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.     

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.     

Computer analysis of 1,25-dihydroxyvitamin D3-receptor regulated promoters: identification of a candidate D3-response element

The receptor for the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] belongs to a superfamily of steroid and triidothyronine (T3) receptors. As yet no 1,25(OH)2D3 response element (DRE) has been identified. Since the T3 and 1,25(OH)2D3 receptors are structurally homologous, we have used the nucleotide sequence of the T3 response element to carry out a computer search of the promoter region of several 1,25(OH)2D3 regulated genes. We report here one candidate DRE from the chick calbindin-D28K gene (AGCCCAATGGCTGAACA) and two candidate DRE's from the rat osteocalcin gene (TCCCCACTGGATGAGCG and CCTGCACTGGGTGAATGA).     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Vitamin D–Mediated Hypercalcemia and Cushing Syndrome as Manifestations of Malignant Pleural Mesothelioma

ObjectiveTo report a case of coincident hypercalcemia and Cushing syndrome arising from mesothelioma.MethodsWe describe the clinical, laboratory, imaging, and pathologic findings of a patient with malignant pleural mesothelioma and elucidate the underlying biologic mechanisms resulting in concurrent overexpression of steroid and polypeptide hormones.ResultsA 62-year-old woman presented with chest discomfort and cough. Radiologic imaging revealed a diffuse pleural-based mass encasing the right lung. There was no invasion into the chest wall, diaphragm, or mediastinum, and there was no distant disease. Laboratory analyses documented hypercalcemia and Cushing syndrome, which were due to ectopic overproduction of 1,25-dihydroxyvitamin D (1,25[OH]₂D) and corticotropin. Surgical resection resulted in normocalcemia with normalization of serum 1,25(OH)₂D and reduction in hypercortisolemia. The extrapleural pneumonectomy specimen revealed overexpression of the 1,25(OH)₂D synthetic enzyme 25- hydroxyvitamin-D-1α-hydroxylase (1α-hydroxylase) and underexpression of the 1,25(OH)₂D catabolic enzyme 24- hydroxylase. Immunohistochemistry and electron microscopy demonstrated corticotropin and secretory granules in the tumor tissue.ConclusionThese findings support the evidence for a paracrine role of vitamin D in the resistance of the human host to antigen. (Endocr Pract. 2008;14:1011-1016)     

Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)₂D₃ and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)₂D₃ enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)₂D₃ treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)₂D₃-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)₂D₃ and did not change that by 1,25(OH)₂D₃ plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)₂D₃-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na⁺,K⁺-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.     

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.     

Metabolism and Action of the Hormone Vitamin D: Its Relation to Diseases of Calcium Homeostasis

Extensive experimental evidence has established a significant role of calciferol in the maintenance of normal calcium homeostasis. Present knowledge indicates that vitamin D₃ must first be converted to 25-OH-D₃ and then to 1,25(OH)₂D₃, the most active known form of the steroid. Many of the factors regulating the rate of production of this last steroid from its precurser have been evaluated, and the concept that vitamin D functions as a steroid hormone seems to be well established.Deranged action of calciferol, caused by impaired metabolism of the steroid or through altered sensitivity of target tissues, may be involved in the pathophysiology of several disease states with abnormal calcium metabolism.It is noted that liver disease, osteomalacia due to anticonvulsant therapy, chronic renal failure, hypophosphatemic rickets, hypoparathyroidism, hyperparathyroidism, sarcoidosis and idiopathic hypercalciuria have possible relation to alterations in metabolism or action of vitamin D.The future clinical availability of 1,25(OH)₂D₃ and other analogs of this steroid may offer potential therapeutic benefit in the treatment of certain of the disease entities discussed.     

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.