Wang Feng-Sheng, Ko Jih-Yang, Weng Lin-Hsiu, Yeh Da-Wei, Ke Huei-Jine, Wu Shin-Long
Department of Medical Research, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Taiwan.
Life Sci. 2009 Nov 4;85(19-20):685-92. doi: 10.1016/j.lfs.2009.09.009. Epub 2009 Sep 25.
Long-term glucocorticoid administration is known to induce bone deterioration. Glycogen synthase kinase-3beta (GSK-3beta) signaling reportedly participates in bone remodeling. This study investigated whether GSK-3beta inhibitor could regulate glucocorticoid-induced inhibition of osteoblast differentiation in vitro or bone mass in vivo.
MC3T3-E1 osteoblasts were treated with kinase-inactive GSK-3beta mutant and 6-bromoindirubin-3'-oxim (BIO) and then exposed to 1microM dexamethasone. Survival and osteoblast differentiation of cell cultures were assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, quantitative RT-PCR, and von Kossa staining. Mineral density, biomechanical properties and microenvironments of BIO- and glucocorticoid-treated rat bone tissues were analyzed using dual-energy X-ray absorptiometry, material testing, and histomorphometry, respectively.
Glucocorticoid decreased levels of phosphorylated Ser9-GSK-3beta and beta-catenin in osteoblast cultures. Kinase-inactive GSK-3beta mutant and BIO treatments attenuated dexamethasone-induced inhibition of beta-catenin, Runx2 abundance, and osteoblast differentiation but suppressed glucocorticoid-induced apoptosis of cell cultures. Exogenous BIO treatment alleviated methylprednisolone-induced impairment of mineral density, biomechanical strength, trabecular bone volume, osteoblast surface, and bone formation rate of rat bone tissue. BIO treatment also attenuated glucocorticoid-induced promotion of osteoclast surface and marrow adipocyte volume in bone tissue. Bone cells adjacent to glucocorticoid-stressed bone tissue displayed strong phosphorylated Ser9-GSK-3beta and beta-catenin immunostaining following BIO treatment.
Inhibition of GSK-3beta abrogated glucocorticoid-induced bone loss by increasing beta-catenin- and Runx2-mediated osteoblast differentiation. Controlling GSK-3beta signaling in bone cells may be a strategy for preventing glucocorticoid-induced osteopenia.
长期应用糖皮质激素会导致骨质恶化。据报道，糖原合酶激酶-3β（GSK-3β）信号通路参与骨重塑过程。本研究旨在探究GSK-3β抑制剂是否能在体外调节糖皮质激素诱导的成骨细胞分化抑制，或在体内调节骨量。
用激酶失活的GSK-3β突变体和6-溴靛玉红-3'-肟（BIO）处理MC3T3-E1成骨细胞，然后将其暴露于1μM地塞米松中。通过末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记、定量逆转录-聚合酶链反应和冯·科萨染色评估细胞培养物的存活和成骨细胞分化。分别使用双能X线吸收法、材料测试和组织形态计量学分析BIO和糖皮质激素处理的大鼠骨组织的矿物质密度、生物力学性能和微环境。
糖皮质激素降低了成骨细胞培养物中磷酸化Ser9-GSK-3β和β-连环蛋白的水平。激酶失活的GSK-3β突变体和BIO处理减弱了地塞米松诱导的β-连环蛋白、Runx2丰度和成骨细胞分化的抑制，但抑制了糖皮质激素诱导的细胞培养物凋亡。外源性BIO处理减轻了甲基泼尼松龙诱导的大鼠骨组织矿物质密度、生物力学强度、小梁骨体积、成骨细胞表面和骨形成率的损害。BIO处理还减弱了糖皮质激素诱导的骨组织破骨细胞表面和骨髓脂肪细胞体积的增加。BIO处理后，与糖皮质激素应激骨组织相邻的骨细胞显示出强烈的磷酸化Ser9-GSK-3β和β-连环蛋白免疫染色。
抑制GSK-3β可通过增加β-连环蛋白和Runx2介导的成骨细胞分化来消除糖皮质激素诱导的骨质流失。控制骨细胞中的GSK-3β信号通路可能是预防糖皮质激素诱导的骨质减少的一种策略。
