Connexin43 potentiates osteoblast responsiveness to fibroblast growth factor 2 via a protein kinase C-delta/Runx2-dependent mechanism

Abstract

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 (PKCdelta) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCdelta translocates to the nucleus, PKCdelta 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 PKCdelta and Runx2 function.

Figure 1.

Synergistic induction of osteocalcin transcription by FGF2 and Cx43. (A) MC3T3 osteoblasts were transiently transfected with the −199OCN LUC reporter plasmid, along with either empty pcDNA vector or with pSFFV-Cx43 expression vector. After serum starvation, the cells were treated for 16 h with the indicate concentration of FGF2 or with vehicle. The samples were then analyzed for luciferase activity. Statistically significant difference relative to the respective paired vehicle-treated control for a, pcDNA or b, Cx43; p < 0.05; c indicates a more than additive (synergistic) response to the combined treatment with FGF2 and Cx43 overexpression. (B) Western blots of whole cell extracts from MC3T3 osteoblasts transiently transfected with pcDNA or pSFFV-Cx43 were probed with anti-Cx43 or anti-GAPDH (load control) antibodies.

Figure 2.

FGF2-Cx43-induced synergism occurs via a Runx2 binding cognate. MC3T3 cells were transiently transfected with (A) −199OCN LUC reporter, (B) OCN CxRE LUC reporter, (C) a p6xOSE2 LUC reporter or (D) a p6xOSE2mut LUC reporter. The cells were cotransfected with expression vector and treated with vehicle or FGF2 (5 ng/ml) as indicated in Figure 1. Statistically significant difference, *p < 0.05; #indicates a more than additive response to the combined treatment with FGF2 and Cx43 overexpression.

Figure 3.

Cx45 overexpression can attenuate the FGF2-Cx43 synergistic activation of osteocalcin transcription. MC3T3 osteoblasts were transiently transfected with (A) −199OCN LUC reporter, (B) a p6xOSE2 LUC reporter, or (C) a p6xOSE2mut LUC reporter. The cells were cotransfected with expression vectors as follows: empty pcDNA, Cx43, Cx45, or both Cx43 and Cx45. The total amount of expression plasmid in each reaction was maintained constant via the addition of empty pcDNA vector. Statistically significant difference, *p < 0.05; #indicates a more than additive response to the combined treatment with FGF2 and Cx43 overexpression.

Figure 4.

Knockdown of Cx43 attenuates the FGF2 responsiveness of the osteocalcin promoter. Transient transfection with siRNA directed against the Cx43 mRNA (Cx43 siRNA) attenuates the FGF2 response of the −199OCN LUC reporter when compared with cells transiently transfected with a nonspecific, scrambled siRNA (SCR siRNA). Inset, a Western blot of whole cell extracts from siRNA-transfected cells probed with anti-Cx43 antibodies. GAPDH antibodies were used as a loading control.

Figure 5.

Modulation of dye coupling by alteration of Cx43 expression or function. MC3T3 osteoblasts were transiently transfected with pcDNA, pSFFV-Cx43, pcDNA-Cx45, or Cx43 siRNA. Forty-eight hours after transfection, gap junctional communication was assessed by the scrape-loading, dye-coupling assay. Top, representative fluorescence images are shown for the Lucifer yellow (left), rhodamine dextran (middle), or merged images (right) after transmission of the Lucifer yellow among transfected cells. Scale bar, 50 μm. Bottom, quantitation of the average distance of transmission of Lucifer yellow from the cells proximal to the scrape line to the most distally Lucifer yellow–positive cell. Data are set relative to the pcDNA-transfected controls. Statistically significant difference from the pcDNA control; *p < 0.05.

Figure 6.

Inhibition of PKCδ reduces Cx43-dependent activation of osteocalcin transcription by FGF2. MC3T3 osteoblasts were transiently transfected with (A) a −199OCN LUC reporter or (B) a p6xOSE2 LUC reporter constructs. In addition, the cells were cotransfected with pcDNA or Cx43 expression constructs. Before treatment with FGF2 (5 ng/ml), the cells were pretreated with 5 μM rottlerin. (C) Cells were transiently cotransfected with p6xOSE2 LUC, pcDNA, or Cx43 expression constructs and a scrambled siRNA expression construct (SCR siRNA) or siRNA construct to knockdown PKCδ expression construct (PRKCD15 siRNA), as indicated. (D) Immunoblot for PKCδ showing the efficacy of the siRNA construct for PKCδ. SCR siRNA represents a scrambled siRNA construct. Cx43 66 siRNA is a construct designed to knock down Cx43 expression and is used as a negative control in this experiment. GAPDH antibodies were used as a loading control. Statistically significant difference, *p < 0.05; #indicates a more than additive response to the combined treatment with FGF2 and Cx43 overexpression.

Figure 7.

Activation of PKCδ by Cx43 and FGF2 synergistically affects Runx2 phosphorylation and DNA-binding activity. (A) Immunoblots of whole cell extracts from MC3T3 cells treated with FGF2 for the indicated number of minutes probed with phospho-PKCδ or total PKCδ antibodies. (B) Immunoblots of whole cell extracts from MC3T3 pcDNA- or Cx43-transfected cells treated with FGF2 for 16 h. The blots were probed with anti-phospho-PKCδ or anti-Runx2 antibodies. Anti-GAPDH antibodies are used as a loading control. (C) Whole cell extracts from pcDNA- or Cx43-transfected cells treated with either vehicle or FGF2 were immunoprecipitated with anti-Runx2 antibodies and subsequently immunoblotted using anti-phospho-serine antibodies. (D) ChIP was performed on cells that were transfected with pcDNA or Cx43 and treated with vehicle or FGF2. The immunoprecipitated material was analyzed by real-time PCR for the presence of the proximal osteocalcin promoter region, using a specific primer set. Data are shown as the abundance of PCR product associated with the bead fraction of the ChIP as a ratio of input DNA and represents the compiled data obtained from three separate experiments. The ChIP data are normalized to the pcDNA vehicle control. (E) A representative agarose gel of the ChIPs showing the PCR product obtained from the immunoprecipitated nucleic acid pulled down with anti-Runx2 antibodies after treatment with or without FGF2 in transfected cells. The same PCR primers to the osteocalcin proximal promoter were used in this experiment. Statistically significant difference, *p < 0.05.

Figure 8.

Treatment with FGF2 causes PKCδ, but not PKCε, to translocate to the nucleus. Immunofluorescence of MC3T3 osteoblasts treated with vehicle (A or C) or 5 ng/ml FGF2 (B or D) for 16 h. PKCδ (A and B) or PKCε (C and D) are shown in green. Nuclear staining by DAPI is shown in blue. The bottom panels are merged images. Arrows indicate robust detection of PKCδ in the nucleus. White scale bar, 10 μm.

Figure 9.

Alteration of Cx43 expression or function alters the nuclear accumulation of phospho-PKCδ in response to FGF2 treatment. Cells were transiently transfected with pcDNA, pSFFV-Cx43, pcDNA-Cx45, or Cx43 siRNA. The transfected cells were subsequently fixed and stained with anti-phospho-PKCδ antibodies, after 30-min treatment with vehicle or FGF2 (5 ng/ml). Phospho-PKCδ staining is shown in green. Nuclear staining by DAPI is shown in blue. Vehicle-treated cells under all conditions show diffuse punctate nuclear staining (small arrows). On treatment with FGF2, the pattern of nuclear staining in some, but not all, cells is altered (large arrows). White scale bar, 10 μm.