Schwann cell dedifferentiation and proliferation is a prerequisite to axonal regeneration in the injured peripheral nervous system. The neuregulin (NRG) family of growth and differentiation factors may play a particularly important role in this process, because these axon-associated molecules are potent Schwann cell mitogens and differentiation factors in vitro. We have examined Schwann cell DNA synthesis and the expression of NRGs and their receptors, the erbB membrane tyrosine kinases, in rat sciatic nerve, sensory ganglia, and spinal cord 0 -30 d postaxotomy. Analysis of NRG cDNAs from these tissues revealed several novel splice variants and showed that cells endogenous to injured nerve express NRG mRNAs. A selective induction of mRNAs encoding the glial growth factor (GGF) subfamily of NRGs occurs in nerve beginning 3 d postaxotomy and thus coincides with the onset of Schwann cell DNA synthesis. In later stages of Wallerian degeneration, however, Schwann cell mitogenesis markedly decreases, whereas elevated GGF expression persists. Of the four known erbB kinases, Schwann cells express both erbB2 and erbB3 receptors over the entire interval studied. Expression of erbB2 and erbB3 is coordinately induced in response to axotomy, indicating that Schwann cell responses to NRGs may be modulated by changes in receptor density. Neuregulin (including transmembrane precursors) and erbB protein are associated with Schwann cells postaxotomy. Thus, in contrast to the concept of NRGs as axon-associated mitogens, our findings suggest that NRGs produced by Schwann cells themselves may be partially responsible for Schwann cell proliferation during Wallerian degeneration, probably acting via autocrine or paracrine mechanisms.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [Löbermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.

Recruitment of hematogenous myelomonocytic cells into injured peripheral nerve is essential for axonal regeneration. The monocyte chemoattractant protein-1 (JE) and melanoma growth stimulatory activity/gro (KC) "immediate early" gene products may be important in this process as these proteins are potent chemoattractants for macrophages and neutrophils, respectively. To test this hypothesis, we examined JE and KC activation in rat sciatic nerve 0-30 days after surgical transection. RT-PCR and in situ hybridization analyses of JE and KC expression demonstrates these mRNAs are present in injured nerve, first being expressed by a cellular subpopulation within the zone of trauma by 1.5 hours after injury. By 16 hours posttransection a subpopulation of JE-positive endoneurial cells is found in the proximal stump and throughout the distal nerve segment, with maximal mRNA accumulation occurring 1 day after injury and expression persisting to 18 days postaxotomy, a period preceding and coincident with macrophage infiltration. In contrast, by 3 days postaxotomy KC expression is markedly diminished, consistent with the limited neutrophilic response to nerve injury. JE expression was also examined in C57BL/Wld(s) mice, which have delayed Wallerian degeneration associated with a failure of macrophage recruitment, and their parental C57BL/6J strain. Although JE mRNA is inducible in sciatic nerve from C57BL/6J mice, these transcripts are undetectable in injured nerve from C57BL/Wld(s) mice. Our findings suggest that activation of the JE locus is at least partially responsible for macrophage invasion of injured peripheral nerve. Furthermore, defective postaxotomy macrophage recruitment in C57BL/Wld(s) mice may involve a failure of JE induction.

Neuregulin-1 (NRG-1) proteins promote Schwann cell survival, differentiation and proliferation during development. High levels of an NRG-like activity are also present in some human peripheral nerve sheath tumors, suggesting that NRG-1 isoforms may be involved in the development of these neoplasms. We examined the expression of NRG-1 and its receptors, the erbB membrane tyrosine kinases, in JS1 cells, a rapidly proliferating line derived from a chemically induced rat malignant peripheral nerve sheath tumor (MPNST). Relative to nontransformed Schwann cells, JS1 cells overexpress the NRG-1 receptor erbB3 and its erbB2 coreceptor; JS1 erbB2 transcripts show no evidence of the activating mutation commonly found in N-ethyl-N-nitrosourea-induced neoplasms. JS1 cells do not express the epidermal growth factor receptor (EGFR), a kinase implicated in the pathogenesis of a major subset of MPNSTs. JS1 cells also express mRNAs encoding multiple alpha and beta isoforms from the glial growth factor and sensory and motor neuron-derived factor NRG-1 subfamilies. Stimulation with NRG-1beta in the presence of forskolin produces a dose-dependent increase in JS1 DNA synthesis. Even in unstimulated JS1 cells, however, erbB2 and erbB3 are constitutively tyrosine phosphorylated. Reducing this constitutive phosphorylation with the specific erbB inhibitor PD158780 markedly impairs JS1 DNA synthesis. These observations support the hypothesis that NRG-1 isoforms and erbB kinases act in an autocrine and/or paracrine fashion to promote mitogenesis in JS1 cells. The absence of EGFR expression in JS1 cells suggests that constitutive activation of the NRG-1/erbB signaling pathway is an alternative means of inducing Schwann cell neoplasia.

We have previously found that adult Schwann cells express receptors for lysophosphatidic acid (EDG2, EDG7) and sphingosine-1-phosphate (EDG5) and that expression of these receptors is significantly upregulated in injured sciatic nerve coincident with postaxotomy Schwann cell proliferation. Based on these observations, we hypothesized that lysophosphatidic acid and/or sphingosine-1-phosphate promote Schwann cell mitogenesis in injured adult nerve. We found that both saturated and unsaturated forms of lysophosphatidic acid, but not sphingosine-1-phosphate, induce DNA synthesis in adult Schwann cells isolated from surgically transected sciatic nerve. Lysophosphatidic acid induces adult Schwann cell DNA synthesis in a dose-dependent manner, acting at 0.1- to 10-microM concentrations. Lysophosphatidic acid-mediated stimulation of adult Schwann cell DNA synthesis occurs via a signaling pathway involving a pertussis toxin-sensitive (G(i)/G(o)) G-protein. Activation of phosphatidylinositol-3-kinase, cAMP-dependent protein kinase A and mitogen-activated protein kinase kinase is also required for lysophosphatidic acid-induced Schwann cell mitogenesis. These findings demonstrate that lysophosphatidic acid promotes proliferation of adult Schwann cells isolated from injured nerve and are consistent with the hypothesis that lysophosphatidic acid promotes in vivo Schwann cell mitogenesis in regenerating peripheral nerve.

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