Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth Emilie Pacary, a Emmanuelle Tixier, a Florence Coulet, b Simon Roussel, a Edwige Petit, a and Myriam Bernaudin a, ⁎ a UMR-CNRS 6185, “Hypoxia and Cerebrovascular Physiopathology” Group, University of Caen, CYCERON, Bd Henri Becquerel, BP 5229, F-14074 Caen cedex, France b INSERM U 525, Faculté de Médecine Pitié-Salpêtrière, 91 Bd de l'hôpital, 75634 Paris, cedex 13, France Received 23 January 2007; revised 28 March 2007; accepted 4 April 2007 Available online 10 April 2007 This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl 2 ) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl 2 ± Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl 2 - induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl 2 decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK path- ways. Moreover, we demonstrate a synergistic effect of CoCl 2 and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons. © 2007 Elsevier Inc. All rights reserved. Keywords: Cell cycle; Neural differentiation; HIF-1; Mesenchymal stem cell; Stem cells; Neurosphere; PC12; Adult bone marrow stem cells; Hypoxia Introduction Adult stem cells, such as mesenchymal stem cells (MSC), may have the potential to acquire mature phenotypes that are different from their physiological fate (Grove et al., 2004). Indeed, in addition to their differentiation into osteoblasts, chondroblasts, adipocytes and hematopoiesis-supporting stromal cells (Pittenger et al., 1999), several in vitro studies demonstrated that MSC have also the ability to differentiate into neuron-like cells (Krabbe et al., 2005) with evidence of immature and mature neuronal functionalities (Hung et al., 2002; Jiang et al., 2003; Kohyama et al., 2001; Pacary et al., 2006; Tropel et al., 2006; Wislet-Gendebien et al., 2005). Despite these findings, MSC differentiation into neurons is still a highly controversial topic and sometimes attributed to an artifact (Bertani et al., 2005; Croft and Przyborski, 2006; Lu et al., 2004; Neuhuber et al., 2004). Anyhow, whatever their ability to differentiate into neurons or not, therapeutic benefit of these cells has been shown in experimental models of cerebral ischemia (Chen et al., 2001), trauma (Mahmood et al., 2001) and Parkinson's disease (Dezawa et al., 2004). Recently, MSC administration has been reported as a feasible and safe therapy in stroke patients (Bang et al., 2005). This clinical preliminary study is very promising but additional fundamental and pre-clinical investigations are required to further evaluate the potential use of MSC as a cellular therapy for stroke. Recent studies showed that hypoxia enhances neurogenesis in vitro and in vivo (Pourie et al., 2006; Studer et al., 2000). One of the hallmarks of the response to hypoxia is the activation of the transcriptional factor hypoxia-inducible factor-1 (HIF-1), which consists of two subunits HIF-1α (120 kDa) and HIF-1β (91– 94 kDa). In normoxia, prolyl-4-hydroxylases (PHDs) are activated and hydroxylate HIF-1α allowing the Von Hippel-Lindau protein (pVHL) to bind to the latter, leading to HIF-1 degradation by the proteasomal pathways. The PHDs contain a ferrous ion (Fe 2+ ) that can be bound by chelators like deferoxamine (DFX) and displaced by heavy metal ions like cobalt. A unique asparaginyl-hydroxylase, called FIH-1 (factor inhibiting HIF-1), contains a Fe 2+ ion and uses O 2 and 2-oxoglutarate as co-factors to hydroxylate asparagine 803 in the HIF-1α protein. This hydroxylation decreases binding of p300/CBP to HIF-1α and prevents the full transcriptional activation of HIF-1 target genes. Therefore, hypoxia, iron chelators www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 35 (2007) 409 – 423 ⁎ Corresponding author. Fax: +33 2 31 47 02 22. E-mail address: bernaudin@cyceron.fr (M. Bernaudin). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.04.002