Subramaniam M, Colvard D, Keeting P E, Rasmussen K, Riggs B L, Spelsberg T C
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
J Cell Biochem. 1992 Dec;50(4):411-24. doi: 10.1002/jcb.240500410.
In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.
在人类中，已知糖皮质激素对骨代谢和功能有显著影响，包括对成骨细胞的重要调节作用。为了有助于理解糖皮质激素对正常人成骨细胞（hOB）的作用机制，对汇合细胞进行了分析，检测糖皮质激素受体（GR）的存在情况，以及糖皮质激素地塞米松（Dex）对快速反应的核原癌基因和骨基质蛋白的晚期反应结构基因表达的影响。还研究了Dex与1,25 - 二羟基维生素D3（1,25 D3）在这些细胞中基因表达上的相互作用。使用功能性受体测定法，在15个不同的细胞株中测得平均11,600个功能性核结合糖皮质激素受体（范围为6,000 - 22,000）。用针对人GR的cDNA探针进行Northern印迹分析，以证明存在一个7Kb的转录本，它可能是这些细胞中GR的mRNA。与先前的研究一致，用Dex处理hOB细胞会增加碱性磷酸酶（AP）的稳态mRNA水平，但对骨钙素（OC）和甘油醛 - 3 - 磷酸脱氢酶（GAPDH）的mRNA水平几乎没有影响。有趣的是，Dex阻断了1,25 D3对OC mRNA水平的诱导，但增强了对AP的诱导。Dex对AP和OC基因表达的上述影响，包括与1,25 D3的相互作用，在蛋白质水平也有体现。还研究了Dex对核原癌基因c - myc、c - fos和c - jun mRNA水平的影响，因为癌蛋白（Fos/Jun）似乎在糖皮质激素对结构基因的延迟调节中起作用。有趣的是，在类固醇处理后1小时内，Dex使非增殖（汇合）hOB细胞中c - myc、c - fos和c - jun mRNA的稳态水平分别比对照（未处理细胞）值增加了3.5倍、10倍和2.0倍。Dex诱导的mRNA水平是短暂的，在类固醇处理后24小时内恢复到基础值。在增殖的hOB细胞中发现了一种减弱但性质相似的反应模式。这些基因在hOB细胞中对糖皮质激素的反应模式类似于禽肝细胞中的反应，但不同于生殖细胞中的反应。这些结果支持了hOB细胞是糖皮质激素靶细胞的理论，并且作为首要事件，糖皮质激素在这些细胞中迅速调节核癌蛋白Fos/Jun的表达。
