Ethnic origin and serum levels of 1alpha,25-dihydroxyvitamin D3 are independent predictors of coronary calcium mass measured by electron-beam computed tomography

BACKGROUND: Blacks have been found to have lower amounts of coronary calcium as well as higher levels of the osteoregulatory steroid 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] than whites. We sought to determine if racial differences in coronary calcium mass could be explained by differences in serum levels of 1,25(OH)2D3. METHODS AND RESULTS: We evaluated standard coronary risk factors, quantified coronary calcium mass with electron-beam computed tomography (EBCT), and measured serum 1,25(OH)2D3 with radioimmunoassay in 283 high-risk subjects (51 [180%] black, 232 [82%] white). Black subjects had lower masses of coronary calcium than whites (14 versus 47 mg; P=.003). Serum 1,25(OH)2D3 levels were slightly higher in blacks (41 versus 38 pg/mL; P=.05). Log 1,25(OH)2D3 levels were inversely proportional to log-transformed calcium mass (r=-.19; P=.001) in both races. Multivariate linear regression demonstrated that both black race (P=.02) and 1,25(OH)2D3 levels (P=.007) contributed inversely and independently to coronary calcium mass. However, an interaction term of racex1,25(OH)2D3 did not significantly contribute to coronary calcium mass, indicating that other undetermined factors in addition to 1,25(OH)2D3 are responsible for ethnic differences in coronary calcium mass. CONCLUSIONS: Both black race and serum levels of 1,25(OH)2D3 are independent negative determinants of coronary calcium mass. Nevertheless, diminished amounts of coronary calcium in blacks are not accounted for by higher 1,25(OH)2D3 levels.     

Partial prevention of active Heymann nephritis by 1 alpha, 25 dihydroxyvitamin D3

The hormone 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunosuppressive effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune-mediated diseases. To test its in vivo activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20-epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 microgram/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7-9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P = 0.06) was reached in the 1,25(OH)2D3 (0.5 microgram/kg) group. The size of the immune deposits also was found to be substantially smaller in the groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium concentration with 2.5 mg/dl above the pretreatment values, and the urinary calcium excretion by a factor of 3-5 during the short treatment time. Treatment with the analogue KH1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosuppressive potency.     

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.     

Serum 1alpha,25-dihydroxyvitamin D3 accumulates into the fracture callus during rat femoral fracture healing

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.     

Synthesis and biological activities of 2 beta-chloro-, 2 beta-fluoro-, and 2 beta-methoxy-1 alpha,25-dihydroxyvitamin D3

Using 1 alpha,2 alpha-oxido-cholesta-5,7-diene-3 beta,25-diol (2) as a starting material, the provitamins of calcitriol with an additional 2 beta-chloro-, 2 beta-fluoro-, and 2 beta-methoxy-substituent (3,4,5) are obtained by transdiaxial opening of the oxirane ring with nucleophiles. An efficient irradiation process is described and used for the synthesis of the 2 beta-substituted calcitriols NS2 (2 beta-Cl), NS6 (2 beta-F), and NS7 (2 beta-OCH3). The affinity of these three vitamin D3 derivatives to the vitamin D receptor (VDR) and was determined. These three A-ring derivatives of 1,25(OH)2D3 were further tested for their proliferation-inhibitory and anti-adipogenic activity and gene regulatoric activity in the vitamin D3-sensitive, murine, mesenchymal cell line C3H10T1/2. The VDR-affinity of the 2 beta-chloro derivative, NS2 (2 beta-Cl), was identical to 1,25(OH)2D3 and its vitamin D binding protein (DBP)-affinity was in the range of 1,25(OH)2D3. NS2 inhibited the proliferation of C3H10T1/2(BMP-4)-cells in the presence of fetal calf serum (FCS) 9 times, and, in the absence of FCS, 111 times lower, as compared with 1,25(OH)2D3. The ID50 dose of adipogenesis-inhibition of NS2 was 13 times higher than the ID50 dose of 1,25(OH)2D3. NS6 (2 beta-F) displayed a slightly higher affinity than 1,25(OH)2D3 to the VDR and DBP-affinity. The proliferation-inhibitory activity in the presence of FCS was 90 times higher, as compared with 1,25(OH)2D3. In the FCS-free proliferation assay NS6 displayed an inhibitory activity in the range of 1,25(OH)2D3. NS6 showed an 5 times higher potency to inhibit (pre)adipocyte-differentiation in C3H10T1/2(BMP-4)-cells than 1,25(OH)2D3. NS7 (2 beta-OCH3) showed the lowest VDR-affinity and the highest DBP-affinity of the tested substances, as compared with 1,25(OH)2D3 (11 times lower and 35 times higher respectively). Its proliferation-inhibitory activity in the FCS-free medium was 9 times and in the FCS-containing assay 67 times lower in comparison with 1,25(OH)2D3. A 1250 times higher NS7-dose was needed to reach the anti-adipogenic potency of 1,25(OH)2D3. All tested substances displayed a similar ability to activate a vitamin D responsive element-regulated reporter gene compared to 1,25(OH)2D3 (NS2 and NS6: 1.3 times higher activity; NS7: 1,4 times lower activity).     

Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D3 production in aging does not involve protein kinase A or C pathways

Intestinal calcium absorption declines with aging as a result of decreased renal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] biosynthesis. At least part of the decline in 1,25-(OH)2D3 may be due to acquired resistance to parathyroid hormone (PTH) stimulation of renal 25-hydroxyvitamin D1-hydroxylase (1-OHase) activity. To test whether aging rats can increase 1,25-(OH)2D3 production in response to PTH, male rats of the same litter were fed a normal Ca diet and were sacrificed at 175-225 g (young rats) or 3 months later at 350-425 g (aging rats). At sacrifice, basal serum 1,25-(OH)2D3 levels (88 +/- 16 versus 49 +/- 8 pg/ml, P < 0.05) and in vitro renal proximal tubule 1-OHase activity (178 +/- 15 versus 77 +/- 5 pmol/mg protein/5 minutes, n = 6, P < 0.001) were lower in aging animals. rPTH-(1-34) (10(-11) or 10(-7) M) increased in vitro 1,25-(OH)2D3 secretion by perifused renal proximal tubules from young but not aging rats. For young and aging rats, rPTH-(1-34) (10(-7) M) increased proximal tubule cAMP-dependent protein kinase (PKA) activity, and lower concentrations (10(-11) M) stimulated translocation of protein kinase C (PKC) activity from cytosolic to soluble membrane proximal tubule cell fractions. The results of this study show that PTH activation of 1,25-(OH)2D3 production may involve both signaling pathways, with the PKC pathway responsive to lower concentrations of the hormone. The acquired resistance to PTH stimulation of 1,25-(OH)2D3 production in aging appears not to involve the hormonal activation of PKA or PKC.     

The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25-hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens

Three experiments were conducted to determine the influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol (vitamin D3), 25-hydroxycholecalciferol [25-(OH)D3], and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in young broiler chicks. Two levels of vitamin A (1,500 and 45,000 IU/kg or 450 and 13,500 microg) were fed in all experiments. In Experiment 1, chicks were fed six levels of vitamin D3 (0, 5, 10, 20, 40, and 80 microg/kg). High dietary vitamin A decreased bone ash (P < 0.001), and increased the incidence of rickets (P < or = 0.02). Linear and quadratic responses to vitamin D3 levels were significant (P < 0.01) for body weight, bone ash, incidence and severity of rickets, and plasma calcium. In Experiment 2, six levels of 25-(OH)D3 (0, 5, 10, 20, 40, and 80 microg/kg) were added to the basal diet. Adding 25-(OH)D3 increased (P < 0.001) body weight, bone ash, and plasma calcium, and decreased rickets and plasma vitamin A. Adding 25-(OH)D3 overcame the reduction in bone ash produced by high dietary vitamin A showing a significant (P < 0.02) interaction. In Experiment 3, six levels of 1,25-(OH)2D3 (0, 2, 4, 8, 16, and 32 microg/kg) were added to the basal diet. High dietary vitamin A increased (P < 0.01) the incidence and severity of rickets. Adding 1,25-(OH)2D3 increased (P < 0.01) body weight, bone ash, plasma calcium, and reduced rickets and plasma and liver vitamin A. Adding 1,25-(OH)2D3 overcame the reduction in bone ash, and the increase in rickets produced by high vitamin A was significant (P < or = 0.05). These results indicate that high dietary vitamin A (45,000 IU/kg) interferes with the utilization of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3, increasing the requirement for each of them. Moreover, 45,000 IU/kg of dietary vitamin A ameliorated the potential toxic effects of feeding high levels of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3 to young broiler chickens. Further work is necessary to find the minimum levels of these vitamins needed to cause these effects.     

In vivo dihydrotachysterol2 metabolism in normal man: 1 alpha- and 1 beta-hydroxylation of 25-hydroxydihydrotachysterol2 and effects on plasma parathyroid hormone and 1 alpha,25-dihydroxyvitamin D3 concentrations

It has recently been shown that in the rat, dihydrotachysterol (DHT) is extensively metabolized in the side-chain in vivo along pathways similar to those of vitamin D. In addition 25-hydroxy-DHT2 [25OHDHT2] is hydroxylated at C1, producing both 1 alpha- and 1 beta- hydroxy compounds. An in vivo study in 1988 demonstrated that in normal adult subjects receiving oral DHT2, plasma 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations fell, but with unchanged plasma PTH levels. Down-regulation of 1,25-(OH)2D3 production by 25-(OH)DHT2 or some other unknown metabolite was also suggested as an explanation for these observations. To investigate whether either of the newly characterized 1 alpha,25- or 1 beta,25-(OH)2DHT2 was formed in vivo in normal man, DHT2 (approximately 1 mg/day, orally) was administered to healthy volunteers (three males and one female). Plasma was analyzed by high performance liquid chromatography and gas chromatography-mass spectrometry, demonstrating the formation of both 1 alpha,25- and 1 beta,25-(OH)2DHT2 in vivo in normal human subjects. Plasma levels of 1,25-(OH)2D3, PTH, ionized and total calcium, inorganic phosphate, and alkaline phosphatase were monitored. The plasma concentrations of DHT2, 25OHDHT2, and 1 alpha,25- and 1 beta,25-(OH)2DHT2 were measured by gas chromatography-mass spectrometry. In all volunteers, plasma ionized calcium increased slightly during DHT2 administration; 1,25-(OH)2D3 and PTH concentrations fell. Plasma levels of DHT2 and its metabolites rose over the same period. The average fall in the level of plasma 1,25-(OH)2D (60-70 pmol/L) was mirrored by a rise in the concentration of 1 alpha,25-(OH)2DHT2 (550 pmol/L). This ratio is appropriate, because it has previously been shown that in a reconstituted COS cell, 1 alpha,25-(OH)2DHT3 has roughly one tenth the potency of 1,25-(OH)2D3. At maximum concentration, the ratios of DHT2/25OHDHT2/1 beta,25-(OH)2DHT2/1 alpha,25-(OH)2DHT2 were approximately 10:1:2:0.1. The concentration of 1 beta,25-(OH)2DHT2 was greater than that of 25OHDHT2, and the ratio of 1 alpha,25- to 1 beta,25-(OH)2DHT2 (1:20) was substantially lower than that in rat plasma (3:10). The data presented here suggest that the active DHT2 metabolite in man is 1 alpha,25-(OH)2DHT2 and that the fall in plasma 1,25-(OH)2D seen during DHT therapy may be partly the result of suppressed PTH secretion.     

Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism

Conflicting results have been reported regarding the efficacy of intermittent versus continuous administration of 1,25(OH)2D3 in renal secondary hyperparathyroidism. To address this issue we examined sham-operated control rats and hyperparathyroid rats with subtotal (5/6) nephrectomy (Nx). The Nx animals (20 to 22 animals per group) were subjected to three treatment protocols: (i) solvent treatment (Nx-solvent); (ii) two i.p. injections of 35 pmol 1,25(OH)2D3 on days 0 and 4 (Nx-bolus); and (iii) continuous infusion of 70 pmol 1,25(OH)2D3 over six days via osmotic minipump (Nx-infusion). All measurements were performed six days after start of treatment. As compared to sham-operated controls, the pre-pro-PTH/beta-actin mRNA ratio was 2.04-fold higher in Nx-solvent. Both modes of administration of 1,25(OH)2D3 resulted in inhibition of PTH mRNA concentrations relative to Nx-solvent. The pre-pro-PTH/beta-actin mRNA ratio was, however, significantly lower (P < 0.05) in Nx-bolus than in Nx-infusion (Nx-bolus 1.26 higher than sham-operated controls; Nx-infusion 1.65 higher than sham-operated controls). Aminoterminal PTH (N-PTH) serum concentrations were higher in Nx-solvent (52 +/- 4 pg/ml) than in sham-operated controls (32 +/- 3 pg/ml, P < 0.01). N-PTH concentrations in Nx-bolus (38 +/- 4 pg/ml) were significantly lower than in Nx-solvent (P < 0.01) and in Nx-infusion (46 +/- 4 pg/ml, P < 0.05). Parathyroid gland weight (microgram/g body wt) was higher in Nx-solvent (1.30 +/- 0.08 pg/ml) than in sham-operated controls (0.79 +/- 0.04 pg/ml, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous up-regulation of vitamin D receptors is tissue specific in the rat

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors (VDR) are expressed in multiple tissues within the body. VDR levels are increased by 1,25(OH)2D3 in intestine and kidney and in numerous cell models. The ability of 1,25(OH)2D3 to affect VDR levels in other target tissues in vivo was studied by assessing VDR levels by the 3H-1,25(OH)2D3 binding assay under varied physiological conditions in the rat. When compared with vitamin D-deficient (-D) controls, rats raised on a normal vitamin D-sufficient (+D) diet showed elevated VDR levels in kidney (391 +/- 53 vs. 913 +/- 76 fmol/g of tissue;p < 0.05), but not in testis, heart, or lung. Up-regulation of the VDR also occurred in kidney of +D rats 1 day after a single 100-ng dose of 1,25(OH)2D3 (454 +/- 43 vs. 746 +/- 113 fmol/mg of DNA; p < 0.05), but no changes were seen in intestine, testis, or lung. Because 1,25(OH)2D3-induced hypercalcemia may independently affect VDR regulation, 1,25(OH)2D3 was infused into -D rats, and normocalcemia was maintained by reduced dietary calcium intake. In this model, the renal VDR was again up-regulated (446 +/- 115 vs. 778 +/- 58 fmol/mg of DNA; p < 0.05), but VDR levels in testis and lung were unaffected. Scatchard analysis and tests of 1,25(OH)2D3 dose (1-100 ng/day for 7 days) and temporal (100 ng/day for 1-7 days) responsiveness further supported the tissue-specific nature of the homologous VDR regulation. Assay of VDR levels by L-1-tosylamido-2-phenylethyl chloromethyl ketone-3H-1,25(OH)2D3 exchange assay ruled out differences in endogenous 1,25(OH)2D3 occupancy as the basis for the observed differences in VDR regulation. Finally, coidentity of the VDR-like sites in kidney versus testis was confirmed by competitive binding analysis comparing their relative affinities for 25(OH)D3 versus 1,25(OH)2D3 (30.5 +/- 6.4 vs. 35.6 +/- 3.6 in kidney and testis, respectively) and by immunoblot analysis using a highly specific monoclonal anti-rat VDR antibody. Thus, under a wide variety of experimental conditions, homologous up-regulation of the VDR occurs in the rat kidney in vivo, but not in several other target tissues which do not regulate plasma calcium homeostasis. Moreover, this differential VDR regulation did not result from secondary changes in plasma calcium, from differential 1,25(OH)2D3 responsiveness in the various tissues, nor from differences in endogenous 1,25(OH)2D3 occupancy of the VDR. These studies thus establish that, in contrast to observations in vitro, the widely described phenomenon of homologous VDR up-regulation in kidney and intestine is not a universal property of 1,25(OH)2D3 target tissues in vivo in the rat.     

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.     

Anti-tumor effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in seborrheic keratosis

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a drug with potent antiproliferative action on keratinocytes that have nuclear receptors for 1,25(OH)2D3. We investigated the effects of 1,25(OH)2D3 on widespread seborrheic keratoses in 51 patients with these tumors. The data indicated that resolution of these tumors was dependent on both tumor size and dose of 1,25(OH)2D3. Among 15 patients treated with a high dose (0.5 microgram/d) of oral 1,25(OH)2D3, the lesions of widespread seborrheic keratoses changed from brown-black papules to brownish papules with erythema and/or crust as early as 2 wk after the start of treatment. The tumors finally developed into an atrophic scar or brownish pigmented macule. Histologically, vacuolation of the spinous cells, vesicle formation, and liquefaction degeneration of the basaloid cells were observed. Numerous lymphocytes had infiltrated in the papillary dermis. Among 36 patients treated with a low dose (0.25 microgram/d) of 1,25(OH)2D3, brownish papules became pale to normal in color and reduced in size, without erythematous change. Histologically, acanthosis of the epidermis was reduced, but degenerative change of the tumor cells was not observed. These data suggest that oral therapy of 1,25(OH)2D3 is an acceptable method well suited to the removal of seborrheic keratoses, especially those that are predominantly small tumors.     

Association of 1 alpha,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], 25-hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1 alpha,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone-occupied VDR. These results were substantiated by studies using baculovirus-expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1 alpha,25-(OH)2D3.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Effect of 1,25 (OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats

In the past, there has been considerable concern that treatment with active vitamin D might accelerate progression independent of hypercalcemia and hypercalcuria. Nevertheless, 1,25(OH)2D3 has known antiproliferative properties and has also been shown to inhibit renal growth. Since glomerular growth is a permissive factor for the development of glomerulosclerosis, we reasoned that 1,25(OH)2D3 might even attenuate progression. To test this working hypothesis we performed two experiments of 8 and 16 weeks duration, respectively, to compare subtotally nephrectomized (SNX) rats treated with ethanol and SNX treated with 1,25(OH)2D3. Control animals were sham operated and pair-fed with SNX animals. 1,25(OH)2D3 (3 ng/100 g body wt/day) was administered by osmotic minipump. 1,25(OH)2D3 had no significant effect on systolic blood pressure and only a transient effect on weight gain. SNX reduced the number of glomeruli (left kidney) from an average of 3.3 x 10(4) to 1.2 x 10(4) per kidney. Mean glomerular volume was 3.87 +/- 0.71 x 10(6) microns 3 in sham operated animals and significantly (P < 0.05) higher (10.1 +/- 1.75 x 10(6) microns 3) in untreated animals 16 weeks after SNX. Glomerular volume was significantly (P < 0.05) less in 1,25(OH)2D3 treated SNX [10.1 +/- 1.75 in ethanol vs. 7.04 +/- 1.78 in 1,25(OH)2D3 treated SNX]. In parallel, there was significantly (P < 0.01) less glomerulosclerosis [glomerulosclerosis index 1.16 +/- 0.14 in the ethanol treated SNX vs. 0.80 +/- 0.16 in SNX treated with 1,25(OH)2D3] in the eight week experiment. Albuminuria was significantly (P < 0.01) lower in 1,25(OH)2D3 treated than in ethanol treated SNX (mean 0.785 mg/24 hr, range 0.43 to 1.80, vs. 3.75 mg/24 hr, 1.29 to 14.2). The morphological data were directionally analogous in a second 16 week experiment. Only slight changes of the vascular sclerosis index and tubulointerstitial index were seen in SNX and were not affected by 1,25(OH)2D3 further. To prove that the effect of 1,25(OH)2D3 was independent of PTH, parathyreoidectomized SNX rats without or with 1,25(OH)2D3 treatment were examined seven days post-SNX. PCNA staining showed suppression of cell proliferation. Furthermore, in situ hybridization for transforming growth factor-B (TGF-beta) showed less vascular and tubular expression in 1,25(OH)2D3 treated rats. We conclude that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy. These effects are independent of PTH. The data document that 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage.     

Selective biological response by target organs (intestine, kidney, and bone) to 1,25-dihydroxyvitamin D3 and two analogues

The hormonally active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates biological responses related to calcium homeostasis, cell differentiation, and immunomodulation in many target cells, including leukemic cells. Most of these responses are dependent upon 1 alpha,25(OH)2D3 interaction with a nuclear receptor protein. Structural analogues of 1 alpha,25(OH)2D3 might allow for separation of biological function, avoiding adverse calcemic effects. This report quantitates intestinal calcium absorption, bone calcium resorption, induction of intestinal and renal calcium-binding protein (CaBP), and occupancy of the intestinal and renal nuclear 1 alpha,25(OH)2D3 receptor in vitamin D-deficient chicks after a single dose of 1 alpha,25(OH)2D3, 1 alpha,25-dihydroxyvitamin-16-ene-23-yne-D3 (analogue V), or 22-[m-(dimethylhydroxymethyl)phenyl]-23,24,25,26,27- pentanor-1 alpha-hydroxy-vitamin D3 (analogue EV). The interaction of these compounds with chick intestinal nuclear 1 alpha,25(OH)2D3 receptor and chick plasma vitamin D-binding protein was determined in vitro; analogues V and EV bound 68% and 62% [1 alpha,25(OH)2D3 receptor] and 8% and 13% (vitamin D-binding protein), respectively, as well as 1 alpha,25(OH)2D3 (100%). 1 alpha,25(OH)2D3 doses (0.075-1.2 nmol) generated responses in intestinal calcium absorption, bone calcium resorption, intestinal CaBP, and renal CaBP. When analogue V (1.2-300 nmol) was administered, increases in bone calcium resorption and renal CaBP were noted. However, a significant response in intestinal calcium absorption and intestinal CaBP appeared only after a 300-nmol dose. Unoccupied nuclear 1 alpha,25(OH)2D3 receptor in the intestine and kidney was determined in vivo after doses of 1 alpha,25(OH)2D3, analogue V, or analogue EV. Doses (0.25-6.0 nmol) of 1 alpha,25(OH)2D3 and analogue EV reduced unoccupied receptor to 24% and 59% (intestine) and to 13% and 41% (kidney), respectively. Analogue V (6.0-600 nmol) decreased unoccupied receptor in the kidney. In the intestine analogue V (300-600 nmol) reduced unoccupied receptor only to 75%. These results confirm that some vitamin D analogues can generate selective biological responses and different levels of target organ receptor occupancy.