Shalhoub V, Conlon D, Tassinari M, Quinn C, Partridge N, Stein G S, Lian J B
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
J Cell Biochem. 1992 Dec;50(4):425-40. doi: 10.1002/jcb.240500411.
To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.
为了解糖皮质激素促进胎鼠颅骨来源的成骨细胞在体外分化以产生类骨矿化结节的机制，我们对一组与成骨细胞生长和分化相关的基因进行了定量分析，这些基因可表征成骨细胞表型的发育情况，分析对象为经糖皮质激素处理的培养物。我们将亚培养细胞对照培养物（结节形成减少）中成骨细胞表达基因的mRNA水平，与持续（35天）暴露于10⁻⁷ M地塞米松（一种合成糖皮质激素）的细胞进行比较，该激素可促进结节形成至通常在原代培养物中观察到的水平。氚标记胸腺嘧啶核苷标记显示，结节间细胞增殖受到选择性抑制，而形成骨结节的细胞的增殖和分化得到促进。在结节形成期，纤连蛋白、骨桥蛋白和c-fos的表达增加。碱性磷酸酶和I型胶原的表达在增殖细胞中最初受到抑制，然后在结节形成后增加，以支持结节的进一步生长和矿化。与结节形成相关的骨钙素在糖皮质激素分化培养物中的表达升高了1000倍。胶原酶基因表达也高于对照组（五倍），在成熟培养物（第35天）中观察到最高水平。此时，还观察到胶原蛋白和转化生长因子β升高，提示基质的周转。糖皮质激素对组蛋白H4（反映细胞增殖）、碱性磷酸酶、骨桥蛋白和骨钙素mRNA水平的短期（48小时）影响显示，其上调或下调取决于成骨细胞表型的发育阶段。通过核转录分析对这些基因转录水平与mRNA水平进行比较表明，糖皮质激素发挥转录和转录后效应。此外，糖皮质激素的存在增强了维生素D3对基因表达的影响。那些被1,25(OH)₂D3上调的基因，地塞米松会使其转录速率增加，而那些被维生素D3抑制的基因，在存在地塞米松和D3时仍受到抑制。我们提出，糖皮质激素促进参与细胞 - 细胞和细胞 - 细胞外基质信号机制的基因表达变化，这些机制支持具有成骨细胞表型发育和骨组织样组织能力的细胞的生长和分化，同时抑制胎鼠颅骨培养物中不能发展为成熟成骨细胞表型的细胞的生长。
