Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)2D3 and for glucocorticoids, and have examined the modulation of the 1,25(OH)2D3 receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)2D3 receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)2D3; this phenomenon occurred in a dose-dependent fashion for triamcinolone (10(-9) to 10(-7) M) with a maximum increase of 1,25(OH)2D3 receptor concentration of approximately equal to twofold. During the culture period, the 1,25(OH)2D3 receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)2D3 receptor concentration was higher in the triamcinolone-treated cells.     

Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture

The effects of high calcium and 1,25-(OH)2D3 on parathyroid cell growth, PTH secretion, and steady-state levels of pre-proPTH mRNA in proliferating bovine parathyroid cells were examined. Cells were established in primary tissue culture and then tested in passages 2 and 5. Cell proliferation was suppressed by 10(-9)-10(-7) M 1,25-(OH)2D3 but not by high calcium (2.5 mM). Cells at passages 2 and 5 were grown to subconfluence and then exposed for 72 h to 2.5 mM calcium or 10(-7) M 1,25-(OH)2D3. Pre-proPTH mRNA was decreased to approximately 50% of control by 2.5 mM calcium compared with 0.3 and 1.0 mM calcium. PTH secretion, as tested by low calcium stimulation for 1 h at the end of 72 h incubation, was inhibited by 50% in cells that had been exposed to high calcium compared with control. Incubation with 10(-7) M 1,25-(OH)2D3 caused a decrease in the levels of pre-proPTH mRNA and PTH release to 50% of control at 72 h. These results suggest that cultured bovine parathyroid cells, at least in early passages, have responses to high calcium and 1,25-(OH)2D3 similar to those in primary nonproliferating cultures studied earlier and that 1,25-(OH)2D3 inhibits the proliferation of parathyroid cells in a dose-responsive fashion.     

1,25-DIHYDROXYVITAMIN D3 AND BREAST CANCER*

Studies by this laboratory have demonstrated the presence of specific, high affinity 1,25-dihydroxyvitamin D3 (1,25-(OH)2, D3) receptors both in surgical specimens of human breast cancer and in breast cancer cells in culture. We report here that 1,25-(OH)2D3 receptors were found in 54% of 230 human primary breast cancers. Although receptor levels are lower than those of oestrogen receptors, using a modified and more sensitive assay method, the apparent receptor concentration is increased without altering the receptor positivity rate. Also in preliminary studies on lymph node metastases and their primary tumours, the receptor positivity rate is higher in the lymph nodes. These findings suggest that metastatic cells may be selected for the presence of 1,25-(OH)2D3 receptors. These data, taken with the evidence that 1,25-(OH)2D3 and several of its metabolites inhibit the growth of human breast cancer cells in culture, exactly analogously with the effects of oestrogens on cancer cell growth in vitro and in vivo, indicate that 1,25-(OH)2D3 or its metabolites could have a role in the ‘hormonal’ therapy of metastatic human breast cancer.     

Actions of calcipotriol (MC 903), a novel vitamin D3 analog, on human bone-derived cells: comparison with 1,25-dihydroxyvitamin D3.

The actions of a novel vitamin D3 analog calcipotriol (MC 903), on human bone-derived cells were compared to those of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both calcipotriol and 1,25-(OH)2D3 inhibited the proliferation of human osteoblast-like cells in a dose-dependent manner (10(-10)-10(-6) M), an effect observed at different cell densities. Lower concentrations of either agent exerted no marked effect on the growth of the cells compared to untreated cultures. Calcipotriol and 1,25-(OH)2D3 were equipotent in stimulating the activity of alkaline phosphatase and the synthesis of osteocalcin in human osteoblast-like cells. The stimulation of alkaline phosphatase activity and osteocalcin synthesis by both compounds was evident by 24 h and was increased progressively up to 96 h in a dose-dependent manner over the concentration range of 10(-10)-10(-6) M. The increment in both proteins was dependent on cell density and was attenuated at higher cell densities. In contrast to these actions, neither calcipotriol nor 1,25-(OH)2D3 (10(-14)-10(-6) M) affected the synthesis of prostaglandin E2. These studies indicate that calcipotriol and 1,25-(OH)2D3 exhibit a similar spectrum of activity on human osteoblast-like cells in vitro.     

1 alpha,25-Dihydroxyvitamin D3 receptors in human thymic and tonsillar lymphocytes

In vitro activated human peripheral blood lymphocytes possess the receptor protein for 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the present study we have examined whether activated lymphocytes that occur in vivo in human thymuses and tonsils also possess receptors for 1,25(OH)2D3. Freshly isolated lymphocyte preparations, from five separate surgical specimens of thymus and tonsil, were depleted of monocytes and examined, before and after fractionation on a density gradient of Percoll, for [3H] 1,25(OH)2D3 binding by means of sucrose density gradient sedimentation, by saturation analysis of the binding, and by DNA-cellulose chromatography. The state of activation of the lymphocyte preparations was determined using [3H] thymidine incorporation, DNA and RNA quantitation (using acridine orange), and by determining the presence or absence of markers of activation (interleukin-2 receptor, transferrin receptor, and HLA-DR molecules). In both the thymic and the tonsillar lymphocyte preparations we detected a 1,25(OH)2D3-binding molecule possessing sedimentation coefficient of 3.3 S and dissociation constant of 10(-10) M as well as DNA binding capability. In thymic lymphocytes, the 1,25(OH)2D3 receptor concentration correlated positively with the number of lymphocytes expressing the transferrin receptor (r = 0.84; p less than 0.05). In addition, in both thymic and tonsillar lymphocytes the concentration of 1,25(OH)2D3 receptors correlated positively with the number of cells in the G1a phase of the cell cycle (r = 0.79, p less than 0.01, and r = 0.88, p less than 0.001 for thymic and tonsillar lymphocytes, respectively). In contrast, the 1,25(OH)2D3 receptor concentration in these preparations did not correlate with the rate of cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-Dihydroxyvitamin D3 metabolism in a human osteosarcoma cell line and human bone cells

The metabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10(-11) M) and pharmacologic (3.5 x 10(-7) M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10(-7) M 1,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spectrometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from 1,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-1,25-dihydroxyvitamin D3; 24-oxo-1,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. Evidence is presented that (1) 1,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11(-11) M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10(-7) M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.     

Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10-25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10-25OHD3, induced differentiation of U937 cells. In these studies, 19/10-25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10-25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10-25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10-25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10-25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10-25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10-25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10-25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1:100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently explained.     

1,25-Dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro

We addressed the question of whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) could directly stimulate cartilage growth in vitro. Pelvic leaflets from chick embryos and scapular growth plates from fetal pigs were organ cultured in serum-free medium in the presence and absence of 1,25-(OH)2D. After 3 days of incubation, 1,25-(OH)2D had increased the pelvic cartilage wet weight 42% and the dry weight 32% above the weight of cartilages incubated in medium alone. 1,25-(OH)2D (10(-9) M-10(-12) M) caused a dose-dependent increase in weight, with maximal increases at 10(-9) M. Furthermore, two deuterized derivatives of 1,25-(OH)2D, 26,27-D6-1,25-(OH)2D3 and 24,26,27-D8-1,25-(OH)2D3, stimulated pelvic cartilage growth in vitro. 26,27-D6-1,25-(OH)2D stimulated increases in growth plate weight above growth plates incubated in medium alone. 26,27-D6-1,25-(OH)2D3 appeared to be potent at lower concentrations than 1,25-(OH)2D on growth plate cartilage. Thus, 1,25-(OH)2D stimulated in vitro growth in two growing cartilage models, the avian pelvic cartilage and the mammalian scapular growth plate cartilage.     

Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures.

Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10(-6)-10(-9) M) and PGE1 (10(-6)-10(-7) M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption was increased (10(-7) M PGE2 and 10(-6) M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that stimulation of 1,25(OH)2D3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis

When primary culture of C75BL6 mouse cortical kidney cells in serum-free medium were incubated with unlabeled 25(OH)D3, they produced a metabolite which co-migrated with authentic 1,25(OH)2D3 and which could be measured by competitive receptor assay. A metabolite co-migrating with authentic 10-oxo-19-nor-25-OH-D3 was also produced. However, when cultures were incubated with 25(OH)D3 for 1 hour or longer, 10-oxo-19-nor-25-OH-D accounted for less than 15% of the total 3H-1,25(OH)2D3 displacement activity. Production of 1,25(OH)2D3 increased with increasing content of the culture, with time of incubation, and with substrate concentration. The apparent Km was 1.4 +/- 0.6 microM and Vmax 2.6 +/- 0.4 pM/mg protein/hr. These cultures possessed a very high level of phosphodiesterase activity, as indicated by their high cyclic AMP (cAMP) response to IBMX. This high phosphodiesterase activity may have been responsible for the lack of stimulation of 1,25(OH)2D3 production by physiologic or near physiologic concentrations of parathyroid hormone (PTH) in the absence of IBMX. However, when IBMX 10(-6) M was present, bPTH 10(-9) M significantly increased production of both cAMP and 1,25(OH)2D3. There was a close correlation between 1,25(OH)2D3 production and cAMP content of the cultures (basal or stimulated). An incubation time of at least 4 hours was required for cAMP to increase 1,25(OH)2D3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH)2D3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of prostaglandin E2 production by vitamin D metabolites in growth zone and resting zone chondrocyte cultures is dependent on cell maturation.

The production of PGE2 by chondrocytes and its regulation by vitamin D metabolites was examined in this study as a function of cell maturation. Costochondral chondrocytes, derived from the resting zone and growth zone cartilage, were grown in culture to fourth passage. At confluence, they were exposed to 10(-8)-10(-11)M 1,25-(OH)2D3 or to 10(-7)-10(-10)M 24,25-(OH)2D3 for either five minutes or 3, 6, 12, or 24 hours. Indomethacin (10(-7)M) was added to one-half of the cultures to block the production of PGE2. The amount of PGE2 released into the media was determined by radioimmunoassay. Both growth zone and resting zone cells produced PGE2 in a time-dependent manner; PGE2 concentration was greater in the resting zone cell cultures. 1,25-(OH)2D3 stimulated PGE2 production by growth zone cells in a dose-dependent manner, significant at 10(-8)-10(-10)M. This effect was observed at 3 hours and remained elevated during the 24 hours of culture. 1,25-(OH)2D3 had no effect on PGE2 production by resting zone cells. However, 24,25-(OH)2D3 (10(-7)-10(-8)M) inhibited PGE2 production from 3-24 hours. No effect was noted when 24,25-(OH)2D3 was added to growth zone cells. Indomethacin reduced PGE2 production to baseline values in all groups examined. The results indicate that chondrocytes in culture produce PGE2. Production is regulated by vitamin D3 metabolites and is cell maturation-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

1a,25-(OH)2D3 Regulates 25-Hydroxyvitamin D3 24R-Hydroxylase Activity in Growth Zone Costochondral Growth Plate Chondrocytes via Protein Kinase C

Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1alpha- and 24R-hydroxylase activities and metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1alpha,25-(OH)2D3 exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10(-9) - 10(-7) M 1alpha,25-(OH)2D3 or 24R,25-(OH)2D, for 24 hours, and 1alpha- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10(-10) - l0(-8) M 1alpha,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1alpha,25-(OH)2D3 (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)2D3-dependent effect on 24,25-(OH)2D3 production was determined by comparing the response to 1alpha,25-(OH)2D3 to the response to 1beta,25-(OH)2D3. 1alpha,25-(OH)2D3 increased 24R-hydroxylase specific activity in a dose-dependent manner, 24,25-(OH)2D3 production by intact cells was also increased. The effect of 1alpha,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1alpha,25-(OH)2D3 caused an increase; 1beta,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours. 1alpha-hydroxylase activity was unaffected by either metabolite at 24 hours. 1alpha,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1alpha,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1alpha,25(OH)2D3-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

Comparative effects of a novel vitamin D analogue MC-903 and 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture

MC-903 is a novel vitamin D analogue which has been shown to promote epidermal cell differentiation but is 100 times less active than 1,25 dihydroxyvitamin D3 (1,25(OH)2D) in causing hypercalcemia. In order to determine the activity of this compound on bone cells, we have compared the effects of MC-903 and 1,25 dihydroxyvitamin D3 (1,25(OH)2D) on parameters of cell proliferation and differentiation in cultured normal human osteoblastic cells derived by migration from trabecular bone fragments. Dose response curves showed that MC-903 was 10 to 100 times less effective than 1,25(OH)2D in stimulating the synthesis of the osteoblast specific protein osteocalcin by human bone cells depending on the basal osteocalcin production. In cells showing high basal osteocalcin synthesis, 1,25(OH)2D (10(-8) M) was 2- to 3-fold more potent than MC-903 (10(-8) M) in inducing osteocalcin from 48 to 96 h of treatment. The greater activity of 1,25(OH)2D over MC-903 was observed in human bone cell cultures with elevated basal osteocalcin levels, indicating that the response to 1,25(OH)2D but not to MC-903 was amplified in cells with the higher osteoblastic characteristics. The effects of MC-903 and 1,25(OH)2D on alkaline phosphatase activity were not markedly different. Transforming Growth Factor-beta (TGF beta) (0.5 ng/mL, 48 h) was found to completely suppress the osteocalcin synthesis induced by 1,25(OH)2D (10(-8) and 10(-9) M), whereas the MC-903-induced osteocalcin synthesis was not affected, suggesting a negative interaction between TGF beta and 1,25(OH)2D but not MC-903 on osteocalcin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

1alpha,25-dihydroxyvitamin D3 induced growth inhibition of PC-3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

BACKGROUND: Prostate cancer growth inhibition by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) is best characterized in the androgen dependent LNCaP cell line, where treatment with this hormone causes cell cycle arrest and apoptosis. 1,25(OH)2D3 also inhibits the growth of PC-3 prostate cancer cells, but not through the induction of G1 arrest or apoptosis. In this study, we have sought to elucidate the mechanism/s involved in PC-3 cell growth inhibition by 1,25(OH)2D3. EXPERIMENTAL METHODS: We determined the effect of transforming growth factor beta (TGFbeta) blocking antibodies on 1,25(OH)2D3 mediated growth inhibition of PC-3 cells. In addition, we also studied the effects of 1,25(OH)2D3 on TGFbeta signaling and receptor expression. Finally, we assessed the role of TGFbeta signaling in the induction of the growth inhibitory protein, insulin like growth factor binding protein 3 (IGFBP-3), by 1,25(OH)2D3. RESULTS: We find that 1,25(OH)2D3 action in PC-3 cells is mediated through at least two distinct pathways, the TGFbeta pathway and the IGFBP-3 pathway. We show that 1,25(OH)2D3 treatment elevates TGFbeta production and signaling, as well as receptor levels, in PC-3 cells. Further, using a blocking antibody against TGFbeta substantially reduces 1,25(OH)2D3 mediated growth inhibition without affecting IGFBP-3 induction, suggesting that IGFBP-3, alone, is insufficient to inhibit the growth of PC-3 cells.     

Relative potencies of 1,25-(OH)(2)D(3) and 19-Nor-1,25-(OH)(2)D(2) on inducing differentiation and markers of bone formation in MG-63 cells.

19-Nor-1,25-(OH)(2)D(2), an analog of 1,25-(OH)(2)D(3), is used to treat secondary hyperparathyroidism because it suppresses parathyroid hormone synthesis and secretion with lower calcemic and phosphatemic activities. 19-Nor-1,25-(OH)(2)D(2) is approximately 10 times less active than 1,25-(OH)(2)D(3) in promoting bone resorption, which accounts in part for the low potency of this analog in increasing serum calcium and phosphorus. Concern that 19-nor-1,25-(OH)(2)D(2) also could be less potent than 1,25-(OH)(2)D(3) on bone formation led to a comparison of the potency of both compounds on osteoblasts. In the human osteoblast-like cell line MG-63, 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) had a similar potency in upregulating vitamin D receptor content and suppressing proliferation. Both sterols caused a similar reduction in DNA content and proliferating cell nuclear antigen protein expression. Time-course and dose-response studies on 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) induction of the marker of bone formation, osteocalcin, showed overlapping curves. The effects on alkaline phosphatase (ALP) activity also were studied in MG-63 cells that had been co-treated with either sterol and transforming growth factor-beta, an enhancer of 1,25-(OH)(2)D(3)-induced ALP activity in this cell line. Transforming growth factor-beta alone had no effect, whereas 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) increased ALP activity similarly. These studies demonstrate that 19-nor-1,25-(OH)(2)D(2) has the same potency as 1,25-(OH)(2)D(3) not only in inducing vitamin D receptor content, osteocalcin levels, and ALP activity but also in controlling osteoblastic growth. Therefore, it is unlikely that 19-nor-1,25-(OH)(2)D(2) would have deleterious effects on bone remodeling.     

Actions of 1,25-dihydroxyvitamin D3 on human mesangial cells.

In the present study, we examined specific binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by an effects of 1,25(OH)2D3 on human mesangial cells (hMC), obtained from healthy portions of tumor-bearing kidneys. Receptors for 1,25(OH)2D3 were characterized by (1) sucrose density gradient analysis, (2) Scatchard analysis, and (3) DNA affinity of the receptor molecule. Specific binding occurred by a single class of macromolecules, sedimenting with 3.5 S in sucrose density gradients (5% to 20%). Receptors showed high affinity (Kd, 1.39 x 10(-10)), and specific binding capacity (Nmax) of 821 binding sites per cell. 1,25(OH)2D3 (10(-6) to 10(-10)) reduced both DNA synthesis (by [3H]thymidine incorporation) and cell growth (by cell counting) throughout the log-phase and confluence. Further evidence of functional effects of 1,25(OH)2D3 on hMC is provided by ultrastructural studies, which showed rapid increase of electron-dense lysosomal particles in hMC exposed to 1,25(OH)2D3. The data identify actions of 1,25(OH)2D3, a molecule with recently recognized immunoregulatory roles, on hMC. The results are consistent with a role of 1,25(OH)2D3 in control of mesangial cell function.