Faculty of Medicine, Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
J Cell Biochem. 2010 May 15;110(2):343-51. doi: 10.1002/jcb.22542.
Large osteoclasts (10+ nuclei), predominant in rheumatoid arthritis and periodontal disease, have higher expression of proteases and activating receptors and also have increased resorptive activity when compared to small (2-5 nuclei) osteoclasts. We hypothesized that large and small osteoclasts activate different signaling pathways. A Signal Transduction Pathway Finder Array was used to compare gene expression of large and small osteoclasts in RAW 264.7-derived osteoclasts. Expression of vascular endothelial growth factor A (Vegfa) was higher in large osteoclasts and this result was confirmed by RT-PCR. RT-PCR further showed that RANKL treatment of RAW cells induced Vegfa expression in a time-dependent manner. Moreover, VEGF-A secretion in conditioned media was also increased in cultures with a higher proportion of large osteoclasts. To investigate the mechanism of Vegfa induction, specific inhibitors for the transcription factors NF-kappaB, AP-1, NFATc1, and HIF-1 were used. Dimethyl bisphenol A, the HIF-1alpha inhibitor, decreased Vegfa mRNA expression, whereas blocking NF-kappaB, AP-1, and NFATc1 had no effect. Furthermore, the NF-kappaB inhibitor gliotoxin inhibited Hif1alpha mRNA expression. In conclusion, VEGF-A gene and protein expression are elevated in large osteoclasts compared to small osteoclasts and this increase is regulated by HIF-1. In turn, Hif1alpha mRNA levels are induced by RANKL-mediated activation of NF-kappaB. These findings reveal further differences in signaling between large and small osteoclasts and thereby identify novel therapeutic targets for highly resorptive osteoclasts in inflammatory bone loss.
大的破骨细胞（有 10 个以上核），在类风湿关节炎和牙周病中更为常见，其蛋白酶和激活受体的表达更高，与小的（2-5 个核）破骨细胞相比，其吸收活性也增加。我们假设大、小破骨细胞激活不同的信号通路。我们使用信号转导通路寻找器阵列比较了 RAW 264.7 衍生的破骨细胞中大、小破骨细胞的基因表达。大的破骨细胞中血管内皮生长因子 A（Vegfa）的表达更高，这一结果通过 RT-PCR 得到了证实。RT-PCR 进一步表明，RANKL 处理 RAW 细胞以时间依赖性方式诱导 Vegfa 表达。此外，高比例大的破骨细胞培养物中条件培养基中 VEGF-A 的分泌也增加。为了研究 Vegfa 诱导的机制，我们使用了转录因子 NF-kappaB、AP-1、NFATc1 和 HIF-1 的特异性抑制剂。HIF-1alpha 抑制剂二甲双酚 A 降低了 Vegfa mRNA 表达，而阻断 NF-kappaB、AP-1 和 NFATc1 则没有影响。此外，NF-kappaB 抑制剂Gliotoxin 抑制了 Hif1alpha mRNA 表达。总之，与小的破骨细胞相比，大的破骨细胞中 VEGF-A 基因和蛋白表达升高，这种增加受 HIF-1 调节。反过来，RANKL 介导的 NF-kappaB 激活诱导了 Hif1alpha mRNA 水平。这些发现揭示了大、小破骨细胞之间信号转导的进一步差异，并确定了炎症性骨丢失中高吸收性破骨细胞的新治疗靶点。
