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.

The growth‐supportive effect of thrombospondin (TSP1) and the expression of TSP1 by human MG‐63 osteoblastic cells are both inhibited by dexamethasone

“…The observation that dexamethasone inhibits TSP expression is based on a number of findings: (a) TSP mRNA expression is inhibited in dexamethasonetreated MG-63 cells, (b) cell surface expression of TSP is decreased by 50 to 70% in the presence of dexamethasone, and (c) secretion of TSP in the culture fluid of dexamethasone-treated MG-63 cells is decreased by 40%. The observation that dexamethasone stimulates alkaline phosphatase activity in human MG-63 osteoblastic cells confirms and extends previous findings obtained with human trabecular osteoblast-like cells [12] and rat ROS 17/2.8 osteoblastic cells [ 131. As previously reported for human trabecular osteoblast-like cells [ 13,191, the increased activity of this enzyme is inversely related to proliferation of human MG-63 cells.…”

“…Glucocorticoid-induced osteoporosis involves reduction of osteoblast proliferation [7-9,131, inhibition of prostaglandin synthesis [9] and inhibition of the expression of bone extracellular matrix proteins including type I collagen [lo], osteocalcin [11,12], osteopontin [14], and fibronectin [ 163. By contrast, glucocorticoids stimulate alkaline phosphatase activity [ 12,131 and bone sialoprotein expression by osteoblastic cells [ 151.…”

“…Osteoblastic cells have receptors for glucocorticoids [8], and cultured mouse MC3T3-El osteoblastic cells have been used to show that the glucocorticoid, dexamethasone, inhibits DNA synthesis and prostaglandin synthesis [9]. In addition, glucocorticoids also affect the expression of bone extracellular matrix proteins including type I collagen [lo], osteocalcin [11,12], alkaline phosphatase [12,13], osteopontin [14], bone sialoprotein [15], and fibronectin [ 161. All of these extracellular matrix proteins undoubtedly play an important role in bone cell morphology, and could be contributing factors to the reduced bone quality and reduced bone formation seen in glucocorticoid-induced osteoporosis.…”

The delayed induction of c-jun in apoptotic human leukemic lymphoblasts is primarily transcriptional

“…The regulation can be purely transcriptional, post-transcriptional, or both [26][27][28][29][30]. Glucocorticoid induces c-jun expression in some cells, but suppresses it in others [25,[31][32][33]. In both AtT-20 pituitary tumor cells and U-937 monocytic leukemia cells, Dex represses the transcription of c-jun [25,34].…”

“…A plasmid pTRIc-jun containing 340 base pairs (bp) c-jun cDNA was constructed. 32 P-labeled antisense c-jun RNA probe was synthesized by T7 promoter-dependent RNA polymerase according to standard in vitro transcription protocols (Ambion, Austin, TX), and purified by gel electrophoresis. RNase protection assay was carried out with a kit from Ambion.…”