1,25-Dihydroxy vitamin D3 inhibits adipocyte differentiation and gene expression in murine bone marrow stromal cell clones and primary cultures

Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10(-12)-10(-8) M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.     

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.     

Therapeutic efficacy of 1alpha,25-dihydroxyvitamin D3 and calcium in osteopenic ovariectomized rats: evidence for a direct anabolic effect of 1alpha,25-dihydroxyvitamin D3 on bone

It is an important question for clinical therapy of osteoporosis with vitamin D metabolites whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone. In this study, female 6-month-old Fischer rats were either ovariectomized (OVX) or sham operated. One month before surgery, all rats were placed on a diet containing 0.25% calcium and were kept on this diet throughout the study. Beginning 3 months post-OVX, groups of OVX rats orally received vehicle, a calcium supplement, low dose (0.025 microg/kg x day) or high dose (0.1 microg/kg x day) 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. By 3 months postsurgery, pretreatment OVX controls had lost 74% and 37% of tibial and vertebral cancellous bone, respectively. Two-way factorial ANOVA showed that a 3-month treatment of osteopenic OVX rats with 1,25-(OH)2D3 dose dependently increased vertebral and tibial cancellous bone mass (P < 0.001 and P = 0.021, respectively) and trabecular width (P < 0.001). Furthermore, 1,25-(OH)2D3 increased serum calcium (P = 0.028) and urinary calcium excretion (P < 0.001) and reduced serum PTH levels (P < 0.001), osteoclast numbers (P < 0.001), and urinary collagen cross-links excretion (P < 0.001). Calcium supplementation alone was without therapeutic effect, and there was no significant two-way interaction between the individual treatment effects of 1,25-(OH)2D3 and calcium on bone mass. These data indicate that the anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats are mediated through a direct activity on bone.     

Antisense oligonucleotides targeted against protein kinase Cbeta and CbetaII block 1,25-(OH)2D3-induced differentiation

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.     

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.     

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.     

Combinational effects of vitamin D3 and retinoic acid (all trans and 9 cis) on proliferation, differentiation, and programmed cell death in two small cell lung carcinoma cell lines

The effects of a combination of vitamin D3 [1,25(OH)2D3] and retinoic acid (RA) on proliferation, differentiation, and apoptosis of the human small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H209 were evaluated. Cell proliferation was inhibited by 1,25(OH)2D3 and RA alone. The combination of 1,25(OH)2D3 and the cis form of retinoic acid resulted in an additive decrease in cell proliferation and the induction of apoptosis in various concentrations. Moreover, 3H-thymidine incorporation was inhibited and the number of viable cells was decreased. The characteristics of the apoptotic cells were examined and confirmed by morphologic analysis, light and electron microscopy, and fluorescence detection. It was concluded that 1,25(OH)2D3 and RA exert additive effects on the inhibition of proliferation and the induction of apoptosis in both the NCI-H82 and the NCI-H209 SCLC cell lines. This finding has important implications for the use of retinoids and 1,25(OH)2D3 in cancer prevention and in the therapy of small cell lung carcinoma.     

A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells: application to the characterization of serum response element binding proteins during osteoclast differentiation

We have developed a new method that allows the purification of large numbers of both authentic osteoclasts (OCs) and in vitro differentiated osteoclast-like cells (OCLs) from rabbits. We characterized the OCLs in terms of the expression of different phenotypic markers of OC differentiation and their ability to resorb bone. The method provides a system for performing biochemical and molecular studies of OC differentiation and function in a single species. We used this system to characterize the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of proteins that bind to the serum response element (SRE) of the c-fos promoter. We found that OCLs and OCs displayed similar SRE-binding activities, including the serum response factor (SRF). This pattern is established in a time-dependent and cell-specific manner in response to long-term treatment of rabbit bone marrow by 1,25(OH)2D3. Thus, 1,25(OH)2D3 can modulate SRF and/or SRF-related protein. This finding may contribute to understanding the role of c-Fos in the regulation of OC differentiation.     

Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulin-induced adipocyte differentiation in 3T3-L1 cells

Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

A group of deltanoids (vitamin D analogs) regulate cell growth and proliferation in small cell carcinoma cell lines

A group of deltanoids has been used for studying the inhibition of cell growth and proliferation in two small cell lung carcinoma (SCLC) in vitro. The biologically active deltanoid, 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3), has functions beyond its classical roles of stimulating calcium transport and serum calcium. It also causes the differentiation of a variety of precursor cells and suppresses growth. Although 1,25(OH)2D3 has an inhibitory effect on growth of certain malignant cells, its hypercalcemic effect has prevented clinical applications. Several new deltanoids, which showed comparable or even greater abilities to induce differentiation and to inhibit proliferation, have been identified. Furthermore, these synthetic deltanoids have been shown to be less effective on calcium metabolism and less hypercalcemic. We have selected four synthetic deltanoids; MC-903, 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E). When compared with 1,25 (OH)2D3, these deltanoids showed considerable potency on cell growth and proliferation in the NCI-H82 and the NCI-H209 SCLC lines. Cells were treated with various concentrations of deltanoids. They inhibited the growth and proliferation of both SCLC cells in vitro in a time-and dose-dependent manner, as determined by cell number and 3H-thymidine uptake. 19-nor-22(E) showed an antiproliferative effect significantly comparable to 1,25(OH)2D3 in the NCI-H82 cell line 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E) inhibited the cell growth in the NCI-H209 cells within the same significance as 1,25 (OH)2D3. The degree of the suppressive effect of the deltanoids was cell line dependent.     

Effect of vitamin D3 and 1,25(OH)2D3 on growth of neoplastically derived cell lines and their alkaline phosphatase activity

Inhibitory effects of 1,25(OH)2D3 and D3 on growth of four neoplastically derived cells were observed in human acute leukemia cell culture CEM-C-1 and CEM-C-7, human cervical carcinoma cell lines C-4-1 and human epithelioid carcinoma cells of cervix HeLa S3K. Concurrently, in dexamethasone-responsive cells C-4-1 and HeLa S3K there was a 1,25(OH)2D3 and D3 induced elevation of alkaline phosphatase with 1,25(OH)2D3 showing the greater effects. It is supposed that vitamins D3-induced alkaline phosphatase activity in malignant cells, which is proposed to be a possible marker of cell differentiation, can be associated with the membrane effects of these vitamins.     

Distribution and metabolism of F6-1,25(OH)2 vitamin D3 and 1,25(OH)2 vitamin D3 in the bones of rats dosed with tritium-labeled compounds

26,26,26,27,27,27-Hexafluo-1,25(OH)2 vitamin D3, the hexafluorinated analog of 1,25(OH)2 vitamin D3, has been reported to be several times more potent than the parent compound regarding some vitamin D actions. The reason for enhanced biologic activity in the kidneys and small intestine appears to be related to F6-1,25(OH)2 vitamin D3 metabolism to ST-232, 26,26,26,27,27,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3, a bioactive 23S-hydroxylated form that is resistant to further metabolism. Since F6-1,25(OH)2 vitamin D3 is considered to prevent osteoporotic decrease in bone mass by suppressing bone turnover, we here compared the distribution and metabolism of [1 beta-3H]F6-1,25(OH)2 vitamin D3 and [1 beta-3H]1,25(OH)2 vitamin D3 in bones of rats by autoradiography and radio-HPLC. In the dosed groups, radioactivity was detected locally in the metaphysis, the modeling site in bones. As compared with the [1 beta-3H]1,25(OH)2 vitamin D3 case, [1 beta-3H]F6-1,25(OH)2 vitamin D3 was significantly retained in this site, and moreover, it mainly persisted as unchanged compound and ST-232. These findings indicate that the reason for the higher potency of F6-1,25(OH)2 vitamin D3 than 1,25(OH)2 vitamin D3 in bones are linked with increased distribution and reduced metabolism.     

Specific-heat critical behavior of the structural random-field system Dy(AsxV1-x)O4

Vitamin D3 derivative 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) exerts various biological effects in cells that possess vitamin D3 receptor (VDR), including enhancement of cell differentiation and inhibition of cell proliferation. These activities of 1,25(OH)2D3 might be responsible for its anti-neoplastic effects, as shown in various experimental systems. The aim of this study was to compare the anti-angiogenic activity of 1,25(OH)2D3, retinoids, and interleukin-12 (IL-12) in an experimental tumor cell-induced angiogenesis assay in mice. Tumor cell-induced angiogenesis assay was performed in x-ray immunosuppressed BALB/c mice by intradermal injections of human tumor cell lines of different origin. The injections resulted within 3 d in a local formation of new blood vessels, and the intensity of angiogenesis correlated with the number of injected cells. Systemic treatment of the mouse recipients with 1,25(OH)2D3 significantly decreased angiogenesis, comparable to the effect of retinoids (all-trans retinoic acid [RA], 9-cis RA and 13-cis RA) and of IL-12. In vitro preincubation of the cells with all compounds (except IL-12) led to the inhibition of their angiogenic capability in vivo. Moreover, combination of 1,25(OH)2D3 and retinoids resulted in a synergistic inhibition of angiogenesis. The results strongly suggest that anti-angiogenic compounds with relatively low toxicity (e.g., 1,25(OH)2D3, retinoids, and IL-12) and their combinations could be beneficial in the treatment of some angiogenesis-associated malignancies.     

Effectiveness of the elcatonin transdermal system for the treatment of osteoporosis and the effect of the combination of elcatonin and active vitamin D3 in rat

The efficacy of percutaneous elcatonin (EC), a hypocalcemic peptide, in the treatment of experimental osteoporosis in rats was evaluated in vivo. Additionally, the effect of the combined use of EC and active vitamin D3 (1,25(OH)2D3) for the treatment was compared with those of three other groups: 1,25(OH)2D3 alone, estradiol plus 1,25(OH)2D3, and a placebo, and low calcium diet (low Ca). The EC transdermal system and the EC plus 1,25(OH)2D3 system, applied to the rat abdominal skin 6 times for 48 h, significantly increased the ash weight and calcium content of the tibia in the rats, compared with those of placebo group (p < 0.05). The EC systems also slightly lowered the alkaline phosphatase activity in plasma of the morbid rats, without a difference in the plasma calcium content. These EC systems were superior to the 1,25(OH)2D3 system and the estradiol plus 1,25(OH)2D3 system in improving osteoporotic parameters. Thus, the EC systems were concluded to be an efficient drug delivery system for Paget's disease and osteoporosis.     

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.     

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.