Modulation of myelomonocytic U937 cells by vitamin D metabolites

Investigation of the effects of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ on the proliferation and differentiation of the human myelomonocytic cell line U937 has been complemented with studies of the effect of the same metabolites on the number of nuclear receptors for 1,25(OH)₂D₃. Both 1,25(OH)₂D₃ and 24,25(OH)₂D₃ inhibit the proliferation of U937 cells in a dose-dependent manner. The concentrations of 24,25(OH)₂D₃ required to produce this effect were 100-times greater than those of 1,25(OH)₂D₃. Inhibition of proliferation was associated with increased expression of the CD14 and 200 kDa 63D3 antigens thus confirming differentiation of U937 towards a more mature cell type.Studies of the nuclear receptor for 1,25(OH)₂D₃ showed that pre-treatment of the cells with 1,25(OH)₂D₃ resulted in an apparent 40% decrease in the number of detectable 1,25(OH)₂D₃ receptors as compared to control U937 cells. This is due to the fact that the 1,25(OH)₂D₃ binds to U937 cell nuclei during culture and thus blocks the subsequent binding of radiolabelled 1,25(OH)₂D₃ used to measure the number of 1,25(OH)₂D₃ receptors. Measurement of the binding of unlabelled 1,25(OH)₂D₃ by radioimmunoassay indicated that pre-treatment of the cells with 1,25(OH)₂D₃ increased the capacity of U937 to bind the hormone, although measurement of these receptors by whole cell assay was prevented by the binding of 1,25(OH)₂D₃ itself. This effect was not observed with 24,25(OH)₂D₃ which was more easily displaced from binding sites by radiolabelled 1,25(OH)₂D₃ and it appears to act through low affinity binding to the 1,25(OH)₂D₃ receptor.     

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.     

Inhibitory effect of nitric oxide on the induction of cytochrome P450 3A4 mRNA by 1,25-dihydroxyvitamin D3 in Caco-2 cells

Cytochrome P450 (CYP)-dependent drug metabolism decreases in vivo and in cultured hepatocytes under various immunostimulatory conditions. Nitric oxide (NO) released during inflammation is presumed to be involved in this phenomenon. CYP3A4, which is abundant in the liver and small intestine and participates in the metabolism of various drugs, is known to be induced by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the colon carcinoma cell line Caco-2. In this study we examined whether NO affected CYP3A4 gene expression induced by 1,25(OH)₂D₃ in Caco-2 cells. Induction of CYP3A4 mRNA by 1,25(OH)₂D₃ was suppressed in a dose-dependent manner by treatment with the NO donors NOR-4 (15–500 μM) or S-nitroso-N-acetyl-penicillamine (30 μM-1 mM), which spontaneously release NO. These results indicated that NO has an inhibitory effect on the induction of CYP3A4 mRNA by 1,25(OH)₂D₃ in Caco-2 cells. Treatment with the guanylate cyclase inhibitor ODQ failed to prevent the inhibition of induction of CYP3A4 mRNA by 1,25(OH)₂D₃. 8-Bromo cGMP had no effect on 1,25-(OH)₂D₃-induced CYP3A4 gene expression. Therefore, the suppression of CYP3A4 mRNA by NO might be mediated through a guanylate cyclase-independent pathway.     

1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)₂D₃ in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)₂D₃ on chondrocytes in OA remains obscure. Effect of 1,25-(OH)₂D₃ on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)₂D₃ activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)₂D₃-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)₂D₃ on MMP13 expression, transfection assays were used to show that 1,25-(OH)₂D₃ activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)₂D₃ in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)₂D₃ activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)₂D₃ to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.     

Effect of 1,25-dihydroxyvitamin D3 on induction of scavenger receptor and differentiation of 12-O-tetradecanoylphorbol-13-acetate-treated THP-1 human monocyte like cells

We investigated the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on the expression of scavenger receptors in human monocytic cell line (THP-1 cells) treated for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces their differentiation into macrophages. The capacity to degrade ¹²⁵I-labeled acetyl low density lipoprotein (LDL) was developed in accordance with macrophage differentiation. The treatment with 10 nM 1,25(OH)₂D₃ for 72 h inhibited the degradation of acetyl LDL by THP-1 macrophages in a dose-dependent manner, suggesting that 1,25(OH)₂D₃ inhibits scavenging function in macrophages. In order to clarify the mechanism of its inhibitory effect on degradation of acetyl LDL, we performed the ligand binding assay using ¹²⁵I-labeled acetyl LDL. Scatchard analysis revealed that 1,25(OH)₂D₃ decreased the number of scavenger receptors without changing the affinity for acetyl LDL. We next examined the effect of 1,25(OH)₂D₃ on the expression of scavenger receptor mRNA. The mRNA of type I scavenger receptor was first detected in THP-1 cells 4 days after the treatment with TPA, the mRNA level increased up to 6 days, and then decreased. The treatment with 1,25(OH)₂D₃ for 72 h dramatically decreased the mRNA levels after the acquisition of macrophage phenotypes as evidenced by nonspecific esterase staining. However, 1,25(OH)₂D₃ did not affect the activity of non-specific esterase nor the induction of interleukin-1β mRNA by lipopolysaccharide in THP-1 macrophages. These findings suggest that 1,25(OH)₂D₃ exclusively decreases the expression of scavenger receptors in TPA-induced THP-1 macrophages without affecting the basic cellular functions as macrophages. © 1995 Wiley-Liss Inc.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-Dihydroxyvitamin D3 in normal human keratinocytes

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)₂D₃ and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)₂D₃ and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)₂D₃ and to all but the highest concentration (10⁻⁶ M) of RA. 1,25(OH)₂D₃ exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10⁻⁹ M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)₂D₃. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)₂D₃ to raise INV levels, possibly by inhibiting 1,25(OH)₂D₃-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)₂D₃, but assessment of a single marker, such as INV protein, may be misleading. J. Cell. Physiol. 174:1–8, 1998. © 1998 Wiley-Liss, Inc.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

The Establishment of a HeLa Cell Demonstrating Rapid Mitogen-Activated Protein Kinase Phosphorylation in Response to 1α,25-Dihydroxyvitamin D3 by Stable Transfection of a Chick Skeletal Muscle cDNA Library

1α,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] phosphorylates the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, within 30 sec in primary cultured chick skeletal muscle cells. MAPK of HeLa cell lines, which had been stably transfected with a cDNA library derived from mRNA of chick skeletal muscle cells, was also rapidly phosphorylated by 1,25-(OH)₂D₃. These cell lines have the potential to be a good tool for further investigation of rapid non-genomic mechanism activated by 1,25-(OH)₂D₃.