Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells

Mn2+ has been shown to promote cell-substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes. These morphological effects of Mn2+ were both time- and dose-dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn(2+)-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place. Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+. Although Mn(2+)-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth-promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn2+ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells. In cells grown in serum-free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata. Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells. These findings are consistent with a hypothesis that Mn(2+)-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects.