Molecular mechanism of osteogenesis-induced osteogenesis

我们最近证实了机械应力(MS)在体外诱导成骨细胞分化和随后的成骨形成，并提示BMP-4可能在这一过程中发挥自分泌/旁分泌因子的作用。我们还发现了100多个被阿尔法女士上调或下调的基因--Adaptin C就是这样一个基因，已知作为包被的小凹/囊泡的成分之一，在内吞作用中发挥作用。原位杂交和免疫组织化学研究表明，在MS负荷作用下，α-Adaptin C基因和蛋白的表达早在3h就已被诱导。这种表达与BMP-4的表达重叠，表明这些细胞最终会分化为成骨细胞。电子显微镜显示，在MS下，细胞对α-Adaptin C呈强阳性的区域，包被的凹坑/囊泡增加。此外，钾耗竭，一种已知的阻断内吞的操作，抑制了MS诱导的成骨细胞分化。这些数据强烈暗示着…更多的是，α-Adaptin C通过刺激EGF受体等分子的内吞作用参与MS诱导的成骨细胞分化。为了进一步研究MS诱导的成骨相关基因，我们还比较了成骨细胞和牙周膜(PDL)细胞的基因表达。我们的工作假设是PDL细胞可能配备了一种抑制钙化的机制，因为PDL在体内永远不会钙化，尽管存在强烈的间歇性多发性硬化症，尽管有报道称其具有成骨潜力。事实上，小鼠细胞系PDL-L2(我们最近建立的)在体外不会在没有超生理性数量的BMP-2的情况下产生矿化结节。我们发现，MSX2基因是阻止PDL-L2细胞向成骨细胞转化的关键调控因子，它是PDL-L2中比成骨细胞丰富得多的基因之一。我们还证明，在OPLL患者的受累韧带中，MSX2的表达受到抑制，这是一种症状依赖的方式。我们进一步证明，MSX2通过直接与Runx2结合和募集HDAC1复合体来抑制Runx2的作用，从而阻止PDL-L2细胞矿化。我们还证明了MS在体外不能矿化PDL-L2细胞，而是促进了矿化。成骨细胞。较少

We have recently demonstrated mechanical stress (MS)-induced osteoblast differentiation and subsequent osteogenesis in vitro, and suggested that BMP-4 may act as an autocrine/paracrine factor in this process. We have also identified more than 100 genes that are either up-or down-regulated by MS. Alpha-adaptin C is one such gene and is known to play a role in endocytosis as one of the constituents of coated pits/vesicles. In situ hybridization and immunohistochemical studies demonstrated that mRNA as well as protein of α-adaptin C was induced as early as 3 hr under MS loading. The expression overlapped with that of BMP-4, suggesting that these cells eventually differentiate into osteoblasts. Electron microscopy demonstrated that coated pits/vesicles increased in those areas where cells, under MS, become strongly positive with α-adaptin C. In addition, potassium depletion, a manipulation known to block endocytosis, inhibited MS-induced osteoblast differentiation. These data strongly sugg … More est that α-adaptin C is involved in the MS-induced osteoblast differentiation via stimulating endocytosis of molecules such as EGF receptors. To further characterize genes involved in MS-induced osteogenesis, we also compared gene expression between osteoblasts and periodontal ligament (PDL) cells. Our working hypothesis was that PDL cells may be equipped with a mechanism by which calcification is suppressed, since the PDL never calcifies in vivo despite esposure to strong intermittent MS and despite their reported osteogenic potential. Indeed, murine cell line PDL-L2 (recently established by us) does not produce mineralized nodules in vitro without superphysiological amounts of BMP-2. We showed that Msx2, one of the genes which are much more abundant in PDL-L2 than in osteoblasts, is a key regulator that prevents PDL-L2 cells from trans-differentiating into osteoblasts. We also demonstrated that Msx2 expression is suppressed in a symptom-dependent manner in affected ligaments of patients with OPLL. We further showed that Msx2 prevents PDL-L2 cells from mineralization by suppressing Runx2 action through directly binding to Runx2 and recruiting HDAC1 complex. We also demonstrated that MS failed to mineralize PDL-L2 cells in vitro, whereas it enhanced mineralization. of osteoblasts. Less