Introduction presence of NGF, PC12 cells undergo mitotic arrest and

Brain Research 885 2000 220–230 www.elsevier.com locate bres Research report Manganese induces neurite outgrowth in PC12 cells via upregulation of a integrins v a a a a b , Pamela Lein , Patrick J. Gallagher , Jeffrey Amodeo , Heather Howie , Jerome A. Roth a Department of Biology , Canisius College, Buffalo, NY 14208, USA b Department of Pharmacology and Toxicology , State University of New York, Buffalo, NY 14214, USA Accepted 12 September 2000 Abstract Previous studies have demonstrated that the divalent cation manganese Mn causes PC12 cells to form neurites in the absence of NGF. Since divalent cations modulate the binding affinity and specificity of integrins, and integrin function affects neurite outgrowth, we tested the hypothesis that Mn induces neurite outgrowth through an integrin-dependent signaling pathway. Our studies support this hypothesis. Function-blocking antisera specific for b integrins block the neurite-promoting activity of Mn by 90–95. Bioassays and biochemical 1 studies with antisera specific for the a , a , or a integrin subunit suggest that the a b heterodimer is one of the principal b integrins v 5 8 v 1 1 mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express a at levels detectable by immunoprecipitation or immunocytochemistry. SDS–PAGE analysis of v biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells, as well as confocal laser microscopy of PC12 immunostained for surface a indicate that Mn increases the surface expression of a integrins. This increase appears to be due in part to v v synthesis of a since specific inhibitors of RNA and protein synthesis block the neurite-promoting activity of Mn. These data indicate that v Mn induces neurite outgrowth in PC12 cells by upregulating a integrins, suggesting that Mn potentially represents an additional v mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.  2000 Elsevier Science B.V. All rights reserved. Theme : Development and regeneration Topic : Process outgrowth, growth cones, and sprouting Keywords : PC12 cell; Manganese; Integrin; Neurite outgrowth; Vitronectin receptor

1. Introduction presence of NGF, PC12 cells undergo mitotic arrest and

differentiate morphologically and biochemically into cells Precise regulation of the rate and direction of neurite that display many properties of differentiated sympathetic outgrowth is essential to the development of functional neurons, including an extensive network of neuronal-like neural circuits both during development and regeneration projections [16]. The mechanisms by which NGF induce [10,11,28]; thus, considerable efforts are being made to neuronal differentiation in these cells involves binding of identify and characterize the mechanisms that control this NGF to trkA receptors [37] which results in sequential process. Rat pheochromocytoma cells PC12 have served activation of ras [27,53], MEK kinase raf [61] and MEK, as a useful model system for this purpose [16]. In the with eventual phosphorylation of the MAP kinases ERK1 and 2 p44 42 [43]. Previous studies in our laboratory [36] have revealed that Mn can similarly induce neurite outgrowth and upregulate the neuronal marker proteins, Corresponding author. Department of Pharmacology and Toxicology, peripherin [2] and GAP-43 [46] in PC12 cells grown in the 102 Farber Hall, State University of New York, Buffalo School of absence of NGF. We have also recently demonstrated that, Medicine, Buffalo, NY 14214, USA. Tel.: 11-716-829-3236; fax: 11- as is true for NGF, Mn-induced neurite outgrowth requires 716-829-2801. E-mail address : jarothbuffalo.edu J.A. Roth. activation of ERK1 and ERK2 [60]. However, there are 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 4 3 - 7 P . Lein et al. Brain Research 885 2000 220 –230 221 also significant differences in the response patterns of concentrations may represent a mechanism for controlling PC12 cells to Mn and NGF. Mn elicits a much more rapid the rate and direction of neurite outgrowth. outgrowth of neurites than NGF hours versus days, respectively and, unlike NGF, Mn does not support neuronal survival [36]. Such observations suggest that

2. Materials and methods