Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)₂D₃ which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)₂D₃. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)₂D₃, 24,25-(OH)₂D₃, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)₂D₃ and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)₂D₃ having a greater effect. 24,25-(OH)₂D₃ caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)₂D₃, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation–dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457–470, 1997. © 1997 Wiley-Liss, Inc.     

A-ring analogues of 1,25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation

1,25-(OH)₂D₃ (1,25) and 24,25-(OH)₂D₃ (24,25) mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms, which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10^-8 M 1,25, 10^-7 M 24,25, or the analogues at 10^-9–10^-6 M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH)₂D₃. Plasma membranes contained increased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)₂D₃. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)₂D₃, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)₂D₃ or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)₂D₃ as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Physiol. 171:357–367, 1997. © 1997 Wiley-Liss, Inc.     

Nongenomic regulation of extracellular matrix events by vitamin D metabolites

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)₂D₃ or 24, 25-(OH)₂D₃ may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25-(OH)D₃ to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-β when incubated with exogenous 1,25-(OH)₂D₃. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.     

1,25-dihydroxyvitamin D3 specifically binds to a human breast cancer cell line (T47D) and stimulates growth

The T47D human breast cancer cell line contains a specific binding protein for 1.25-(OH)₂D₃, with 15000 sites per cell. The Kd (1.1 × 10^?10 M) and sedimentation coefficient on sucrose gradients (3.7S) are the same as those reported for the 1,25-(OH)₂D₃ receptor in other tissues. Other vitamin D₃ metabolites bound to the receptor with an order of affinities 1,25-(OH)₂D₃ > 1,24,25-(OH)₃D₃ > 25-OHD₃ > 24,25-(OH)₂D₃ > D₃. A new analogue 1β,25-(OH)₂D₃ was only as effective as 24,25-(OH)₂D₃ at displacing the hormone from the receptor. Cell growth was stimulated in a dose dependent manner by the addition of 1,25-(OH)₂D₃ (up to 0.8 nM) to the medium. A higher concentration of hormone was without effect.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)₂D₃ and 1,25-(OH)₂D₃ regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)₂D₃ affecting primarily growth zone (GC) cells and 24,25-(OH)₂D₃ affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)₂D₃ has been shown to increase phospholipase A₂ activity in GC, while 24,25-(OH)₂D₃ has been shown to decrease phospholipase A₂ activity in RC. Stimulation of phospholipase A₂ in GC caused an increase in PKC, whereas inhibition of phospholipase A₂ activity in RC cultures increased both basal and 24,25-(OH)₂D₃-induced PKC activity, suggesting that phospholipase A₂ may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A₂ inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A₂ activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A₂ inhibitors decreased both basal and 1,25-(OH)₂D₃-induced PKC activity in GC. When phospholipase A₂ activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)₂D₃-induced PKC activity in RC and increased 1,25-(OH)₂D₃-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A₂ activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A₂ activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A₂. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

1α ,25-Dihydroxyvitamin D3 activates T cells of tumor bearers through protein phosphatase 2A

 The sterol 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can inhibit T cell activation as well as restore the functional competence of suppressed T cells. The present studies determined whether 1,25(OH)₂D₃ had a differential effect on the activation of normal T cells or of suppressed T cells from mice bearing Lewis lung carcinoma tumors. Normal spleen cell proliferation in response to immobilized anti-CD3 was unaffected by the lower doses of 0.1 – 10 nM 1,25(OH)₂D₃, and was inhibited by the higher dose of 100 nM 1,25(OH)₂D₃. In contrast, 1,25(OH)₂D₃ increased proliferation and interferon γ secretion by T cells of tumor bearers in response to stimulation through T cell receptor/CD3. Assessment of mechanisms associated with the 1,25(OH)₂D₃ stimulation of tumor-bearer T cells implicated protein phosphatase 2A (PP-2A). First, PP-2A activity of spleen cells from tumor bearers was reduced compared to that of normal spleen cells but was increased by 1,25(OH)₂D₃. Second, 1,25(OH)₂D₃ stimulation of tumor-bearer T cell proliferation was dependent on this PP-2A activity as it was blocked by doses of okadaic acid that selectively inhibit PP-2A. These results suggest that 1,25(OH)₂D₃ preferentially enhances the responsiveness of immunosuppressed T cells from tumor bearers to TCR/CD3 stimulation by restoring PP-2A activity. Received: 7 November 1996 / Accepted: 2 January 1997     

Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: Existence of a paracrine system for 1,25 dihydroxycholecalciferol

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecal-ciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)₂D₃ synthesis was studied by measuring the conversion of [³H] 25(OH)D₃ to [³H] 1,25(OH)₂D₃. The purified product was tested for its ability to compete with synthetic [³H] 1,25(OH)₂D₃ for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogeneous 1,25(OH)₂D₃ were evaluated by the increase in 25(OH)D₃-24 hydroxylase activity. Furthermore the expression of 1,25(OH)₂D₃ receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)₂D₃. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D₃-24 hydroxylase response to 1,25(OH)₂D₃ were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)₂D₃ and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)₂D₃ produced locally by neighboring immature ones. © 1994 wiley-Liss, Inc.     

1,25-Dihydroxyvitamin D3 and agents that increase intracellular adenosine 3′, 5′-monophosphate synergistically inhibit fibroblast proliferation

Summary Agents that increase intracellular cAMP (cAMP elevating agents) and 1,25(OH)₂D₃ inhibit the proliferation of many cell types. We investigated the combined effect of 1,25(OH)₂D₃ and cAMP elevating agents on exponentially growing mouse 3T3 fibroblasts. The following cAMP elevating agents were used: theophylline and pentoxyfilline, which inhibit cAMP-dependent phosphodiesterase; prostaglandin E₂ which activates adenylate cyclase by a receptor-mediated mechanism; forskolin, which directly stimulates adenylate cyclase; and the cell permeable cAMP analogs 8-bromo cAMP and N⁶ benzoyl cAMP. 1,25(OH)₂D₃ and cAMP elevating agents were added to exponentially growing fibroblasts cultured in 96-well microtiter plates and cell number was monitored 3–7 d later. 1,25(OH)₂D₃ and the cAMP elevating agents as single agents inhibited the growth of the 3T3 cells. The combined treatment of the fibroblasts with 1,25(OH)₂D₃ and the cAMP elevating agents resulted in an antiproliferative effect that was more than additive. The synergistic interaction depended on the dose of 1,25(OH)₂D₃ and was apparent already at 10⁻⁸ M of the hormone. The specificity of the effect of 1,25(OH)₂D₃ was demonstrated by the finding that 24,25-dihydroxyvitamin D₃, a vitamin D metabolite with low affinity for the vitamin D receptor, did not affect the antiproliferative effect of cAMP elevating agents. From the synergistic interaction between 1,25(OH)₂D₃ and the cell permeable cAMP analogs, we infer that the site of interaction between the two signaling pathways is distal to the cAMP generating and degrading machinery.     

Studies of the internalization of vitamin D3 metabolites by cultured osteogenic sarcoma cells and their application to a non-chromatographic cytoreceptor assay for 1,25-dihydroxyvitamin D3

Cultured osteogenic sarcoma (OS) cells have been used here to study the internalization of 1,25(OH)₂D₃ and other major metabolites of D₃ by cells. Intact OS cells incubated for 1h at 37°C in medium containing [³H]1,25(OH)₂D₃ at low concentrations (0.16 to 1.6nM) take up and retain this hormone with high affinity (K_d=3.3×10^?10M) similar to that found for the hormone-receptor interaction in cytosol preparations. Vitamin D₃ and its major metabolites such as 25(OH)D₃ or 24,25(OH)₂D₃, even at supraphysiological concentrations, are not internalized by the cells when small amounts of plasma D binding protein (DBP) or human alpha-globulin are added to the incubation medium. This phenomenon can be exploited to develop a non-chromatographic cytoreceptor assay for 1,25(OH)₂D₃.     

Platelet-rich plasma and 1,25(OH)2 vitamin D3 synergistically stimulate osteogenic differentiation of adult human mesenchymal stem cells

Mesenchymal stem cell proliferation and differentiation are regulated by growth factors and systemic hormones in a concerted manner. We have examined the effects of platelet-rich plasma (PRP) and its interaction with 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human mesenchymal stem cells (hMSCs). PRP stimulated cell proliferation in a manner inversely proportional to the dose. Exposure to PRP inhibited hMSC osteogenic differentiation. Co-treatment with PRP and 1,25(OH)₂D₃, however, synergistically stimulated osteogenic differentiation. Serial co-treatment with PRP and 1,25(OH)₂D₃ showed that this synergistic effect was due to the modulation of a 1,25(OH)₂D₃-induced differentiation by PRP rather than vice versa.     

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.     

Bone turnover in rats treated with 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3

Female rats were given 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 0.25 g per 100 g body weight (bw), 25-hydroxyvitamin D₃ (25(OH)D₃), 1.7 g/100 g bw or 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) 1.7 g/100 g bw, subcutaneously three times a week for 12 weeks. Traditional variables pertaining to calcium homeostasis and growth, i.e. blood and urine calcium (Ca) and phosphate (P), serum levels of vitamin D₃ metabolites parathyroid hormone, (PTH), calcitonin (CT), prolactin (PRL) and growth hormone (GH) were measured every four weeks. This data pool was correlated with bone matrix turnover parameters, i.e. serum levels of alkaline phosphatase (ALP) and urinary hydroxyproline (u-HYP) excretion. After 12 weeks of treatment, 1,25(OH)₂D₃ significantly enhanced serum total and ionized Ca, urine Ca and urine P, and also diminished urine cAMP due to reduced renal function (creatinine clearance). However, 25(OH)D₃ administration had no such impact. 24,25(OH)₂D₃ opposed the effect of 1,25(OH)₂D₃ after 12 weeks by significantly augmenting serum P and diminishing serum levels of total Ca and ionized Ca. Cross sectional group analyses showed that criculating levels of ALP were directly related with serum 1,25(OH)₂D₃ and inversely related to serum 24,25(OH)₂D₃ and CT. Total u-HYP and per cent non-dialysable HYP (ndHYP) were reciprocally and positively correlated with serum PRL, respectively. However, no such relations were observed with serum GH.It appears that rats with elevated circulating levels of 1,25(OH)₂D₃ exhibit increased bone resorption, while augmented 24,25(OH)₂D₃ is associated with the opposite. Apparently, high bone turnover (i.e. reduced total urinary HYP and enhanced ndHYP) is associated with high serum PRL.     

Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDRs) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element–reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)₂D₃, showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)₂D₃ on fibroblasts treated with transforming growth factor β1 (TGFβ1), considered a driver of many fibrotic disorders, we found that 1,25(OH)₂D₃ inhibited TGFβ1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)₂D₃ also inhibited TGFβ1 stimulation of α-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFβ1-treated fibroblasts. Finally, we examined how 1,25(OH)₂D₃ affects epithelial–mesenchymal transformation of lung epithelial cells upon exposure to TGFβ1. We showed that the TGFβ1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)₂D₃. These observations suggest that under TGFβ1 stimulation, 1,25(OH)₂D₃ inhibits the pro-fibrotic phenotype of lung fibroblasts and epithelial cells.     

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.     

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.