It has long been known that nerves stimulate growth and maintenance of skeletal muscles in ways not dependent on physical contacts, but numerous attempts to identify and characterize the myotrophic agent(s) secreted by nerves have been unsuccessful. We here suggest that products of the neuregulin gene may be these agents. The neuregulins are a family of proteins made by alternative splicing of a single transcript to give as many as 15 protein products. One member of this family, glial growth

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... tor 2 (rhGGF2) is a very potent stimulator of myogenesis in L6A1 myoblasts, giving a maximal stimulation of cell fusion and creatine kinase elevation at a concentration of 1 ng/ml (18 pM). The stimulation of myogenesis is not rapid, but it is prolonged, continuing over a period of at least 6 days. The effects of rhGGF2 are additive with those of insulin-like growth factor I (IGF-I) or its analog R3-IGF-I, suggesting that the actions of these two myotrophic agents differ in at least one rate-limiting step. We have observed one possible difference; unlike the IGFs, rhGGF2 does not induce elevation of the steady state level of myogenin mRNA. The myotrophic actions of nerves have been known for a long time, and it has been widely recognized that nerves stimulate muscle formation and maintenance in ways not entirely dependent on direct physical contact. A review by Gutman (1) 20 years ago discusses a large body of early evidence for the existence of "long term maintenance regulations not mediated by nerve impulses" and states that "motor, sensory, or central neurons can supply the . . . agent." Subsequent searches for such myotrophic agents have not been successful (at least twice transferrin was isolated when a "muscle trophic factor" or "sciatin" was sought (2, 3)), but we have recently uncovered a strong candidate for the role of the myotrophic agent from nerve cells, glial growth factor 2 (GGF2 1 ) and presumably its fellow members of the neuregulin family of neurotrophic proteins. GGF2 is one of a recently characterized family of closely related products of a single gene, made by alternative splicing to give as many as 15 distinct protein products (4 -7). The members of this family have a number of different names, including heregulin, neu differentiation factor, acetylcholine receptor inducing activity, and glial growth factors. Most contain transmembrane and cytoplasmic domains; they are primarily glycosylated, transported to the cell surface, and reside as transmembrane proteins (although there is some hydrolytic processing of a portion within the cell) (8). This group also found that cell-associated molecules can be released by regulated proteolytic cleavage; for example, phorbol esters cause this release. A new member, sensory and motor neuron-derived factor, has recently been added to the neuregulin family (9); like GGF2, it lacks transmembrane and cytoplasmic regions and is presumably secreted by cells that express it. Most attention has been focused on the actions of these agents in the nervous system, but they also have major effects on growth and development of the mammary epithelial cells (10, 11) , and knockout experiments have demonstrated that they are essential for early development of the heart (12). Insofar as we are aware, the only studies of effects of neuregulins on skeletal muscle have been concerned with induction of the acetylcholine receptor (13, 14) ; actions on other aspects of myogenic differentiation have not been reported. Two of the current authors have previously published an abstract describing stimulatory actions of rhGGF2 on a human muscle cell line (15); this prompted the current study, and it demonstrates that the response we see in L6A1 myoblasts is not limited to that cell line. EXPERIMENTAL PROCEDURES Materials-Unless otherwise specified, all cell culture materials were purchased from Life Technologies, Inc. rhGGF2 was prepared from stably transfected CHO cells essentially as described by Marchionni et al. (7). IGF-I was a gift from Ciba-Geigy, R3-IGF-I a gift from GroPep, and the myogenin probe from E. N. Olson. All materials used in the creatine kinase assay were purchased from Sigma. Cell Cultures and Analyses-L6A1 myoblasts were plated in DMEM containing 10% horse serum and 1% chick embryo extract. Standard cultures were plated at 1.2 ϫ 10 5 cells/35-mm dish for creatine kinase or cell number determinations, and 1 ϫ 10 6 cells/100-mm dish for RNA isolations. After incubation overnight, the cultures were washed with DMEM before the addition of rhGGF2 or other agents in DMEM containing 0.05% bovine serum albumin (pretested as free of mitogenic activity). To quantitate cell proliferation, cells were trypsinized and counted in a Model ZBI Coulter Counter 24 to 48 h after the addition of growth factors. For measurement of the extent of cell differentiation, at 2 to 6 days, the cell monolayers were washed with phosphate-buffered saline, 0.25 ml of 0.05 M glycylglycine (pH 6.75) was added, and the dishes were stored at Ϫ70°C. The cultures were thawed on ice just prior to assay, the cells were scraped off, and the cell lysate was assayed for creatine kinase using a NAD-coupled microtiter assay (16), which allows simultaneous measurement of CK activity of as many as 96 samples, and for which all calculations are fully automated. DNA in