The NC1 Domain of Type IV Collagen Promotes Axonal Growth in Sympathetic Neurons through Interaction with the 1B1 InteglSn

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (~<24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inacfive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen Iv's effects to those of laminin reveal~ that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize fl~ integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the ot~/31 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin. XTRACELLULAR matrix (ECM) 1 molecules influence numerous aspects of neural development, including cellular migration, process outgrowth, synaptogenesis, and myelination (reviewed in Sanes, 1989). The most potent and effective protein within neural ECM appears to be laminin, which has been shown to be involved in nerve regeneration in situ (Sandrock and Matthew, 1987). Tissue culture studies reveal that laminin can increase the number of processes extended by neurons in vitro and/or the rate at which they grow (reviewed by Reichardt et al., 1989; Sanes, 1989; Sephel et al., 1989), and can also participate in the guidance of growth cones (Hammarback et al., 1988). Additionally, laminin may play a role in neuronal morphogenesis since its actions have recently been shown to be processspecific. That is, laminin has been shown to selectively modulate axonal but not dendritic growth in sympathetic (Lein and Higgins, 1989) and hippocampal neurons in vitro (Lein, P. L, G. A. Banker, and D. Higgins, manuscript in preparation). 1. Abbreviations used in this paper: ECM, extracellular matrix; PIV, pepsinized collagen IV. In addition to laminin, the peripheral nervous system contains other ECM components such as collagen IV, nidogen/entactin, fibronectin, and heparan sulfate proteoglycan. That these other molecules play significant roles in neuronal development and regeneration is suggested by demonstrations that laminin alone is not sufficient to explain either migration or process outgrowth and guidance phenomena in situ (Reichardt et al., 1989; Sanes, 1989). One ECM protein which may be an important modulatory factor in the development and regeneration of the peripheral nervous system is collagen IV. Type IV collagen, which is synthesized by glial ceils (Carey et al., 1983), is present in both fiber tracts and ganglia of the peripheral nervous system (Bunge et al., 1989). Within the peripheral nervous system, collagen IV serves as a structural component of the endoneurial basement membrane, providing tensile strength as well as a scaffold with which other basement membrane components can associate (Timpl, 1989). However, recent evidence indicates that collagen IV can also directly affect cellular functions. In nonneuronal cells, it regulates attachment, migration, and morphological differentiation (Kleinman et al., 1982a; Aumailley and Timpl, 1986; Chen and Little, 1987; © The Rockefeller University Press, 0021-9525/91/04/417/12 $2.00 The Journal of Cell Biology, Volume 113, Number 2, April 1991 417-428 417 on July 9, 2017 jcb.rress.org D ow nladed fom Herbst et al., 1988; Chelberg et al , , 1989). CoUagen IV also promotes neuritic outgrowth in select neuronal cell type s (Carbonetto et al., 1983; Hall et al., 1987; Tomaselli et al., 1987; Politis, 1989; Turner et al., 1987). The domains of the molecule responsible for this latter activity, as well as the identity of the processes affected, have not been previously defined. To further elucidate the role of collagen IV in the peripheral nervous system, the present study was initiated to (a) define the domain(s) of the collagen IV molecule which affect neuritic outgrowth; (b) characterize collagen IV's effects on axonal and dendritic development in sympathetic neuron in vitro; and (c) compare these effects to those induced by the well-characterized neurite-promoting factor, laminin. In addition, we sought to identify the receptor for collagen IV in peripheral neurons. Previous in vitro studies indicate that the neurite-promoting activities of collagen IV and laminin are mediated primarily by receptors of the fll integrin family (Hall et al., 1987; Tomaselli et al., 1987, 1988). The recent development of antisera specific for individual ~ integrin subunits has led to the identification of the o~/31 integrin as a receptor that mediates process outgrowth on laminin in human neuroblastoma cells (Clegg et al., 1989); and the at/3~ integrin as a dual collagen IV/laminin receptor in PC12 cells (Turner et al., 1989; Tawil et al., 1990; Tomaselli et al., 1990). In this study, we demonstrate that an mAb (3A3) specifc for the c~1/3~ integrin inhibits collagen IV but not laminin-induced process outgrowth in primary cultures of sympathetic and dorsal root sensory neurons. These data suggest that, for neurons of the peripheral nervous system, the u~fl~ integrin serves primarily as a collagen IV receptor and a different integrin mediates responses to laminin. Since mAb 3A3 has also been shown to retard axonal regeneration in the sciatic nerve in situ (Toyota et al., 1990), our observations suggest a regulatory role for collagen IV in the growth of axons of peripheral neurons. Materials and Methods ECM Proteins and Enzymatic Digests Collagen 1V and laminin were purified from the murine Engelbreth-HolmSwarm tumor as previously described (Kleinman et al., 1982b). Laminin was radiolabeled by the Iodogen method (Haas and Bright, 1985), using Na125I (28/zCi//~l) from Amersham Corp. (Arlington Heights, IL) and Iodogen from Pierce Chemical Co. (Rockford, IL). Type IV collagen and collagen IV-derived peptides were radioiodinated as described by Markwell (1982) using Nal25I (10 #Ci/#l) from ICN K & K Laboratories Inc. (Plainview, NY) and IODO-BEADS from Pierce Chemical Co. Human plasma fibronectin was purchased from Bethesda Research Laboratories (Gaithersburg, MD); human serum fibrinogen was obtained from CalbiochemBehring Corp. (La Jolla, CA). Rat tail tendon type I collagen was purified according to the procedure of Bornstein (1958). Previously described methods were used to prepare the NC1 (Timpl et al., 1985) and 7S domains (Timpl et al., 1981) of native type IV collagen. Pepsinized type IV collagen depleted of the 7S domain was prepared as described by TLmpl et al. (1983). The purity of all collagen IV peptides was assessed by SDS-PAGE and by diode array multi-wavelength analysis spectrophotometry downstream from a C18 ttbond pac reverse phase column.