Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells

Abstract

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.