Evidence that estrogens modulate activity and increase the number of 1,25-dihydroxyvitamin D receptors in osteoblast-like cells (ROS 17/2.8).

A detailed understanding of the mechanism of action of estrogen on bones is still lacking. The present study was designed to examine possible modulation by 17 beta-estradiol (E2) on the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and on vitamin D receptors (VDR) in the ROS 17/2.8 osteoblast-like cell line. Cells were grown in phenol-red free medium supplemented with charcoal-stripped fetal calf serum (FCS). Total cellular VDR were measured in cell homogenates after extraction with a KCl hypertonic buffer. VDR-binding capacity doubled in the presence of 10 nM E2 (16.2 +/- 2.3 vs. 7.0 +/- 1.3 fmol/mg protein, respectively; P less than 0.01), while the Kd for 1,25-(OH)2D3 did not change (approximately 0.1 nM). Tamoxifen alone had no effect on VDR, while it completely abolished the E2-induced increase in VDR, indicating that the effect was specific for E2 and estrogen receptor mediated. 1,25-(OH)2D3 inhibited cell proliferation, determined by [3H] thymidine incorporation to DNA, in a dose-dependent fashion between 0.01-100 nM. The inhibitory effect of 1,25-(OH)2D3 on cell proliferation was significantly augmented in the presence of E2 (10 nM). 1,25-(OH)2D3 increased osteocalcin secretion to the medium by the cells in a dose-dependent fashion between 0.01-100 nM. In the presence of E2 (10 nM), maximal osteocalcin secretion in response to 1,25-(OH)2D3 was 3.5-fold higher than that in response to 1,25-(OH)2D3 alone. These results indicate that E2 modulates 1,25-(OH)2D3 activity in osteoblast-like cells, and that this effect can be attributed to an increase in VDR.     

Modulation by vitamin D status of the responsiveness of rat bone to gonadal steroids

We have previously demonstrated that gonadal steroids stimulate [3H]thymidine incorporation and creatine kinase specific activity in skeletal tissues. In the present study we report that in 20-day-old vitamin D-deficient Wistar-derived rats, 17 beta-estradiol (E2; 5 micrograms/rat) or testosterone (50 micrograms/rat) failed to stimulate [3H]thymidine incorporation into diaphyses of long bones and that the response to these hormones in terms of increased creatine kinase specific activity was less than half the value in normally fed rats. Two daily ip injections of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3; 0.5 ng/g BW], but not 24,25-(OH)2D3 (5 ng/g BW), partially restored the biological responses to E2 in bone of 21-day-old vitamin D-deficient female rats. Vitamin D deficiency did not impair the responsiveness to gonadal steroids in the epiphysis of long bones, uterus, or prostate, in contrast to its effect on diaphysis. In 21-day-old normally fed female rats, neither vitamin D metabolite enhanced the response to E2. When cultures of rat epiphyseal cells were treated daily for 5 days with either 1,25-(OH)2D3 (1 nM) or 24,25-(OH)2D3 (10 nM), followed by E2 (30 nM) for 24 h, creatine kinase activity was significantly higher than in cultures treated daily for 5 days with vehicle alone, and then with E2. The same treatment of rat embryo calvaria bone cells showed that 1,25-(OH)2D3, but not 24,25-(OH)2D3, significantly increased the creatine kinase activity response to E2. These findings suggest that vitamin D metabolites selectively affect the biological responses of skeletal tissues to gonadal steroids.     

1,25-Dihydroxyvitamin D3 stimulates the secretion of insulin-like growth factor binding protein 3 (IGFBP-3) by cultured human osteosarcoma cells.

Several types of specific insulin-like growth factor binding proteins have been reported. These binding proteins are produced by peripheral tissue-derived cells and they modulate the functions of insulin-like growth factors. In this study, we investigated both the secretion of insulin-like growth factor binding protein 3 (IGFBP-3) from a human osteosarcoma cell line MG63, and the effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the production of this binding protein. The beta subunit of IGFBP-3 was detected in perinuclear cytoplasm of MG63 cells by immunocytochemical study. Immunoblotting and SDS-PAGE analysis revealed that both 150KD MW entire molecules and 40-60KD MW beta subunit molecules of IGFBP-3 were present in cell-conditioned media. 1,25-(OH)2D3 stimulated the production of the IGFBP-3 molecule by MG63 cells. The concentration of IGFBP-3 in conditioned media began to rise at 12 hours after the addition of 10(-8) M of 1,25-(OH)2D3 and reached peak level at 48 hours. Dose-dependent effects of 1,25-(OH)2D3 were demonstrated. The its maximum effect was observed at 10(-10) M. The concentration of IGFBP-3 in cytosol also increased at a 10(-10) M concentration of 1,25-(OH)2D3. We conclude from these results that human osteosarcoma cells MG63 produce the IGFBP-3 molecule and that 1,25-(OH)2D3 stimulates the production of this protein. These data suggests that the synergistic effects of 1,25-(OH)2D3 on the action of IGF-I on osteoblastic cells, which we reported previously, may be modulated by locally produced IGFBP-3.     

Estrogen synthesis by osteoblast cell lines.

Estrogens play a central role in modulating bone turnover and in the postmenopausal female are formed almost exclusively by peripheral conversion of sex steroid precursors derived from the adrenals. In this study we have demonstrated that three human osteoblastic cell lines [HOS, U20S (HTB96) and MG63] possess the enzymes necessary for estrogen synthesis and metabolism. Aromatase, estradiol 17 beta-hydroxysteroid dehydrogenase (reductive and oxidative) and estrone sulfatase activities were measured in whole cell monolayers over a 20 h period by isotopic assay techniques. Significant aromatase activity was detected in all three cell lines ranging from 1.8 +/- 0.2 fmol/20 h/10(6) cells (mean +/- S.D., n = 3) for MG63 cells to 51 +/- 1.5 fmol/20 h/10(6) cells for HOS cells. The specific aromatase inhibitor, 4-hydroxyandrostenedione (1 mumol/L) completely inhibited aromatase activity in these cells. Two of the cell lines, HOS and MG63, had significant estradiol 17 beta-hydroxysteroid dehydrogenase activity with oxidative (32.7 +/- 1.9 and 1068.4 +/- 40.2 fmol/20 h/10(6) cells respectively) predominant over reductive activity (1.6 +/- 0.4 and 38.7 +/- 1.8 fmol/20 h/10(6) cells). All three cell lines were able to hydrolyse estrone sulfate to estrone with activities ranging from 13.3 +/- 1.5 fmol/20 h/10(6) cells for U20S cells to 482.2 +/- 3.7 fmol/20 h/10(6) cells for MG63 cells. Since estrogen has been implicated as a critical factor in the modulation of bone resorption and formation, the regulation of skeletal estrogen production, particularly at the time of the menopause, is likely to be an important mechanism by which bone volume is determined in physiological and pathological states.     

Calcium antagonizes 1,25-dihydroxyvitamin D3 inhibition of breast cancer cell proliferation

The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited proliferation of two breast cancer cell lines (HT-39 and MCF-7) in a dose-dependent manner. We showed that 10 nM 1,25-(OH)2D3 inhibited HT-39 cell growth by 26.3 +/- 3.2% and MCF-7 proliferation by 19.5 +/- 8.6% (mean +/- SE). When both cell lines were cultured in the presence of medium containing varying concentrations of calcium, analysis of cell growth revealed that lowering the medium Ca2+ concentration from 1.3 to 0.1 mM inhibited cell proliferation of HT-39 cells by 20.0 +/- 2.3% and that of MCF-7 cells by 13.4 +/- 3.4% (mean +/- SE). Raising medium Ca2+ from 1.3 to 2.6 mM stimulated cell proliferation of HT-39 cultures by 51.8 +/- 2.3% and that of MCF-7 cells by 13.3 +/- 3.3% (mean +/- SE). When HT-39 cells were grown in medium containing different calcium levels and dosed with 10 nM 1,25-(OH)2D3, we found that the inhibitory action of 1,25-(OH)2D3 was modified. HT-39 cells in medium containing 0.1 mM calcium were more potently inhibited by 10 nM 1,25-(OH)2D3 (increased by 74% relative to that in cells in 1.3 mM calcium). Moreover, the inhibitory action of 1,25-(OH)2D3 (10 nM) on HT-39 cells grown in 2.6 mM calcium media was decreased by 68% relative to that in cells in 1.3 mM extracellular calcium. The phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB) stimulated HT-39 proliferation by 30.1 +/- 4.1%. Cells treated with PDB showed increased cell mitotic index, but unlike extracellular calcium, PDB had no effect on the dose response of 1,25-(OH)2D3 to inhibit HT-39 cell growth. We show that 1,25-(OH)2D3 action on malignant cell proliferation is dependent on the extracellular calcium concentration. The data suggest that the effect of calcium to antagonize 1,25-(OH)2D3 on HT-39 cells is not shared by all agents (e.g. PDB) that increase HT-39 cell proliferation.     

Effect of 1,25-(OH)2-vitamin D3 on growth, homologous receptor and c-myc regulation in C3H/10T1/2 cells

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor concentration, cell proliferation, and the steady-state level of c-myc mRNA were examined in the C3H/10T1/2 mouse embryo fibroblasts, before and after exposing the cells to 1,25-(OH)2D3. The non-transformed, logarithmically growing C3H/10T1/2 Cl 8 cells contained a high concentration of 1,25-(OH)2D3 receptor (164 fmol/mg of protein). An up-regulation of the 1,25-(OH)2D3 receptor and a potent inhibition of cell growth were observed by exposing the cells to 10 nM 1,25-(OH)2D3. The concentration of 1,25-(OH)2D3 receptor in the two chemically transformed, tumorigenic cell lines. C3H/10T1/2 Cl 16 and C3H/10T1/2 TPA 482, was 218 and 63 fmol/mg of protein, respectively. In the two transformed cell lines, 10 nM 1,25-(OH)2D3 had only negligible effect on cell growth. In the Cl 16 cells, an up-regulation of the 1,25-(OH)2D3 receptor was demonstrated, but only a weak up-regulation was found in the TPA 482 cells by the 1,25-(OH)2D3 treatment. No major changes were found in c-myc mRNA levels by the 1,25-(OH)2D3 treatment. Despite inhibition of cell growth, the steady-state level of c-myc mRNA was slightly induced (35%, mean) in the Cl 8 cells compared to control cells. In the transformed cells, no consistent change of the c-myc level was found. In contrast to earlier reports, we did not find any correlation between the 1,25-(OH)2D3 receptor and c-myc level, nor did we find any decrease of c-myc mRNA by 1,25-(OH)2D3 treatment in the C3H/10T1/2 fibroblasts.     

Age and gender specific stimulation of creatine kinase specific activity by gonadal steroids in human bone-derived cells in culture

We previously reported a non-enzymatic method for isolation of human bone cells in culture that display osteoblastic features and respond to 1,25 dihydroxy vitamin D (1,25) and to parathyroid hormone (PTH). The present study was undertaken to analyze the response of cultured human bone cells to 17beta-estradiol (E2) and to dihydrotestosterone (DHT) as a function of gender and age. Cultured human bone cells, obtained from biopsies during orthopedic surgery, were divided into four groups defined by gender and age: pre- and post-menopausal healthy non-osteoporotic women that were not under hormone replacement therapy (HRT) and mature (<55-year-old) and older (>60-year-old) men. We found gender specific responses to gonadal steroids using the specific activity of the brain type (BB) isozyme of creatine kinase (CK) as a response marker. Constitutive levels of CK activity did not change with age or gender and the enzyme extracted from cells from the different sexes and ages did not respond to either progesterone (P) or to 1,25. CK from the different cells responded to gonadal steroids in a gender specific manner, i.e. CK from female derived cells responded to E2 only and the enzyme from male derived cells responded to DHT only. In female derived cells the response to E2 declined significantly with age, while the response to DHT in CK from male derived cells did not vary with age. This may be due to either decreased proportion of mature osteoblasts and/or their differentiation state and/or changes in the levels of estrogen receptor(s), coactivators or corepressors in these cells. These results extend our knowledge of human osteoblast biology (beyond murine cells) and are therefore more relevant for developing models for treatment of human metabolic bone diseases such as post-menopausal osteoporosis.     

Epidermal growth factor and calcitriol synergistically induce osteoblast maturation

Calcitriol (1,25(OH)₂D₃) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.     

Specificity of steroid binding in New World primate B95-8 cells with a vitamin D-resistant phenotype.

We recently described the existence of a competitive binding component in vitamin D-resistant New World primate cells that has a relatively low affinity (Kd, approximately 10(-8) M) but high capacity for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] compared to that possessed by the vitamin D receptor (VDR). Here we show that this binding component is capable of binding a vitamin D3 metabolite other than 1,25-(OH)2D3 as well as steroid hormones structurally disparate from vitamin D3 sterols. We studied the binding of [3H]1,25-(OH)2D3 and [3H]25-hydroxyvitamin D3 ([3H]25OHD3) in extracts of the vitamin D-resistant marmoset lymphoblastic cell line B95-8 in the presence and absence of potential competitive ligands, including 25OHD3, 1,25-(OH)2D3, 17 beta-estradiol, testosterone, and progesterone, at concentrations ranging from 1-100 nM. Compared to extracts containing the authentic VDR, extracts of B95-8 cells bound 180% more 1,25-(OH)2D3 and 12-fold more 25OHD3 on a weight basis. The affinity of this binder for 25OHD3 was 2.2 times as great as its affinity for 1,25-(OH)2D3. Further, at concentrations approaching the Kd of this binder for 1,25-(OH)2D3, 25OHD3 was 3 times more effective than 1,25-(OH)2D3 in competing with [3H]1,25-(OH)2D3 for binding. This binder eluted from a Sephadex G-100 column with an apparent mol wt of 58 kilodaltons, and pooled elution fractions from the column encompassing this mol wt range were capable of inhibiting binding of 1,25-(OH)2D3 to the VDR by 65%. Competitive steroid binding analyses showed estradiol to be at least as effective as 1,25-(OH)2D3 in inhibition of [3H]1,25-(OH)2D3 binding; homologous binding studies with 17 beta-estradiol as labeled and competitive ligand demonstrated that concentrations of the gonadal steroid that successfully displaced [3H]1,25-(OH)2D3 also displaced 17 beta-[3H] estradiol. Using [3H]25OHD3 as the labeled ligand and a more extensive array of competitive ligands, the rank order of steroid binding was 25OHD3 > 1,25-(OH)2D3 > or = estradiol = progesterone = testosterone. These results suggest that the phenotype of steroid hormone resistance in New World primates may result from the overexpression of an intracellular 58-kilodalton protein(s) that interferes with the steroid-receptor interaction by competing for ligand binding.     

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.     

Association between the expression of the c-myc oncogene mRNA and the expression of the receptor protein for 1,25-dihydroxyvitamin D3

Studies in lymphocytes have indicated similarities in the state of activation, the time kinetics, and the pathologic states associated with the expression of the c-myc oncogene, and the expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor protein. Here, we have sought evidence for an association between c-myc and the 1,25-(OH)2D3 receptor protein in mammalian cells other than lymphocytes. Comparing two rat osteogenic sarcoma cell lines, one that produces constitutively relatively high levels of the 1,25-(OH)2D3 receptor protein (ROS 17/2.8) and one in which the 1,25-(OH)2D3 receptor protein is practically undetectable (ROS 2/3), we found that the 1,25-(OH)2D3 receptor-expressing cell line also expressed c-myc mRNA. In contrast, the cell line in which the 1,25-(OH)2D3 receptor was undetectable did not express c-myc mRNA. Furthermore, we transfected mouse skin fibroblasts (NIH 3T3) with a recombinant plasmid carrying the human c-myc oncogene. We found a dramatic increase in the 1,25-(OH)2D3 receptor concentration in five separate clonal lines of NIH 3T3 cells transfected with the c-myc-carrying plasmid compared to their nontransfected counterparts or to NIH 3T3 fibroblasts transfected with the vector plasmid alone. The receptor protein of the transfected cells exhibited biochemical characteristics indistinguishable from those of classical receptors for 1,25-(OH)2D3. The increased expression in the transfected cells appeared specific for the receptor for 1,25-(OH)2D3; receptors for sex steroids were not detected in the nontransfected NIH 3T3 cells and remained undetectable after transfection with c-myc. Moreover, the level of the glucocorticoid receptor protein, which was expressed in the nontransfected cells, did not change upon transfection with c-myc.     

Combined effects of dexamethasone and 1,25-dihydroxyvitamin D3 on parathyroid hormone secretion in cultured bovine parathyroid cells

The present studies investigate the effects of glucocorticoids on the function of the parathyroid glands using primary cultures of bovine parathyroid cells. Treatment of parathyroid cell cultures with dexamethasone for 48 h caused a dose-dependent stimulation of PTH secretion. The minimal concentration of dexamethasone required for a significant stimulation of PTH secretion was 0.1 nM. The stimulatory effect of dexamethasone on the secretion of PTH was found within 12 h of treatment with 100 nM dexamethasone. The steroids deoxycorticosterone and cortexolone, which do not have glucocorticoid activity were without effect of PTH secretion. Since glucocorticoids may modulate the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in other tissues, additional studies were performed to evaluate the interactions of glucocorticoids and 1,25-(OH)2D3. Addition of 1,25-(OH)2D3 to parathyroid cell cultures for 48 h significantly suppressed PTH secretion. In the presence of dexamethasone, however, 1,25-(OH)2D3 also significantly decreased PTH secretion, although it did not reduce PTH secretion to control levels. The treatment of parathyroid cell cultures with 100 nM dexamethasone did not affect the parathyroid cell content of 1,25-(OH)2D3 receptors. In summary, these studies indicate that glucocorticoids significantly increase the secretion of PTH in vitro. This stimulatory effect can be inhibited by 1,25-(OH)2D3. The parathyroid gland is an additional site of physiological antagonism of glucocorticoids and 1,25-(OH)2D3.     

1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture

In this study we demonstrate the presence of specific, high-affinity receptors for 1,25-dihydroxyvitamin D3 in malignant melanoma. Receptors are present both in cultured melanoma cells and in melanoma tumor tissue produced by inoculation of cells into athymic rats. The receptor sediments at 3.25 on sucrose density gradients, possesses a preferential affinity for 1,25-(OH)2D3 and has an apparent Kd of 0.18 nM by Scatchard analysis. We also demonstrate that human melanoma cells are responsive to 1,25-(OH)2D3 in vitro. Inclusion of 1,25-(OH)2D3 in the culture medium produced a marked increase in cell doubling time. This inhibitory effect of the hormone on melanoma cell proliferation was dose-related and represents the first demonstration of a 1,25-(OH)2D3 mediated action on tumor cells.     

Down-regulation of androgen receptor by progestins and interference with estrogenic or androgenic stimulation of mammary carcinoma cell growth

The regulatory influence of medroxyprogesterone acetate (MPA) on estrogen and androgen receptors of the human breast cancer cell lines MCF-7 and EFM-19 was explored in conjunction with the growth-promoting properties of these steroids. In the absence of steroidal stimulation, up to 1 microM MPA had no effect on the proliferation of the MCF-7 cell strain used and of EFM-19 cells. Under stimulation with 10 nM 17 beta-estradiol or 1 microM dihydrotestosterone, dose-dependent inhibition of the cell proliferation rates by 0.1-1 microM MPA was observed. Binding of MPA to the androgen receptor (Kd = 2.1 nM) but not to the estrogen receptor was demonstrable. During incubation of MCF-7 or EFM-19 cells with 1 microM MPA for 7 days, the estrogen and androgen receptor contents were down-regulated by approximately 50% and 60%, respectively. Likewise, the number of androgen-binding sites was reduced to 35% of the untreated controls after incubation of MCF-7 cells with 1 microM synthetic progestin R5020 for 7 days. The results indicate down-regulation of estrogen and androgen receptors by progestins in the absence of stimulatory effects on the proliferation of mammary carcinoma cells.     

Regulation of cell growth, c-myc mRNA, and 1,25-(OH)2 vitamin D3 receptor in C3H/10T1/2 mouse embryo fibroblasts by calcipotriol and 1,25-(OH)2 vitamin D3.

Calcipotriol is a synthetic 1,25-(OH)2D3 analogue with high affinity for the 1,25-(OH)2D3 receptor, but with a lower affinity than 1,25-(OH)2D3 for vitamin D binding protein in serum. The inhibitory action of calcipotriol and 1,25-(OH)2D3 on proliferation of C3H/10T1/2 mouse embryo fibroblasts was examined in the non-transformed cell line Cl 8 and in the two transformed, tumorigenic cell lines Cl 16 and TPA 482. Upon exposure to 10 nmol/l calcipotriol or 1,25-(OH)2D3, the proliferation of Cl 8 cell line was almost completely suppressed, whereas both hormones had no effect on the cell lines Cl 16 and TPA 482. Calcipotriol was at least as effective as 1,25-(OH)2D3 in inducing up-regulation of the 1,25-(OH)2D3 receptor. Displacement studies showed no difference between calcipotriol and 1,25-(OH)2D3 in the affinity for the receptor present in Cl 8 or Cl 16 cell extracts. Furthermore, the inhibition of cell growth in Cl 8 cells by calcipotriol was not accompanied by any consistent change in the steady-state expression of c-myc mRNA. In conclusion, calcipotriol had potent growth inhibitory effect on the non-transformed cell line similar to 1,25-(OH)2D3. In the transformed cell lines, calcipotriol did not inhibit proliferation despite potent up-regulation of the 1,25-(OH)2D3 receptor.     

Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17beta-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (x2) of 17beta-estradiol (0.01 microM) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 microM) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17beta-estradiol (0.01 microM), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17beta-estradiol significantly increased (x1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor gamma2 was up-regulated by 17beta-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 beta-estradiol (0.01 microM) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 microM) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor gamma2 expression (estrogens).     

1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.     

Liarozole acts synergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity

1Alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the proliferation of many cancer cells in culture, but not the aggressive human prostate cancer cell line DU 145. We postulated that the 1,25-(OH)2D3-resistant phenotype in DU 145 cells might result from the high levels of expression of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) induced by treatment with 1,25-(OH)2D3. As this P450 enzyme initiates 1,25-(OH)2D3 catabolism, we presumed that a high level of enzyme induction could limit the effectiveness of the 1,25-(OH)2D3 antiproliferative action. To examine this hypothesis we explored combination therapy with liarozole fumarate (R85,246), an imidazole derivative currently in trials for prostate cancer therapy. As imidizole derivatives are known to inhibit P450 enzymes, we postulated that this drug would inhibit 24-hydroxylase activity, increasing the 1,25-(OH)2D3 half-life, thereby enhancing 1,25-(OH)2D3 antiproliferative effects on DU 145 cells. Cell growth was assessed by measurement of viable cells using the MTS assay. When used alone, neither 1,25-(OH)2D3 (1-10 nM) nor liarozole (1-10 microM) inhibited DU 145 cell growth. However, when added together, 1,25-(OH)2D3 (10 nM)/liarozole (1 microM) inhibited growth 65% after 4 days of culture. We used a TLC method to assess 24-hydroxylase activity and demonstrated that liarozole (1-100 microM) inhibited this P450 enzyme in a dose-dependent manner. Moreover, liarozole treatment caused a significant increase in 1,25-(OH)2D3 half-life from 11 to 31 h. In addition, 1,25-(OH)2D3 can cause homologous up-regulation of the vitamin D receptor (VDR), and in the presence of liarozole, this effect was amplified, thus enhancing 1,25-(OH)2D3 activity. Western blot analyses demonstrated that DU 145 cells treated with 1,25-(OH)2D3/liarozole showed greater VDR up-regulation than cells treated with either drug alone. In summary, our data demonstrate that liarozole augments the ability of 1,25-(OH)2D3 to inhibit DU 145 cell growth. The mechanism appears to be due to inhibition of 24-hydroxylase activity, leading to increased 1,25-(OH)2D3 half-life and augmentation of homologous up-regulation of VDR. We raise the possibility that combination therapy using 1,25-(OH)2D3 and liarozole or other inhibitors of 24-hydroxylase, both in nontoxic doses, might serve as an effective treatment for prostate cancer.     

The involvement of calcium ions in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) was found to suppress growth of human leukemic cells (HL-60), and to induce the differentiation of these cells to monocyte-like cells. The purpose of the present study was to examine the role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells. Incubation of the HL-60 cells with 1,25(OH)2D3 (10(-7) M) for 4 days caused a significant inhibition of 50% of cell growth. The number of differentiated cells increased simultaneously from 24 x 10(3) +/- 2 x 10(3) in the controls to 658 x 10(3) +/- 32 x 10(3) in the 1,25(OH)2D3 (10(-7) M)-treated cells. The role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells was first studied by changing the available calcium in the medium and by measuring the effect of 1,25(OH)2D3 on intracellular Ca2+ levels. Limitation of the available Ca2+ by means of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or verapamil enhanced the inhibitory effect on proliferation and decreased the number of differentiated cells obtained by 1,25(OH)2D3 alone. These effects could be abolished by restoring the Ca2+ levels. The role of the intracellular free Ca2+ ions in the effect of 1,25(OH)2D3 was further illustrated by measuring the intracellular Ca2+ levels. The intracellular free Ca2+ concentration in 1,25(OH)2D3 (10(-7) M)-treated HL-60 cells rose significantly from 117.0 +/- 6.3 nM in the untreated HL-60 cells to 145.0 +/- 7.5 nM in the treated cells (p less than 0.02). Addition of verapamil moderated the increase in intracellular free Ca2+ (125.0 +/- 5.2 nM) obtained by 1,25(OH)2D3 alone. Thus the elevation of intracellular free Ca2+ caused by 1,25(OH)2D3 treatment may be involved in the effect of the hormone on the HL-60 cells.