Fibroblasts That Proliferate Near Denervated Synaptic Sites in Skeletal Muscle Synthesize the Adhesive Molecules Tenascin(J1), N-CAM, Fibronectin, and a Heparan Sulfate Proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)