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.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D₃ was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D_28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca²⁺ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M_R = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D_28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D_28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.     

Screening of Vitamin D activity (VDA) of Solanum glaucophyllum leaves measured by radioimmunoassay (RIA)

The ingestion of Solanum glaucophyllum (SG) causes a calcinosis of cattle named Enteque Seco (ES). The toxic principle is the 1,25-(OH)₂D₃, mainly conjugated as glycoside. This study aims to validate a simple novel method of evaluation of the VDA of SG leaves. Aqueous extracts of SG were purified using C₁₈ minicolumns and assayed by RIA with an antibody raised in rabbits by injection of the acid—C22, 1-(OH)Vitamin D₃. Data were expresed as glycoside equivalent to 1,25-(OH)₂D₃ in ng/g of dry leaves. We compared this data with 1,25-(OH)₂D₃ levels measured, in the same samples, by liquid chromatography (HPLC) after enzyme cleavage. This procedure involved the incubation of SG leaves with rumen fluid, followed by C₁₈-OH solid phase extraction. The 1,25-(OH)₂D₃ fraction was run by HPLC and detection was achieved using a photodiode array detector. Data were expressed as micrograms of 1,25-(OH)₂D₃/g dry leaves. A significant regression of 1,25-(OH)₂D₃ levels (Y) as a function of glycoside RIA 1,25-(OH)₂D₃ equivalents (X) was found: Y = 12.02 + 0.35X [R = 0.81; P = 0,0002; N = 15], allowing us to conclude that this novel assay could be used to estimate the amount of this active principle contained in SG leaves.     

Functional involvement of calcium in the homologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in osteoblast-like cells

In several cell types 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) causes up-regulation of its receptor. The present study demonstrates that in the osteoblast-like cell line UMR 106 this up-regulation is inhibited by two different calcium channel blockers (nitrendipine, verapamil). Also with chelating extracellular calcium (EGTA) and by inhibition of calcium release from intracellular stores (TMB-8) comparable results were obtained. These findings indicate that calcium is functionally involved in this cellular response to the steroid hormone 1,25(OH)₂D₃. Moreover, data obtained with EGTA show that the 1,25(OH)₂D₃ receptor level is closely regulated by the extracellular calcium concentration.     

Novel insights in the immune function of the vitamin D system: synergism with interferon-beta

The 1,25(OH)₂D₃ analog, TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)₂D₃), has an interesting dissociation profile between its potent immunomodulatory and its calcemic effects in vivo. The strong immunomodulatory potency of TX527 is reflected by its ability to attenuate experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). At present most MS patients are being treated with systemic IFN-β administration. The aim of this study was to investigate whether combining IFN-β with TX527 could empower its EAE-protective effects. We evaluated also combinations with the standard immunosuppressant cyclosporin A (CsA). EAE was induced in SJL mice by PLP immunization, treatment was started 3 days before disease induction. The TX527 + IFN-β combination resulted in significant disease protection which was superior to the effect of both treatment separately. No disease amelioration, even aggravation, was obtained with the IFN-β + CsA combination. By adding TX527 to the IFN-β + CsA combination near complete protection from EAE was achieved (100% protection from paralysis, mean maximal score of 1.8 ± 1.5, both p < 0.05 versus controls and all individual treatments). From these data we conclude that adding TX527 to an IFN-β and/or CsA treatment results in clear additional immunomodulatory effects in EAE prevention and is therefore a potentially interesting candidate to be considered in clinical intervention trials in MS.     

Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca2+-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation.     

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.     

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Vitamin D analogues in the treatment of psoriasis.

Psoriasis is a chronic hyperproliferative skin disease in which inflammatory and immunologic processes may play important pathophysiologic roles. Recently the skin has been identified as a target tissue for vitamin D. Because 1,25-dihydroxy vitamin D3 inhibits epidermal proliferation and promotes epidermal differentiation, it has been introduced for the treatment of psoriasis vulgaris. In addition to 1,25-(OH)2-D3, synthetic vitamin D3 analogues have undergone clinical evaluation. Calcipotriol (INN) (calcipotriene [USAN]) has been studied most extensively. Compared with 1,25-(OH)2-D3, calcipotriol is about 200 times less potent in its effects on calcium metabolism, although similar in receptor affinity. Topical calcipotriol 50 micrograms/g applied twice daily is efficacious and safe for the treatment of psoriasis. Because topical calcipotriol is slightly more efficacious than betamethasone 17-valerate and dithranol, calcipotriol should be considered a first line drug in the management of psoriasis. These results illustrate that it is possible to separate the vitamin D effects on the cellular level from those on calcium metabolism not only in vitro, but also in a clinical setting.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.