Oxygen-Induced Transcriptional Dynamics in Human Osteoblasts Are Most Prominent at the Onset of Mineralization CLAUDIA NICOLAIJE, JEROEN VAN DE PEPPEL, AND JOHANNES P.T.M. VAN LEEUWEN * Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Oxygen tension plays an important role in the regulation of cellular processes. During hematopoietic stem cell (HSC) differentiation, HSCs migrate from one stem cell niche to the next, each with a different oxygen tension that determines which signaling pathways are on and off, determining the differentiation stage of the cell. Oxygen tension influences osteoblast differentiation and mineralization. Low oxygen levels inhibit matrix formation and mineralization. We were interested in the regulatory mechanisms that underlie this inhibition and wondered whether a switch in oxygen tension could have varying effects depending on the differentiation phase of the osteoblasts. We performed an oxygen tension switch phase study in which we switched osteoblasts from high to low oxygen tension during their 3 week differentiation and mineralization process. We performed microarray expression profiling on samples collected during this 3 week period and analyzed biochemical and histo-chemical endpoint parameters to determine the effect of a switch in oxygen levels on mineralization. We found that low oxygen tension has the most profound impact on mineralization when administered during the period of matrix maturation. Additionally, a large set of genes was regulated by oxygen, independent of the differentiation phase. These genes were involved in cell metabolisms and matrix formation. Our study demonstrates that variation in oxygen tension strongly affects gene expression in differentiating osteoblasts. The magnitude of this change for either expression levels or the number of regulated probes, depends on the osteoblast differentiation stage, with the phase prior to the onset of mineralization being most sensitive. J. Cell. Physiol. 228: 1863–1872, 2013. ß 2013 Wiley Periodicals, Inc. Oxygen is an important regulatory factor in numerous cellular processes, from stem cell differentiation to pancreatic development and cardiomyogenesis (Fraker et al., 2009; Mazumdar et al., 2010; van Oorschot et al., 2011). In the bone marrow environment, oxygen tension plays an important role regulating hematopoietic (HSC) and mesenchymal stem cell (MSC) differentiation (Eliasson and Jonsson, 2010; Raheja et al., 2010). The oxygen level in the bone marrow and its surrounding bone tissue ranges somewhere between 1% and 10% under normal in vivo conditions and can be as low as 0.1% at fracture sites (Chow et al., 2001; Harrison et al., 2002). MSC and osteoblast differentiation has been extensively studied in cell culture experiments performed under traditional culture conditions, using 20% O 2 . More recently performed studies show that hypoxic culture conditions cause a delay in osteoblast differentiation and mineralization, leaving the cells in a more stem-like state (Salim et al., 2004; D’Ippolito et al., 2006; Utting et al., 2006; Fehrer et al., 2007). In addition, hypoxic conditions are often found at the side of fractures and inside the callus during repair (Epari et al., 2008; Lu et al., 2008). Here they play an important role in repair, since a hypoxic state leads to the activation of VEGF expression, initiating essential vessel formation in the affected region (Steinbrech et al., 1999), which in the end leads to re-oxygenation. Prolonged hypoxia at fracture sites leads to a decrease in fracture healing rates (Heppenstall et al., 1976). More recently it has also been shown that hypoxia responsive MSCs have a greater differentiation potential than unresponsive MSCs (Nagano et al., 2010). This indicates that hypoxia can be used to improve fracture repair rates and bone healing. More detailed knowledge about the regulatory processes underlying these hypoxic conditions, which potentially mimic in vivo conditions more closely, will provide novel insights into the processes that regulate MSC and osteoblast differentiation and the role of oxygen in those processes. This knowledge will be of invaluable use in the field of fracture healing and tissue engineering, but also provides us with new insights and possibilities when studying bone marrow stem cells niches and MSC-HSC interaction. In a previous study, we used our human pre-osteoblast SV-HFO model to study osteoblast differentiation under low oxygen tension and concluded that low oxygen tension decreases matrix mineralization by inhibiting osteoblast differentiation at an early stage (Nicolaije et al., 2011). SV-HFO cells undergo a 3 week differentiation and mineralization process that can be divided into three phases; a differentiation phase (days 0–5; Phase 1), the matrix maturation phase in which matrix is produced (days 5–12; Phase 2) and the mineralization phase that results in a mineralized matrix (days 12–19; Phase 3). We hypothesized that the impact of oxygen tension on osteoblast differentiation is dependent on the stage of osteoblast differentiation. Following this hypothesis, the aim of the current study was to investigate the impact of oxygen tension on gene expression against the backdrop of osteoblast differentiation. In order to study this, we performed oxygen switch experiments during the three phases of osteoblast differentiation. We collected data from cells that were continuously cultured under 20% (high) or 2% (low) oxygen to determine long term effects, as well as data from cells that were Additional supporting information may be found in the online version of this article. *Correspondence to: Johannes P.T.M. van Leeuwen, Erasmus MC, Department of Internal Medicine, Room Ee585d, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: j.vanleeuwen@erasmusmc.nl Manuscript Received: 19 September 2012 Manuscript Accepted: 6 February 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 4 March 2013. DOI: 10.1002/jcp.24348 ORIGINAL RESEARCH ARTICLE 1863 Journal of Journal of Cellular Physiology Cellular Physiology ß 2013 WILEY PERIODICALS, INC.