Department of Orthopaedics, University of Maryland, School of Medicine, Baltimore, MD, USA.
J Bone Miner Res. 2013 Jun;28(6):1468-77. doi: 10.1002/jbmr.1867.
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.
间隙连接蛋白 43（Cx43）在成骨细胞功能和骨量积累中起着至关重要的作用，但 Cx43 缝隙连接传递的第二信使的身份、这些第二信使的靶标以及它们如何调节成骨细胞功能在很大程度上仍然未知。我们已经表明，成骨细胞中 Cx43 表达的改变可以通过调节 runt 相关转录因子 2（Runx2）的转录活性来影响对成纤维细胞生长因子 2（FGF2）的反应性。在这项研究中，我们研究了磷脂酶 Cγ1/肌醇多磷酸盐/蛋白激酶 C δ（PKCδ）级联在 Cx43 依赖性 MC3T3 成骨细胞对 FGF2 的转录反应中的贡献。磷脂酶 Cγ1、肌醇多磷酸激酶，它生成肌醇 1,3,4,5-四磷酸（InsP₄）和 InsP₅，以及肌醇六磷酸激酶 1/2 的表达敲低和/或功能抑制，生成肌醇焦磷酸盐，阻止了 Cx43 通过 Runx2 增强 FGF2 诱导信号的能力。相反，过表达磷脂酶 Cγ1 和肌醇六磷酸激酶 1/2 增强了 FGF2 对 Runx2 的激活作用，以及 Cx43 过表达对这种反应的影响。这些途径的破坏阻止了 PKCδ 的核积累和 PKCδ 与 Runx2 的 FGF2 依赖性相互作用，从而降低了 Runx2 的转录活性。这些数据表明，FGF2 信号涉及肌醇多磷酸级联，包括肌醇六磷酸激酶（IP6K），并证明 IP6K 调节 Runx2 和成骨细胞基因表达。此外，这些数据表明，水溶性肌醇多磷酸盐是 Cx43 依赖性扩增成骨细胞对 FGF2 反应的介质，并表明这些低分子量第二信使可能是通过 Cx43 间隙连接传递的成骨细胞功能的生物学相关介质。
