatively spliced sequences that neighbor binding domains can play significant roles in the interaction of the domain with cell-surface receptors of dermal fibroblasts. Comparison of RET 5 responses with those of RET 8 cells has identified changes in adhesive mechanisms as cells undergo “aging” processes. Attachment and microfilament bundle formation were far more effective for RET 5 cells than for RET 8 cells on any of the Hep II fragments. Conversely, RET 8 cells were far more sensitive to an RGDS-containing peptide in their medium on Cell I fragments than RET 5 cells. These results together indicate that in vivo aging leads to greater dependence upon cell-surface integrin binding and less dependence upon heparan sulfate proteoglycan binding for responses on FN matrices. When RET 5 cells entered senescence ( in vitro aging), they also became much more sensitive to peptide A. On several fragments and on intact pFN, RET 8 cells generated very thick stress fibers that were observed only on one fragment with RET 5 cells. Therefore, aging either in vivo or in vitro leads to changes in the dependence upon two different receptor activities directed to fibronectins responsible for adhesion of human dermal fibroblasts.