Asta Pirskanen

Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells

Hormone-dependent accumulation of specific binding sites for 1,25(OH)2D3 and changes in human 1,25-dihydroxy-vitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH)2D3 were quantified by incubating cultured intact cells with [3H]1,25(OH)2D3 at 37 degrees C. Based on the uptake of 1,25(OH)2D3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH)2D3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH)2D3, and maximal binding was achieved with 0.2-0.7 nM 1,25(OH)2D3. Treatment with 1,25(OH)2D3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH)2D3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH)2D3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH)2D3.

Homologous and heterologous regulation of 1,25-dihydroxyvitamin D-3 receptor mRNA levels in human osteosarcoma cells

The heterologous regulation of hormone receptors is well described in the hormone receptor literature. We were interested in determining whether human 1,25-dihydroxyvitamin D-3 receptor (hVDR) and glucocorticoid receptor (GR), members of the steroid/thyroid hormone receptor family, are heterologously regulated by other steroids and related hormones. We used human osteosarcoma cells (MG-63) and measured hVDR and GR mRNA levels after androgen, estrogen, glucocorticoid, progesterone, thyroid hormone, vitamin A and vitamin D treatments. Each hormone, except androgen and progesterone, was capable of increasing hVDR mRNA levels like the natural ligand in human osteosarcoma cells. On the other hand, GR gene expression was not affected by these hormones. To study whether the cells responded to the 1,25(OH)2D3-treatment with changes in differentiation and proliferation, we also studied c-myc and c-fos gene expression. Both genes were only regulated by 1,25(OH)2D3. 1,25(OH)2D3 slightly increased the accumulation of c-fos mRNA within 4-12 h from the hormone addition, while the increase in c-myc mRNA appeared at 24 h.

Modulation of 1,25(OH)2D3-induced osteocalcin synthesis in human osteosarcoma cells by other steroidal hormones

We have previously shown that osteocalcin synthesis is readily induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in MG-63 human osteosarcoma cells (Mahonen et al. (1990) Biochim. Biophys. Acta 1048, 30-37). In the present study, the regulation of osteocalcin synthesis by other hormones of the steroid-thyroid hormone family (retinoic acid, 17 beta-estradiol, triiodothyronine, and dexamethasone) was examined. We found that the other hormones alone had no effects on medium osteocalcin and osteocalcin mRNA concentrations by 96 h of treatment. Compared with 1,25(OH)2D3, however, the combination of 1,25(OH)2D3 with dexamethasone resulted in a greatly reduced medium osteocalcin concentration. Also estradiol and triiodothyronine diminished the stimulatory effect of 1,25(OH)2D3. In contrast, the combination of 1,25(OH)2D3 with retinoic acid resulted in an increased medium osteocalcin concentration. The inhibition of osteocalcin synthesis by dexamethasone and triiodothyronine was accompanied by decreased osteocalcin mRNA levels. Retinoic acid and estradiol, however, did not influence the 1,25(OH)2D3-induced osteocalcin mRNA levels. To examine the specificity of the hormonal effects, the activity of alkaline phosphatase was determined. Both baseline and 1,25(OH)2D3-stimulated alkaline phosphatase activity was found to be inhibited by all other hormones. These results suggest that the steroidal hormones specifically affect osteocalcin synthesis in osteoblastic bone cells, and that complex interactions occur at the level of transcription and/or translation resulting in each case in a finely adjusted rate of osteocalcin synthesis.

Affinity of 22-oxa-1,25(OH)2D3 for 1,25-dihydroxyvitamin D receptor and its effects on the synthesis of osteocalcin in human osteosarcoma cells

A new 1,25-dihydroxyvitamin D3 analog, 22-oxa-1,25(OH)2D3, which may have pharmaceutical use, e.g., in the treatment of psoriasis, was studied using cultured MG-63 human osteosarcoma cells. We found that the new compound binds to 1,25-dihydroxyvitamin D receptors and regulates receptor mRNA levels like the natural ligand. Our results also indicate that 22-oxa-1,25(OH)2D3 induces the synthesis of osteocalcin and the activity of alkaline phosphatase in MG-63 cells through a receptor-mediated process identically with 1,25(OH)2D3.

State of methylation of the human osteocalcin gene in bone‐derived and other types of cells

DNA methylation is a general mechanism of controlling tissue‐specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone‐derived (MG‐63, U2‐Os, SaOs‐2) and other types (normal lymphocytes, A‐498, Hep G2) of cells. Reverse transcription–polymerase chain reaction (RT‐PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG‐63 and induced in SaOs‐2 but not in U2‐Os osteoblast‐like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single‐copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A‐498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5‐azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG‐63 cells. In gel mobility shift assays, human vitamin D receptor and the AP‐1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression. J. Cell. Biochem. 66:404–412, 1997.

Use of Non-Collagen Markers in Osteoporosis Studies

SummaryClinical and research laboratories routinely measure various hormonal and nonhormonal parameters of calcium and phosphorus metabolism, and markers of bone turnover. Such measurements may help clinical decision-making relating to metabolic bone disease and osteoporosis. Molecular biological and cell-culture techniques are being used in basic biochemical research on bone-cell metabolism. Results may aid understanding of normal and abnormal regulation of the bone-cell metabolism, and thus provide further insights relating to the diagnosis and prevention of osteoporosis.

Hormonal regulation of vitamin D receptor levels and osteocalcin synthesis in human osteosarcoma cells

Summary1,25(OH)2D3 was found to regulate its own receptor levels via an increase in corresponding mRNA levels in human osteoblast-like osteosarcoma cells (MG-63). In addition, exposure of the cells for 24h to dexamethasone, estradiol, retinoic acid, or triiodothyronine resulted in a dose-dependent accumulation of hVDR mRNA. Combination of 1,25(OH)2D3 with any other hormone used in this study did not result in an additive increase in hVDR mRNA levels. Progesterone or dihydrotestosterone did not influence hVDR mRNA levels. Of the studied hormones, only 1,25(OH)2D3 was alone able to stimulate the synthesis and secretion of osteocalcin. Compared with 1,25(OH)2D3, the combination of 1,25(OH)2D3 and retinoic acid resulted an increased synthesis of osteocalcin. In contrast, the combination of 1,25(OH)2D3 with dexamethasone, estradiol, or triiodothyronine diminished the stimulatory effect of 1,25(OH)2D3. A complex interaction of several different hormone receptors seems to occur within the regulatory regions of hVDR and osteocalcin genes, or at the level of translation, resulting, in each case, a finely adjusted vitamin D receptor and osteocalcin expression.

Affinity of MC 903 for 1,25-dihydroxyvitamin D receptor and its effects on the synthesis of osteocalcin in human osteosarcoma cells

MC 903 is a new structural analog of the naturally occurring, biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. MC 903 and 1,25(OH)2D3 have shown similar receptor binding properties and comparable effects on leukemic cell differentiation. However, MC 903 is at least 100 times less potent in influencing calcium metabolism than 1,25(OH)2D3. We have therefore studied, how MC 903 competes for the binding sites of 1,25(OH)2D3, influences the 1,25(OH)2D3 induced synthesis of the most abundant bone non-collagenous protein, osteocalcin, and induces the activity of alkaline phosphatase in MG-63 human osteosarcoma cells. We found that the new compound binds to 1,25(OH)2D3 receptors and regulates receptor mRNA levels essentially like the natural ligand. Our results also indicate that MC 903 induces the synthesis of osteocalcin and the activity of alkaline phosphatase in MG-63 cells through a receptor-mediated process almost identically with 1,25(OH)2D3. Growth of the MG-63 cells was inhibited slightly more with MC 903 than with 1,25(OH)2D3.