1,25-Dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro

We addressed the question of whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) could directly stimulate cartilage growth in vitro. Pelvic leaflets from chick embryos and scapular growth plates from fetal pigs were organ cultured in serum-free medium in the presence and absence of 1,25-(OH)2D. After 3 days of incubation, 1,25-(OH)2D had increased the pelvic cartilage wet weight 42% and the dry weight 32% above the weight of cartilages incubated in medium alone. 1,25-(OH)2D (10(-9) M-10(-12) M) caused a dose-dependent increase in weight, with maximal increases at 10(-9) M. Furthermore, two deuterized derivatives of 1,25-(OH)2D, 26,27-D6-1,25-(OH)2D3 and 24,26,27-D8-1,25-(OH)2D3, stimulated pelvic cartilage growth in vitro. 26,27-D6-1,25-(OH)2D stimulated increases in growth plate weight above growth plates incubated in medium alone. 26,27-D6-1,25-(OH)2D3 appeared to be potent at lower concentrations than 1,25-(OH)2D on growth plate cartilage. Thus, 1,25-(OH)2D stimulated in vitro growth in two growing cartilage models, the avian pelvic cartilage and the mammalian scapular growth plate cartilage.     

1,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice.

The active form of vitamin D, 1,25(OH)2D3, can prevent various forms of experimentally induced autoimmune disorders. The aim of this study was to confirm these findings in NOD mice that spontaneously develop an autoimmune type of diabetes mellitus. Therefore, the effect of a long-term 1,25(OH)2D3 treatment on the incidence of insulitis, the histological lesion preceding diabetes, was studied. Forty-three NOD mice were treated with 1,25(OH)2D3 (5 micrograms/kg) i.p. every other day from age 21 days on, when no insulitis was present yet. At day 100, 16 control mice receiving the treatment vehicle (arachis oil) had an incidence of insulitis of 75%, whereas only 41% of the 1,25(OH)2D3-treated animals developed insulitis (P < 0.025). Calcemia, determined 24 h after the last 1,25(OH)2D3 injection was 2.5 +/- 0.1 mM, which was higher than in control animals (2.3 +/- 0.1 mM), but was well tolerated. Cellular immunity, as assessed with the mixed lymphocyte reaction performed at day 100, was not impaired significantly. This study demonstrates that long-term treatment with high doses of 1,25(OH)2D3 is able to decrease the incidence of insulitis in spontaneous autoimmune diabetes without major side effects.     

1,25-Dihydroxyvitamin D3 receptors and human colon adenocarcinoma.

Epidemiological evidence suggests that dietary calcium and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) might have a protective effect against colorectal cancers. Since the presence of receptors is required for steroid action, specific 1,25-(OH)2D3 receptors (RD3) were investigated in biopsies taken at different levels of the colon. The study involved 90 biopsies from patients operated on for colorectal adenocarcinoma. They were paired biopsies from adenocarcinoma tissue and adjacent normal mucosa. In addition, 26 normal intestinal mucosa biopsies from patients without cancer were examined. RD3 receptors were assayed in tissue extract by the dextran-coated charcoal technique and also characterized by sucrose density gradient sedimentation. Scatchard analysis showed a single class of specific high affinity-low capacity sites binding for 1,25-(OH)2D3. The incidence of RD3 was 86 per cent in normal mucosa (n = 77) and lower in carcinoma (n = 34), for which the incidence decreased significantly (P less than 0.001) from right colon (58 per cent) to left colon (37 per cent) and rectum (19 per cent). These data suggest that the normal colon is a potential target organ for 1,25-(OH)2D3 which might modulate calcium transport in the colon. Loss of receptivity to 1,25-(OH)2D3 is associated with malignant transformation.     

Short-term course of 1,25(OH)2D3 stimulates osteoblasts but not osteoclasts in osteoporosis and osteoarthritis

Summary We investigated the effect of short-term, 1,25-dihydroxyvitamin D₃ therapy (4 μg/day for 4 days) on calcium metabolism in 27 postmenopausal women (11 cases with osteoporosis and 16 cases with osteoarthritis). Bone mass at the axial and appendicular skeleton was higher in osteoarthritis than in osteoporosis. Initial values of calcium metabolism were similar. Osteoporotic and osteoarthritic patients responded with a similar significant increase in serum osteocalcin (+61% and +54%, respectively), fasting urinary calcium excretion (+178% and +124%, respectively) and 24 hour calcium excretion (+148% and +142%, respectively). Parathyroid hormone (PTH) levels decreased significantly in both groups (−30% and −18%, respectively). Osteoclastic bone resorption, evaluated by urinary hydroxyproline excretion, was not stimulated in either group. We conclude that in osteoporosis and also in osteoarthritis (1) 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulation of osteoblast function is similar in production of osteocalcin; (2) the vitamin D target tissues react adequately to 1,25(OH)₂D₃ stimulation; (3) short-term high dose of 1,25(OH)₂D₃ does not stimulate bone resorption; and (4) the differences in bone mass between osteoarthritis and osteoporosis are not related to an alteration of the responsiveness to stimulation by 1,25 (OH)₂D₃.     

Increased vitamin D receptor level enhances 1,25-dihydroxyvitamin D3-mediated gene expression and calcium transport in Caco-2 cells.

Altered vitamin D receptor (VDR) level has been proposed to explain differences in intestinal responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We tested whether the enterocyte VDR level influences 1,25(OH)2D3-mediated gene expression and transepithelial calcium (Ca) transport in the human intestinal cell line Caco-2. Cells were stably transfected with a human metallothionein (hMT) IIA promoter-human VDR (hVDR) complementary DNA (cDNA) transgene that overexpressed hVDR in response to heavy metals. In MTVDR clones, induction of 25-hyroxyvitamin D3-24-hydroxylase (24-OHase) messenger RNA (mRNA) expression by 1,25(OH)2D3 (10(-9) M, 4 h) was correlated to metal-induced changes in nuclear VDR level (r2 = 0.99). In MTVDR clones, basal VDR level was 2-fold greater and 1,25(OH)2D3-mediated Ca transport (10(-7) M, 24 h) was 43% higher than in parental Caco-2 cells. Treatment of MTVDR clones with Cd (1 microM, 28 h) increased VDR level by 68%, significantly enhanced 1,25(OH)2D3-mediated Ca transport by 24%, and increased accumulation of calbindin D9K mRNA by 76% relative to 1,25(OH)2D3 alone. These observations support the hypothesis that the enterocyte VDR level is an important modulator of intestinal responsiveness to 1,25(OH)2D3.     

1,25-Dihydroxyvitamin D3 metabolism in a human osteosarcoma cell line and human bone cells

The metabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10(-11) M) and pharmacologic (3.5 x 10(-7) M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10(-7) M 1,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spectrometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from 1,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-1,25-dihydroxyvitamin D3; 24-oxo-1,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. Evidence is presented that (1) 1,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11(-11) M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10(-7) M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.     

1,25-Dihydroxyvitamin D3 receptors and their relationship to histological features in renal cell carcinoma

The present studies were carried out in 11 human renal cell carcinomas to determine the presence of a receptor specific for an active form of vitamin D, 1,25-dihydroxyvitamin D3. Saturation and Scatchard analyses of the cytosol receptor for 1,25-dihydroxyvitamin D3 showed that nine tumors had a detectable level of the receptor (two fmol/mg. protein). The equilibrium dissociation constant of these receptors ranged between 46 and 380 pM and the binding capacity also ranged between 3.5 and 12.7 femtomol/mg. protein. Sucrose density gradient analysis of the specific binders revealed that the tumors had a receptor protein appearing as a single 3.6S peak. Two tumors which had only a trace of the receptor were high grade solid tumors consisting mainly of spindle or pleomorphic cells. Nine tumors possessing 1,25-dihydroxyvitamin D3 receptor consisted of clear and/or granular cells. Thus, the absence of the receptor was only accompanied by low differentiated sarcomatoid tumors with poor prognosis. However, so far, the amount of the receptor in the receptor-positive tumor did not relate to the other clinical and pathological features of the patients.     

Effects of 1,25-Dihydroxyvitamin D3 and Vitamin D3 on the Expression of the Vitamin D Receptor in Human Skeletal Muscle Cells

Calcified Tissue International - Vitamin D receptor (VDR) expression and action in non-human skeletal muscle have recently been reported in several studies, yet data on the activity and expression...     

Evidence forin vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes

Summary Mammalian cells increase net expression of 1,25(OH)₂D₃ receptors after exposure to physiological concentrations of 1,25(OH)₂D₃ in vitro. we examined specific binding of 1,25(OH)₂D₃ by human monocytes before and after daily administration of 1.5–2 ug 1,25(OH)₂D₃ p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (N_max) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH)₂D₃ treatment respectively. The results suggest (a) up-regulation of 1,25(OH)₂D₃ receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulationin vivo.     

23(S)25(R)-1,25-Dihydroxyvitamin D3-lactone, a naturally occurring metabolite of 1,25-dihydroxyvitamin D3, inhibits osteoclast-like cell formation in human bone marrow cultures

We have used a human bone marrow culture system that forms multinucleated cells (MNCs), 50% of which express the osteoclast phenotype, to examine the 23(S)25(R)-1,25-dihydroxyvitamin D₃-26,23-lactone (1,25-D₃-lactone) on osteoclast-like cell formation. The 1,25-D₃-lactone is a vitamin D₃ metabolite that has recently been detected in human serum under physiological conditions at concentrations of approximately 131 pg/ml (3 × 10⁻¹⁰ M) and can inhibit bone resorption induced by 1,25-dihydroxyvitamin D₃ (1,25-D₃) in vivo and in vitro. We examined the effects of the 1,25-D₃-lactone on the formation of MNC that cross-reacted with 23C6 monoclonal antibody (23C6-positive MNC), which preferentially binds to osteoclasts. All metabolites of 1,25-D₃ except the 1,25-D3-lactone increased both total and 23C6-positive MNC formation in a dose-dependent manner. In contrast, the 1,25-D₃-lactone inhibited both total and 23C6-positive MNC formation, whether the cultures were treated with 1,25-D₃, parathyroid hormone, or interleukin-1β, all potent stimulators of MNC formation. This inhibitory action of 1,25-D₃-lactone on MNC formation was very similar to the inhibitory effects of calcitonin. These data suggest that (1) 1,25-D₃-lactone is a potent natural inhibitor of formation of cells with the osteoclast phenotype at physiological concentrations and (2) the inhibition of these cells by 1,25-D₃-lactone may not result solely from its competitive binding to the 1,25-D₃ receptor. Received: April 24, 1997 / Accepted: May 30, 1997     

Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin-D28k and the 1,25-dihydroxyvitamin D3 receptor.

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.     

1a,25-Dihydroxyvitamin D3 stimulates alkaline phosphatase activity and inhibits soft-tissue proliferation in implants of bone matrix

To test the importance of vitamin D metabolites on intramuscular implants of demineralized bone, four-month-old rats were given either 1a,25-(OH)2D3 or 24R,25-(OH)2D3, or a combination of both metabolites, and sacrificed at intervals ranging from five to 35 days after implantation. Histologically there was a reduced ingrowth of mesenchymal cells into the implanted matrix cylinders in the presence of 1a,25-(OH)2D3; the reduction was followed by decreased total DNA and protein values until the 16th experimental day. At 35 days postimplantation, the quantity of new bone was the same in all treated groups. However, 1a,25-(OH)2D3 increased the alkaline phosphatase activity 60%-110% (depending on the denominator used). The metabolite 24R,25-(OH)2R3 had no effect on cell growth or the alkaline phosphatase activity. These results provide evidence for the inhibitory effect of 1a,25-(OH)2D3 on mesenchymal cell growth and its stimulatory effect on osteoblasts, which are responsible for increased alkaline phosphatase activity and new bone formation in vivo.     

Tryptophan missense mutation in the ligand-binding domain of the vitamin D receptor causes severe resistance to 1,25-dihydroxyvitamin D.

In this study, two related young children, brother and sister, exhibited severe vitamin D-resistant rickets without alopecia. Sequence analysis of the total vitamin D receptor (VDR) cDNA from skin fibroblasts revealed a substitution of the unique tryptophan of the VDR by arginine at amino acid 286 (W286R). Cultured skin fibroblasts of the two patients expressed normal-size VDR protein (immunocytochemistry and Western blotting) and normal length VDR mRNA (Northern blotting). But, these fibroblasts, as well as COS-7 cells transfected with the W286R mutant, failed to bind 3H 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The tryptophan substitution did not affect VDR trafficking toward the nucleus but abolished the 24-hydroxylase gene response to 1,25(OH)2D3, even at 10(-6) M concentrations. In conclusion, this case report of a new family with hereditary vitamin D-resistant rickets (HVDRR) emphasizes the crucial role of the VDR tryptophan for ligand binding and for transactivation of 1,25(OH)2D3 target genes. It clearly shows the clinical significance of this VDR amino acid for calcium homeostasis and bone mineralization. This observation suggests further that the presence of a stable VDR-bound ligand may not be obligatory for normal hair follicle development.     

1,25-Dihydroxyvitamin D3 but not cinacalcet HCl (Sensipar/Mimpara) treatment mediates aortic calcification in a rat model of secondary hyperparathyroidism.

BACKGROUND: Calcitriol treatment of secondary hyperparathyroidism (HPT) in chronic kidney disease (CKD) patients can lead to increased serum calcium and phosphorus, which have been associated as risk factors for vascular calcification. Cinacalcet HCl (Sensipar/Mimpara) {(alphaR)-(-)-alpha-methyl-N-[3-[3-(trifluoromethylphenyl)propyl]-1-napthalenemethanamine hydrochloride} lowers serum parathyroid hormone (PTH), calcium, phosphorus and calcium-phosphorous (CaxP) product in stage 5 CKD dialysis patients; however, its effects on vascular calcification are unknown. METHODS: Cinacalcet HCl (10 or 1 mg/kg, p.o. gavage), 1,25-dihydroxyvitamin D(3) (0.1 microg, s.c, calcitriol) or the combination was administered daily for 26 days in a rat model of secondary HPT [5/6 nephrectomy]. After dosing, aortic calcification was determined using the von Kossa staining method. Serum PTH and blood chemistries were determined on days 0, 26 and 0, 14, 26, respectively, prior to and after dosing. RESULTS: Calcitriol-treated rats had moderate to marked aortic calcification, whereas no significant calcification was observed in vehicle- or cinacalcet HCl-only treated groups. Co-administration of cinacalcet HCl with calcitriol did not attenuate the calcitriol-mediated increase in CaxP product or calcitriol-mediated aortic calcification. Both calcitriol and cinacalcet HCl therapy significantly reduced serum PTH levels. Calcitriol significantly elevated serum calcium, serum phosphorous and CaxP product above pretreatment levels, or those seen with vehicle or cinacalcet HCl. Cinacalcet HCl (10 or 1 mg/kg) decreased serum ionized calcium and decreased calcitriol-induced hypercalcaemia. CONCLUSION: Cinacalcet HCl and calcitriol both effectively reduce PTH, albeit via different mechanisms, but unlike calcitriol, cinacalcet HCl did not produce hypercalcaemia, an increased CaxP product or vascular calcification.     

Up-Regulation of Inducible Nitric Oxide (NO) Synthase and NO Production in HL-60 Cells Stimulated to Differentiate by Phorbol 12-Myristate 13-Acetate Plus 1,25-Dihydroxyvitamin D3 Is Not Obtained With Dimethylsulfoxide Plus 1,25-Dihydroxyvitamin D3

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] augments the action of either prostaglandin E₁ (PGE₁) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)₂D₃ with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1α, IL-6, tumor-necrosis factor (TNF)-α, PGE₂, and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)₂D₃ (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)₂D₃ could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 μM) blocked the synergistic interaction between 1,25(OH)₂D₃+ PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE₂ (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)₂D₃+ PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)₂D₃ treatments. Our findings suggest that interaction between 1,25(OH)₂D₃ and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE₂. Received: 13 March 1997 / Accepted: 14 November 1997     

1,25-Dihydroxyvitamin D3 effects on collagen and DNA synthesis in periosteum and periosteum-free calvaria

1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is essential for normal growth and mineralization, but its direct effects on various aspects of bone formation remain controversial. 1,25(OH)₂D₃ was studied for its effects on DNA, collagen and noncollagen protein synthesis, and alkaline phosphatase activity (APA) in the periosteum and periosteum-free bone from 21-day fetal rat calvariae. 1,25(OH)₂D₃(0.01 to 10 nM) inhibited the incorporation of ³H-proline into collagenase-digestible protein (CDP) and the percent of collagen synthesized, and, at 10 nM, APA in the periosteum-free bone. 1,25(OH)₂D₃ inhibited type I collagen without affecting other collagen types. In contrast, 1,25(OH)₂D₃ at 10 nM caused a small but significant stimulation of the incorporation of ³H-thymidine into acid-insoluble residues (DNA) and on DNA content; both effects were exclusively observed in the periosteum. Hydroxyurea did not modify the inhibitory effect of 1,25(OH)₂D₃ on ³H-proline incorporation into CDP. These studies indicate that 1,25(OH)₂D₃ stimulates periosteal DNA synthesis but inhibits type I collagen synthesis and APA in the periosteum-free bone.