We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L-and N-types of voltage-dependent Ca 2؉ channel blockers, diltiazem and -conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca 2؉ -dependent manner. A transient elevation in intracellular Ca 2؉ level was observed upon MSH stimulation. These findings suggest that extracellular Ca 2؉ influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.Nervous system function is dependent upon highly specific connections that form between neurons during development. The patterning and specificity of these connections requires neurite extension toward the proper targets guided by the growth cone in response to environmental signals. However, the process that involves signal-induced morphological changes resulting in coordinated cytoskeletal remodeling in the specialized growth cone is poorly understood (1).The semaphorins/collapsins are a large family of structurally distinct secreted and transmembrane proteins characterized by the presence of a conserved sema domain of about 500 amino acids (2-4). Chick collapsin-1 and its mammalian homolog, mouse semaphorin D (MSD) 1 (initially named sema III), act in vitro as collapsing factors on growth cones and as selective chemorepellents for subpopulations of spinal and cranial, sensory, and motor axons (5, 6). Thus, semaphorins are thought to be involved in the axon guidance mechanism during neuronal development. To investigate the mechanism of neural network formation, we identified several novel semaphorins (7-9). One of them, mouse semaphorin H (MSH) is structurally similar to MSD and acts as a chemorepellent on sensory axons (9, 10). To investigate the signaling pathway for secreted semaphorin MSH, we used PC12 cells (a clone derived from a pheochromocytoma tumor of the rat adrenal medulla), which are known to differentiate into neuronal cells in response to NGF.Studies on PC12 cells have demonstrated that a variety of extracellular signals can lead to neurite outgrowth and morphological differentiation. In this cell line, the signaling pathway via the NGF receptor is the most well characterized (11-13). Ligand binding to its tyrosine kinase receptor (Trk) causes activation of Ras-dependent MAPK cascades resulting in cellular differentiation and neurite outgrowth (14).In this study, we have found that MSH induces neurite outgrowth in PC12 cells through a Ras-M...

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FIG. 2.Time course of Na ϩ -dependent Ca 2ϩ uptake in VC and NO. Na ϩ -dependent Ca 2ϩ uptake of both VC (s) and NO (OE) was determined by subtracting the values obtained in Na ϩ -containing buffer from the Na ϩ -free choline buffer. Each point represents the mean value Ϯ SEM (n ϭ 6). Two-way-ANOVA showed a significant difference of Ca 2ϩ uptake with time (P Ͻ 0.05) and Ca 2ϩ uptake of NO was significantly different when compared to VC (P Ͻ 0.001).