The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade

Abstract

Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP₄) and InsP₅, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

Fig. 1

PLCγ1 mediates the basal and Cx43-dependent potentiation of Runx2 activity by FGF2. Luciferase reporter assays were performed on cells co-transfected with a Runx2-reponsive luciferase reporter (p6xOSE2-Luc) and pSFFV-neo empty vector (grey bars) or pSFFV-Cx43 expression vector (black bars) and treated with vehicle (Veh) or FGF2 (10ng/ml, 4h). Cells were treated with the PLC inhibitor (A) U73122 (2μM), (B) ET-18-OCH3 or (C) transfected with the dominant negative PLCγ1 Y783F or PKCδ K376R constructs. (D) Quantitative real time RT-PCR amplification of PLCγ1 and PLCγ2 from MC3T3 osteoblast RNA. (E) Western blot of whole cell extracts from FGF2 treated cells probed with anti-phospho-PLCγ1 (Tyr783) antibodies. Anti-GAPDH antibodies were used as a load control. (F) Luciferase reporter assays (p6xOSE2-Luc reporter) and western blot (anti-PLCγ1 or anti-GAPDH antibodies) of cells transfected with scrambled siRNA (SCR, 50nM) or PLCγ1 siRNA (PLCG1, 50nM). (G) Luciferase reporter assays (p6xOSE2-Luc or p6xmutOSE2-Luc reporters) of cells co-transfected with pSFFV-neo empty vector (grey bars) or pSFFV-Cx43 expression vector (black bars) along with a wild type PLCγ1 or empty vector construct and treated with vehicle (Veh) or FGF2 (10ng/ml, 4h), as above. For all luciferase assay graphs, cells transfected with pSFFV-neo empty vector are shown with grey bars, cells transfected with pSFFV-Cx43 expression vector are shown with black bars. Graphs depict mean + s.d. for a representative experiment performed in triplicate wells, n ≥ 3. For real time PCR, * p-value < 0.05. For luciferase reporter assays, * p-value < 0.05 relative to the corresponding FGF2-treated control.

Fig. 2

Contribution of DAG lipase or InsP₃ Receptors to the basal or Cx43-potentiated FGF2 response of a Runx2 reporter. Luciferase reporter assays were performed on cells co-transfected with p6xOSE2-Luc and pSFFV-neo empty vector (grey bars) or pSFFV-Cx43 expression vector (black bars) and treated with vehicle (Veh) or FGF2 (10ng/ml, 4h) in the presence or absence of (A) the DAG lipase inhibitor RHC-80267 (20μM) or (B) the InsP₃ Receptor inhibitor 2-APB (50μM). Graphs depict means + s.d. for a representative experiment performed in triplicate wells, n = 3. * p-value < 0.05 relative to the corresponding FGF2-treated control.

Fig. 3

Involvement of higher order inositol polyphosphates in the Cx43-dependent potentiation of Runx2 activity. (A) A schematic of the pathway by which PLC activity can yield higher order inositol polyphosphates and inositol pyrophosphates. (B) Luciferase reporter assays were performed on cells co-transfected with p6xOSE2-Luc and pSFFV-neo empty vector (grey bars) or pSFFV-Cx43 expression vector (black bars) and scrambled siRNA (SCR, 50nM) or IPMK siRNA (IPMK, 50nM) and then treated with vehicle (Veh) or FGF2 (10ng/ml, 4h). Inset, western blot of whole cell extracts from cells transfected with scrambled or IPMK targeted siRNA probed with anti-IPMK and anti-GAPDH antibodies. (C) Luciferase reporter assays were performed as above in cells treated with vehicle (Veh) or FGF2 (10ng/ml, 4h) in the presence or absence of the IP6K inhibitor TNP (5μM). (D) Quantitative real time RT-PCR of IP6K1, IP6K2 and IP6K3 in confluent cultures of MC3T3 cells. (E) Luciferase reporter assays were performed as above in cells treated with 50nM scrambled siRNA, IP6K1 siRNA or IP6K2 siRNA. Quantitative real time RT-PCR (IP6K1 and IP6K2 primers, respectively) of cells transfected with 50nM scrambled siRNA, IP6K1 siRNA or IP6K2 siRNA. For luciferase assay graphs, cells transfected with pSFFV-neo empty vector are shown with grey bars, cells transfected with pSFFV-Cx43 expression vector are shown with black bars. (F) Luciferase reporter assays were performed on cells co-transfected with p6xOSE2-Luc and pSFFV-neo empty vector (grey bars) or pSFFV-Cx43 expression vector (black bars) along with a IP6K1 expression vector, IP6K2 expression vector or an empty vector and treated with vehicle (Veh) or FGF2 (10ng/ml, 4h), as above. For all luciferase assay graphs, cells transfected with pSFFV-neo empty vector are shown with grey bars, cells transfected with pSFFV-Cx43 expression vector are shown with black bars. Graphs depict means + s.d. for a representative experiment performed in triplicate wells, n = 3. For real time PCR, * p-value < 0.05. For luciferase reporter assays, * p-value < 0.05 relative to the corresponding FGF2-treated control.

Fig. 4

The Nuclear translocation of PKCδ and the physical interaction of Runx2 and PKCδ require IP6K1. (A) An immunoprecipitation was performed on whole cell extracts from cells treated with 10ng/ml FGF2 for 0, 5 or 30 minutes with anti-total PKCδ, anti-phospho-PKCδ (Thr505) or anti-phospho-PKCδ (Ser643) antibodies. Subsequently, western blots of the bead fractions from these immunoprecipitation were probed with anti-Runx2 antibodies, n=2. (B) An immunoprecipitation with anti-total PKCδ antibodies was performed on whole cell extracts from cells treated with 10ng/ml FGF2 for 0 or 30 minutes in the presence or absence of 5μM TNP. The western blots were then probed with anti-Runx2 antibodies. Top, the eluted bead fraction from the immunoprecipitation; bottom, 1/10^th of the input fraction. (C) Western blots of nuclear extracts from cells transfected with 50nM SCR siRNA, IP6K1 siRNA or IP6K2 siRNA and treated with 10ng/ml FGF2 for 0, 5 or 30 minutes. Blots were probed with anti-phospho-PKCδ (Thr505) or Lamin A/C antibodies (load control).

Fig. 5

Inhibition of IP6Ks reduces the expression of Runx2-regulated osteoblast genes. MC3T3 cells were treated with vehicle (DMSO) or 5μM TNP for 4 hours, prior to isolation of RNA for quantitative real time RT-PCR for collagen Iα1 (Col1a1), osteocalcin, Osterix or Runx2. * p-value < 0.05 relative to the corresponding Veh-treated control, n = 3.

Fig. 6

Model of Cx43 potentiated Runx2 activity via an inositol pyrophosphate second messenger following FGF2 stimulation. Upon binding to its receptor (FGFR) in one cell, FGF2 activates PLCγ1, generating DAG and InsP₃ (IP3). Subsequently, the activity of IPMK and IP6K1 leads to the production of inositol polyphosphates and pyrophosphates, such as InsP₆ (IP6) and InsP₇ (IP7). The InsPs activate PKCδ, which translocates to the nucleus where it interacts with Runx2, increasing its transcriptional activity and driving the expression of osteoblast genes. In addition, we speculate that these small, water soluble second messengers may be communicated to adjacent cells via Cx43 containing gap junctions. In the coupled cell, PKCδ, which is locally recruited to the Cx43-containing gap junction channel, can re-initiate signaling in this cell, independent of direct stimulation by FGF2, resulting in a potentiation of the response among coupled cells.