Biochemical and Biophysical Research Communications 377 (2008) 400–406 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.145 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc In addition to a well known effect on angiogenesis, recent data have demonstrated that low oxygen tension favours neurogenesis. Indeed, hypoxia enhances proliferation, survival, and dopaminer- gic differentiation of neuronal precursors in vitro [1]. In addition, mild transient hypoxia in the newborn rat was shown to trigger substantial neurogenesis [2]. Oxygen deprivation initiates a wide range of adaptative responses and this process is largely controlled by the transcrip- tional factor hypoxia-inducible factor-1 (HIF-1), which consists of two subunits HIF-1a and HIF-1b. Whereas HIF-1b does not respond to changes in oxygen tension, HIF-1a is rapidly degraded in normoxia. Indeed, in the presence of O 2 , Fe 2+ and 2-oxoglutarate, HIF-1a is hydroxylated on prolines by prolyl-4-hydroxylases (PHs) which allows the binding of von Hippel–Lindau protein (pVHL) and the subsequent HIF-1a degradation through the ubiquitin–protea- some pathway. An asparaginyl-hydroxylase FIH-1 (Factor inhib- iting HIF-1), requires also oxygen, iron and the co-factor 2-oxo- glutarate to hydroxylate an asparagine on HIF-1a protein. This hydroxylation, under normoxic conditions, prevents the transcrip- tional activation of HIF-1 target genes. Therefore, hypoxia by inacti- vating PHs and FIH-1 stabilizes HIF-1a and allows the activation of hypoxia responsive elements (HREs) in HIF-1 target genes. As PHs and FIH require iron to be active, HIF-1 stabilization and activation can also be obtained with iron chelators (such as desferroxamine, DFX) and heavy metal ions (such as cobalt) [3]. More recently, it has been shown that HIF-1 activation can be induced also in vitro with PH inhibitors such as 2-oxoglutarate mimetics [4]. HIF-1a may be an essential mediator in the hypoxia-induced neurogenic effect since HIF-1a has been shown to be essential for normal brain development [5] and for the production of dopami- nergic neurons [6]. In addition, EPO (erythropoietin) and VEGF (vascular endothelial growth factor), two well known HIF-1 tar- gets, enhance neurogenesis both in vitro and in vivo [7]. In this context, we hypothesized that pharmacological acti- vation of HIF-1 could promote neuronal differentiation. To test this hypothesis, we previously studied the effect of two HIF-1- activation mimicking agents, cobalt chloride (CoCl 2 ) and DFX, on neuronal differentiation of bone-marrow-derived mesenchymal stem cells (MSC) [8,9]. Our results indicated that HIF-1 activation, provoked by CoCl 2 or DFX, induced morphological and expres- sion changes of MSC in accordance with neuronal differentiation [8,9]. Interestingly, both treatments were potentiated by Y-27632, an inhibitor of ROCK which is known to play a role in neuronal development [10]. Our hypothesis was further validated by our data showing that CoCl 2 /Y-27632 co-treatment promoted also neuronal differentiation of neurospheres and PC12 cells [9]. Here, we used a PH inhibitor, FG-0041, which is a small molecule that has some structural features with 2-oxoglutar- Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage Emilie Pacary * , Edwige Petit, Myriam Bernaudin CERVOxy team “Hypoxia and cerebrovascular pathophysiology”, UMR CI-NAPS 6232 Université de Caen Basse-Normandie, Université Paris-Descartes, CEA, CNRS, Centre CYCERON, Bd Henri Becquerel, BP5229, 14074 CAEN cedex, France article info abstract Article history: Received 15 September 2008 Available online 09 October 2008 This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, Red_Close]is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells.een_Open FG-0041/Y-27632 Green_Close][Red_OpenFG- 0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation. © 2008 Elsevier Inc. All rights reserved. Keywords: Mesenchymal stem cell Prolyl hydroxylase Neural differentiation PC12 HIF-1 * Corresponding author. Address: The National Institute for Medical Research, Division of Molecular Neurobiology, Mill Hill, London NW7 1AA, UK. Fax: +44 208 816 2109. E-mail address: emiliepacary@hotmail.com (E. Pacary).