Matemba Stephen F, Lie Anita, Ransjö Maria
Department of Odontology, Division of Oral Cell Biology, Umeå University, SE 901 87, Umeå, Sweden.
J Cell Biochem. 2006 Oct 1;99(2):528-37. doi: 10.1002/jcb.20866.
Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.
核因子κB受体激活剂配体（RANKL）在破骨细胞生成过程中至关重要，但破骨细胞分化所涉及的信号传导事件远未完全明确，其他信号可能在破骨细胞生成中发挥作用。缝隙连接细胞间通道提供了一种更直接的细胞间通讯途径，它允许相邻细胞交换第二信使、离子和细胞代谢产物。在此，我们研究了缝隙连接通讯在小鼠骨髓培养物破骨细胞生成中的作用。在骨髓基质细胞和成熟破骨细胞中检测到了缝隙连接蛋白连接蛋白43（Cx43）的免疫反应位点。通过刮擦加载荧光素黄染料转移技术证明，羧苄青霉素（CBX）在功能上阻断了缝隙连接通讯。CBX对甲状旁腺激素（PTH；10 nM）或福司可林（FSK；1 microM）刺激的7至8天骨髓培养物中形成的抗酒石酸酸性磷酸酶（TRAP）阳性多核细胞数量产生剂量依赖性抑制（显著≥90 microM）。此外，CBX（100 microM）显著抑制前列腺素E2（PGE2；10 microM）和1,25(OH)2-维生素D3刺激的小鼠骨髓培养物中的破骨细胞分化。因此，定量实时聚合酶链反应（PCR）分析表明，CBX下调了破骨细胞表型标志物的表达，但对RANK、RANKL和骨保护素（OPG）mRNA表达没有任何显著影响。然而，结果表明CBX显著抑制小鼠骨髓培养物中RANKL刺激（100 ng/ml）的破骨细胞生成。综上所述，我们的结果表明，信使分子的缝隙连接扩散与破骨细胞分化中RANKL下游的信号通路相互作用。需要进一步研究来确定其中的确切机制和涉及的分子靶点。
