Gatchalian C L, Schachner M, Sanes J R
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Cell Biol. 1989 May;108(5):1873-90. doi: 10.1083/jcb.108.5.1873.
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)
四种黏附分子，腱生蛋白（J1）、神经细胞黏附分子（N-CAM）、纤连蛋白和一种硫酸乙酰肝素蛋白聚糖，在大鼠骨骼肌去神经支配后于突触部位附近的细胞间隙中积聚（Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420 - 431）。我们现在探究哪些细胞合成这些分子，以及这种合成是如何被调控的。电子显微镜显示，去神经支配2天后，单核细胞开始选择性地积聚在突触周围的细胞间隙中。通过超微结构和免疫组织化学标准，这些细胞被鉴定为成纤维细胞；[3H]胸腺嘧啶核苷放射自显影显示它们的积聚是由局部增殖导致的。电子显微镜免疫组织化学表明，N-CAM与成纤维细胞表面相关联，而腱生蛋白（J1）与邻接成纤维细胞的胶原纤维相关联。使用免疫荧光和免疫沉淀方法，我们发现从去神经支配肌肉的突触周围区域分离出的成纤维细胞在体外合成N-CAM、腱生蛋白（J1）、纤连蛋白和一种硫酸乙酰肝素蛋白聚糖。因此，在突触部位附近的细胞间隙中选择性增殖的成纤维细胞很可能是去神经支配肌肉中黏附分子细胞外沉积物的细胞来源。为了阐明可能在体内调控这些分子积聚的因素，我们分析了培养的成纤维细胞中腱生蛋白（J1）和纤连蛋白的表达。来自去神经支配肌肉无突触区域的成纤维细胞，以及皮肤、肺和3T3成纤维细胞在培养中积累高水平的腱生蛋白（J1）和纤连蛋白，表明在这方面突触周围成纤维细胞并非独一无二。然而，当首次置于培养中时，去神经支配肌肉的成纤维细胞比受神经支配肌肉的成纤维细胞携带更多的腱生蛋白（J1），这表明成纤维细胞对该分子的表达受肌肉的神经支配状态调控；培养几天后这种差异不再明显。在3T3细胞中，腱生蛋白（J1）在增殖培养物中积累量高，在汇合培养物中降低，并在通过低密度重新接种或损伤汇合单层刺激增殖的细胞中重新激活。因此，腱生蛋白（J1）的合成与有丝分裂活性平行调控。相比之下，在体内去神经支配后增加幅度较小的纤连蛋白水平，在受神经支配和去神经支配肌肉的成纤维细胞以及增殖和静止的3T3细胞中相似。（摘要截选至400字）
