Osteoblast Proliferation or Differentiation Is Regulated by Relative Strengths of Opposing Signaling Pathways ANGELA RAUCCI, PAOLA BELLOSTA, ROBERTA GRASSI, CLAUDIO BASILICO, * AND ALKA MANSUKHANI * Department of Microbiology, New York University School of Medicine, New York, New York Skeletal development requires the correct balance of osteoblast proliferation, survival, and differentiation which is modulated by a network of signaling pathways and transcription factors. We have examined the role of the AKT (PKB), and ERK1/2 signaling pathways in the osteoblast response to FGFs, which inhibit differentiation, and to IGF-1 and Wnt signaling, which promote it. Using osteoblastic cell lines as well as primary calvarial osteoblasts, we show that ERK1/2 and AKT have distinct effects in FGF-induced osteoblast proliferation and differentiation. ERK1/2 is a primary mediator of FGF-induced proliferation, but also contributes to osteoblast differentiation, while AKT is important for osteoblast survival. Signaling by IGF-1, that promotes osteoblast differentiation, strongly activates AKT and weakly ERK1/2, while the opposite results are obtained with FGF, which inhibits differentiation. By introducing a constitutively active form of AKT, we found that increased AKT activity drives osteoblasts to differentiation. Increasing the AKT signal in osteoblasts that harbor FGFR2 activating mutations, found in Crouzon (342Y) and Apert (S22W) syndromes, is also able to drive differentiation in these cells, that normally fail to differentiate. Wnt signals, that promotes differentiation, also induce AKT phosphorylation, and cells expressing active AKT have increased levels of stabilized beta-catenin, a central molecule in Wnt signaling. Our results indicate that the relative strengths of ERK and AKT signaling pathways determine whether osteoblasts are driven into proliferation or differentiation, and that the effects of AKT may be due, in part, to synergy with the Wnt pathway as well as with the Runx2 transcription factor. J. Cell. Physiol. 215: 442–451, 2008. ß 2007 Wiley-Liss, Inc. The flat bones of the skull are formed by the process of intramembranous ossification during which mesenchymal cells condense directly to osteoprogenitors and undergo an orderly spatial and temporal pattern of differentiation into mature, bone forming osteoblasts. These cells secrete a matrix (osteoid), which is eventually mineralized to form the calvarial flat bones that are separated by flexible sutures (Morriss-Kay and Wilkie, 2005). Activating mutations in FGF receptors are the cause of several human autosomal dominant craniofacial disorders whose unifying feature is craniosynostosis, or premature fusion of the cranial sutures. In craniosynostosis disorders, the regulated progression of osteoblast proliferation, differentiation and apoptosis is perturbed. While it is clear that excessive or unregulated FGF signaling gives rise to defects in bone formation, the specific osteoblast response that causes the defect is not well defined (Marie et al., 2005; Ornitz, 2005). We and others have previously shown that FGF signaling has a different effect on mature and immature osteoblasts. Immature osteoblasts are induced to proliferate and the proliferative effect is lost as the cells differentiate (Debiais et al., 1998; Mansukhani et al., 2000). Osteoblasts in differentiating conditions continuously exposed to FGF undergo increased apoptosis and their differentiation is blocked. Similar results are produced by the constitutive expression of the craniosynostosis-linked mutations FGFR2/S252W (Apert) or FGFR2/C342Y (Crouzon) (Mansukhani et al., 2000). In this study we have examined the contribution of the signal transduction pathways ERK and AKT in the proliferation, differentiation and apoptosis of osteoblasts, and in the FGF response. The ERK MAP kinase signal transduction pathway is activated by growth factors and is associated with cell proliferation, differentiation and survival. The PI3-kinases are important signal transducers of responses to hormones and growth factors. AKT (PKB), is a downstream target of phosphphatidylinositol-3-kinase (PI3-K) and is a critical mediator of cell proliferation and survival (Datta et al., 1999; Brazil and Hemmings, 2001; Brazil et al., 2004). AKT can phosphorylate a variety of targets leading to activation or inhibition of their function. The anti-apoptotic effects of AKT activation are linked to its ability to inhibit pro-apoptotic proteases (caspase-9), inactivation of the Forkhead/FOXO transcription factors that activate several pro-apoptotic molecules, and by inhibiting the Bcl-2 family protein, BAD (Brazil and Hemmings, 2001; Tran et al., 2003). Furthermore, the AKT pathway is known to play an important role in bone development (Liu et al., 2007; Peng et al., 2003). Abbreviations: MAPK; mitogen-activated protein kinase; FBS; fetal bovine serum; BrdU; bromodeoxyuridine; PBS; phosphate-buffered saline; SD; standard deviation. Contract grant sponsor: NIAMS; Contract grant number: AR051358. Angela Raucci’s present address is San Raffaele Scientific Institute, DIBIT, Chromatin Dynamics Unit, via Olgettina 58, 20132 Milano, Italy. Paola Bellosta’s present address is Department of Biology, The City College, Convent Avenue at 138th Street, New York, NY 10031. *Correspondence to: Claudio Basilico or Alka Mansukhani, Department of Microbiology, New York University School of Medicine, New York, NY 10016. E-mail: basilc01@med.nyu.edu; mansua01@med.nyu.edu Received 21 May 2007; Accepted 11 September 2007 DOI: 10.1002/jcp.21323 ORIGINAL ARTICLE 442 Journal of Journal of Cellular Physiology Cellular Physiology ß 2007 WILEY-LISS, INC.