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Vol. 20, Issue 11, 2697-2708, June 1, 2009

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Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD 21201

Submitted October 29, 2008; Revised March 20, 2009; Accepted March 24, 2009
Monitoring Editor: Asma Nusrat

In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKC) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKC translocates to the nucleus, PKC and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKC and Runx2 function.

Address correspondence to: Joseph P. Stains (jstains{at}umoa.umm.edu)

Abbreviations used: ChIP, chromatin immunoprecipitation; DAG, diacylglycerol; Cx43 and Cx45, connexin43 and 45, respectively; FGF2, fibroblast growth factor 2; FGFR, fibroblast growth factor receptor; IOD, integrated optical density; PKC, protein kinase C delta.