Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein Marco Pedrazzi . , Monica Averna * . , Bianca Sparatore , Mauro Patrone , Franca Salamino , Manuela Marcoli , Guido Maura , Chiara Cervetto , Daniela Frattaroli , Sandro Pontremoli , Edon Melloni , 1 Department of Experimental Medicine (DIMES) University of Genova, Genova, Italy, 2 Centre of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy, 3 Dipartimento di Scienze e Innovazione Tecnologica, DiSIT, University of Piemonte Orientale ‘‘Amedeo Avogadro’’, Alessandria, Italy Abstract Background: Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca 2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. Principal Findings: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca 2+ -dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1 (130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. Conclusion: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1. Citation: Pedrazzi M, Averna M, Sparatore B, Patrone M, Salamino F, et al. (2012) Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein. PLoS ONE 7(8): e44518. doi:10.1371/journal.pone.0044518 Editor: Christopher Mark Norris, Univ. Kentucky, United States of America Received June 18, 2012; Accepted August 8, 2012; Published August 31, 2012 Copyright: ß 2012 Pedrazzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Italian Ministry of Education, University and Research (PRIN 2008), the University ‘‘A. Avogadro’’ and the University of Genoa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: monica.averna@unige.it . These authors contributed equally to this work. Introduction The different effects exerted by extracellular HMGB1 on specific target cells have been related to the ability of this protein to interact with alternative cell surface receptors, such as the receptor for advanced glycation end products (RAGE) and the toll- like receptors (TLRs) 2 and 4 [1,2]. However, a number of observations indicate that HMGB1 can also act as a co-stimulating accessory protein without association with these receptors [3–5]. In this respect, we have demonstrated previously that HMGB1 administered to erythroleukemia cells, in the presence of hexamethylenebisacetamide, or to epidermoid carcinoma cells, in the presence of epidermal growth factor, induced an increased cell calcium influx leading to activation of erythroid differentiation or cell motility, respectively [6,7]. Although we excluded the involvement of RAGE in these cell responses to HMGB1, the receptor responsible for the elevation of [Ca 2+ ] i promoted by HMGB1 was not identified [8]. Recently, it has been demon- strated that HMGB1-TLR4 signaling activates the Ca 2+ conduc- tance of the glutamate gated NMDAR contributing to acute and chronic seizures in mouse models [9]. Glutamate is a major neuromediator of the CNS and its effect on the influx of Ca 2+ through NMDAR regulates enzyme activities and protein trafficking required for neuronal development and synaptic plasticity [10]. NMDAR is also a crucial effector in acute and chronic neurological diseases through glutamate excitotoxicity caused by intracellular Ca 2+ overloading [10,11]. Moreover, NMDAR has been shown to operate outside the CNS in bone, heart, lung, skin, and several endocrine and hematopoietic cells [12,13]. On the basis of these observations, here we have explored PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e44518 1,2 1,2 1,2 1,2 3 1,2 1,2 1 1 1 12