Vitamin D receptor genotype is associated with radiographic osteoarthritis at the knee.

Osteoporosis and osteoarthritis are age-related disorders of the skeleton with genetic components. Low bone density is a risk factor for osteoporotic fracture while osteoarthritis is associated with increased bone density. The 1,25-dihydroxyvitamin D3 receptor (VDR) gene locus was previously found to be associated with bone density. We therefore studied the relationship between radiographic osteoarthritis at the knee and VDR genotype in a population-based sample (n = 846), using molecular haplotyping of anonymous intragenic DNA polymorphisms. Radiographic osteoarthritis was defined using the Kellgren score, which is based on the assessment of osteophytes and joint space narrowing (JSN). We show that one VDR haplotype allele is significantly overrepresented in individuals with knee osteoarthritis and associated with a 2.27-fold increased relative risk (95% confidence interval 1.46, 3.52). Adjustment for bone density at the femoral neck did not change these results, indicating that the association is not mediated by bone density. The association appeared to be largely explained by the presence of osteophytes rather than JSN. Our results indicate a role of the VDR gene in the pathogenesis of osteophytes while linkage disequilibrium with another nearby gene, i.e., the collagen type IIa1 gene encoding the most abundant protein in cartilage, might contribute to the association.

Inhibition of breast cancer cell growth by combined treatment with vitamin D3 analogues and tamoxifen.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has potential to be used as an antitumor agent, but its clinical application is restricted by the strong calcemic activity. Therefore, new vitamin D3 analogues are developed with increased growth inhibitory and reduced calcemic activity. In the present study, we have examined the antiproliferative effects of four novel vitamin D3 analogues (CB966, EB1089, KH1060, and 22-oxa-calcitriol) on breast cancer cells, either alone or in combination with the antiestrogen tamoxifen. The estrogen-dependent ZR-75-1 and estrogen-responsive MCF-7 cell lines were used as a model. It was shown that, with EB1089 and KH1060, the same growth inhibitory effect as 1,25-(OH)2D3 could be reached at up to 100-fold lower concentrations, whereas CD966 and 22-oxa-calcitriol were nearly equipotent with 1,25-(OH)2D3. The growth inhibition by the vitamin D3 compounds could be augmented by combined treatment with tamoxifen. At the maximal effective concentrations of the vitamin D3 compounds, the effect of combined treatment was addictive (MCF-7 cells) or less than additive (ZR-75-1 cells). Tamoxifen increased the sensitivity of the cells to the vitamin D3 compounds 2- to 4000-fold, which was expressed by a shift to lower median effective concentration values. Thereby, the vitamin D3 compounds may be used at even lower dosages in combination therapy with tamoxifen. A major problem of tamoxifen therapy is the development of tamoxifen resistance. We have observed that tamoxifen-resistant clones of ZR-75-1 cells retain their response to the vitamin D3 compounds. Regulation of the growth-related oncogene c-myc (mRNA level) and the estrogen receptor (protein level) were studied but appeared not to be related to the antiproliferative action of the vitamin D3 compounds. Together, our data point to a potential benefit of combination therapy with 1,25-(OH)2D3 or vitamin D3 analogues and tamoxifen for the treatment of breast cancer.

Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D-3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells.

The osteoblast-like osteosarcoma cell line UMR-106 has been shown to possess high-affinity receptors for 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Also, these cells metabolize 1,25-(OH)2D3 to more polar metabolites. As previously demonstrated (Pols, H.A.P., et al. (1987) Biochim. Biophys. Acta 931, 115-119) the time course of specific binding of 1,25-(OH)2D3 in intact UMR-106 cells was found to be characterised by (a) an ascending phase, representing association with receptor, (b) a maximum at 90-120 min and (c) a rapid descending phase, closely associated with a decrease of medium 1,25-(OH)2D3 due to the metabolism of the hormone. The purpose of the present study was to investigate further the self-induced metabolism of 1,25-(OH)2D3 in relation to the homologous up-regulation of its receptor in these cells. Inhibition of metabolism of 1,25-(OH)2D3 with ketoconazole resulted, after a lag-time of about 90 min, in a sharp increase of receptor accumulation. This increase in receptor level in the presence of ketoconazole was blocked by coincubation with cycloheximide and actinomycin D. Preincubation experiments with unlabeled 1,25-(OH)2D3 showed that the elevation of hormone binding was 1,25-(OH)2D3-concentration dependent (ED50 200-300 pM). Addition of ketoconazole during these preincubations resulted in an even more pronounced accumulation of receptors, whereby the ED50 (50-60 pM) was comparable with the dissociation constant of the 1,25-(OH)2D3 receptor (41.3 +/- 4.3 pM). In summary, these data support the concept that the self-induced metabolism of 1,25-(OH)2D3 has a dual effect: (1) directly, by the regulation of the cellular concentration of and, consequently, receptor occupancy by the active form of vitamin D and (2) indirectly by its ability to modulate the ligand-dependent regulation of the 1,25-(OH)2D3.

Effect of ketoconazole on metabolism and binding of 1,25-dihydroxyvitamin D-3 by intact rat osteogenic sarcoma cells.

The antifungal imidazole, ketoconazole, was tested for effects on 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) metabolism and binding in intact osteoblast-like osteogenic sarcoma cells (UMR-106). Ketoconazole inhibited the C-24 oxidation of 1,25-(OH)2D3 in a dose-dependent manner. Furthermore, inhibition of 1,25-(OH)2D3 metabolism by ketoconazole resulted, after a lag time of 2 h, in a sharp increase of receptor-bound 1,25-(OH)2D3. The data suggest that the self-induced 1,25-(OH)2D3 metabolism may play an important role in controlling the intracellular levels of and, consequently, receptor occupancy by the active form of vitamin D. Furthermore the results are compatible with the existence of a homologous up-regulation of the 1,25-(OH)2D3-receptor.

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3.

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.

Circulating and liver-bound salt-resistant hepatic lipases in the golden hamster.

The serum of male golden hamsters was found to contain a circulating triacylglycerol hydrolase activity (serum lipase). In vitro, the enzyme activity was slightly activated by 1 M NaCl (+20%) and inhibited by rat serum (-29%). The hamster liver contained an enzyme with similar characteristics (liver lipase). This enzyme was released into the circulation after intravenous administration of heparin. Both lipase activities were further characterized and compared. The serum lipase had a pH optimum of 9, which was higher than that of the liver enzyme (pH 8.0). The serum enzyme did not bind to Sepharose-heparin columns in contrast to the liver lipase, which could be eluted from the column with 0.75 M NaCl. A polyclonal antibody preparation raised against the heparin-releasable salt-resistant lipase from rat liver inhibited both the hamster serum enzyme and the liver enzyme completely. The affinity of the antibodies towards the hamster enzymes was lower than the affinity towards the rat liver enzyme, but similar with that towards the hamster enzymes in the serum and the liver. A panel of five monoclonal antibodies raised against the rat enzyme did not bind either of the hamster enzymes. If the hamsters were fed a normal lab chow, the lipase activity in the serum amounted up to 110 +/- 20 mU (mean +/- S.D., n = 16) per ml serum (about 600 mU per animal), the liver contained 200 +/- 41 mU per g tissue (total about 800 mU per animal). In animals fed a cholesterol-enriched diet, the serum activity increased by 82% and the liver activity by 27%.

Growth hormone restores hepatic lipase mRNA levels but the translation is impaired in hepatocytes of hypothyroid rats.

During hypothyroidism, hepatic lipase (HL) activity is decreased. The low HL may be due to thyroid hormone insufficiency or to the concomitant fall in growth hormone (GH) activity. We studied HL expression in hepatocytes freshly isolated from hypothyroid rats with and without additional GH-substitution. In all animals HL mRNA was detected by RT-PCR in the hepatocytes, but not in the non-parenchymal cells. In hypothyroid cells HL mRNA levels were reduced by 40%, and the in vitro secretion of HL-activity and HL-protein was decreased by about 50%. In cells from GH-substituted hypothyroid rats, HL mRNA level was normalised, but the secretion of HL remained low. The specific enzyme activity of secreted HL was similar under all conditions. The discrepancy between HL mRNA and HL secretion in GH-supplemented rats may be due to (post)translational effects. Therefore we studied the HL synthesis and maturation in hepatocytes from hypothyroid and GH-substituted rats. Pulse-labelling experiments with [(35)S]methionine showed that the incorporation of [(35)S]methionine into HL protein was lower both in hypothyroid cells and in GH-supplemented cells than in control cells. During the subsequent chase, the intracellular processing and transport of newly synthesized HL protein in the hepatocytes from hypothyroid rats, whether or not supplemented with GH, was similar to control cells. We conclude that in livers of hypothyroid, GH-substituted rats translation of HL mRNA is inhibited despite restoration of HL mRNA levels.

Evidence for coordinated regulation of osteoblast function by 1,25-dihydroxyvitamin D3 and parathyroid hormone.

From several animal studies and clinical observations it became evident that at target tissue level 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and parathyroid hormone (PTH) must act in an interrelated manner. In the present study we examined the interaction between 1,25-(OH)2D3 and PTH in the target cell of these hormones in bone, the osteoblast. In addition we studied the role of PTH-activated signal pathways. The three osteoblastic cell lines UMR 106, ROS 17/2.8 and MG-63 were used as model systems. In UMR 106 cells 1,25-(OH)2D3 and PTH caused a synergistic up-regulation of the vitamin D receptor (VDR) which was accompanied by a synergistic induction of VDR mRNA expression whereas in both ROS 17/2.8 and MG-63 cells no interaction was observed. In UMR 106 cells the effect of PTH on homologous up-regulation of VDR could be mimicked by the cAMP agonist forskolin and by dibutyrylic-cAMP. Phorbol ester activation of protein kinase C reduced basal as well as 1,25-(OH)2D3-induced up-regulation of VDR. 1,25-(OH)2D3 induced 24-hydroxylase activity in UMR 106 and MG 63 cells and, in contrast to VDR regulation, in both cell lines PTH and 1,25-(OH)2D3 synergistically induce 24-hydroxylase activity. Similar to VDR regulation the effect of PTH was mimicked by activation of cAMP production whereas protein kinase C activation reduced the induction by 1,25-(OH)2D3. Finally, we examined the interaction with respect to osteocalcin synthesis. In ROS 17/2.8 and MG-63 cells 1,25-(OH)2D3 stimulated osteocalcin production. In ROS 17/2.8 cells PTH as well as stimulation of cAMP production by forskolin enhanced 1,25-(OH)2D3-induced osteocalcin production whereas, as we have shown previously, activation of protein kinase C does not change 1,25-(OH)2D3-stimulated osteocalcin production. In MG-63 cells neither PTH nor forskolin significantly changed 1,25-(OH)2D3 induction of osteocalcin synthesis. From the present study it can be concluded that indeed at target cell level 1,25-(OH)2D3 and PTH act in a coordinated manner. On basis of the potentiation of 1,25-(OH)2D3 action by PTH in osteoblasts together with the previously reported inhibition of PTH-stimulated cAMP production by 1,25-(OH)2D3 we postulate a negative feedback-loop at target cell level. The activation of the cAMP pathway results in an enhancement of the 1,25-(OH)2D3 action whereas the protein kinase C pathway attenuates the 1,25-(OH)2D3 action. Finally, the present study provides a basis for the indications from in vivo observations about an interrelated action of 1,25-(OH)2D3 and PTH at the target cell. More generally it demonstrates on the basis of analyses of endogenous cellular responses evidence for an interplay between receptor-activated pathways of peptide and steroid hormones.

Role of extracellular calcium in the regulation of 1,25-dihydroxyvitamin D3 formation in cultured human keratinocytes.

Cultured normal human keratinocytes (NHK) provide a useful experimental model for studies of processes occurring during terminal differentiation, since the extent of keratinocyte maturation can be manipulated experimentally by modulation of extracellular calcium concentration. When NHK are maintained in low calcium (0.06 mM) medium they proliferate but do not stratify. Raising the level of calcium to 1-2 mM results within a few hours in induction of keratinocyte differentiation. Results of the present study show that formation of 1,25-(OH)2D3 is higher in NHK grown at 0.06 mM than in NHK grown at 1.6 mM calcium concentration. After 2 h exposure of low calcium cultures to 1.6 mM calcium the 1,25-(OH)2D3 production starts to decrease. On the other hand, exposure of cells cultured in 1.6 mM calcium medium to 0.06 mM calcium concentration induced already within 4 h an increase in 1,25-(OH)2D3 formation which was not accompanied by a decrease in cornified envelope formation. Thereby, the present study demonstrated that calcium can regulate 1,25-(OH)2D3 formation independently of changes in keratinocyte differentiation.

Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes.

The 1 alpha,25-dihydroxyvitamin D3 (VD3)-dependent stimulation of osteocalcin (OC) and osteopontin (OP) gene transcription in bone tissue is mediated by interactions of trans-activating factors with distinct VD3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for recognition by distinct hormone receptor homo- and heterodimers. However, the exact composition of endogenous VD3- induced complexes recognizing the OC- and OP-VDREs in osteoblasts has not been definitively established. To determine the identity of these complexes, we performed gel shift immunoassays with nuclear proteins from ROS 17/ 2.8 osteoblastic cells using a panel of monoclonal antibodies. We show that VD3- inducible complexes interacting with the OC- and OP-VDREs represent two distinct heterodimeric complexes, each composed of the vitamin D receptor (VDR) and the retinoid X receptor-alpha (RXR). The OC- and OP-VDR/RXR alpha heterodimers are immunoreactive with RXR antibodies and several antibodies directed against the ligand-binding domain of the VDR. However, while the OC-VDRE complex is also efficiently recognized by specific monoclonal antibodies contacting epitopes in or near the VDR DNA-binding domain (DBD) (between amino acids 57-164), the OP-VDRE complex is not efficiently recognized by these antibodies. By systematically introducing a series of point-mutations in the OC-VDRE, we find that two internal nucleotides of the proximal OC-VDRE half-site (nucleotide -449 and -448; 5'-AGGACA) determine differences in VDR immunoreactivity. These results are consistent with the well established polarity of RXR heterodimer binding to bipartite hormone response elements, with the VDR recognizing the 3'-half-element. Furthermore, our data suggest that the DBD of the VDR adopts different protein conformations when contacting distinct VDREs. Distinctions between the OC- and OP-VDR/RXR alpha complexes may reflect specialized requirements for VD3 regulation of OC and OP gene expression in response to physiological cues mediating osteoblast differentiation.

Age-associated changes in ultrasound measurements of the calcaneus in men and women: the Rotterdam Study.

Broadband ultrasound attenuation (BUA) and speed of sound (SOS) of the os calcis were measured in a sample of 1405 persons (628 men), aged 55-93 years, from the cohort of the Rotterdam Study, a population-based study of diseases in the elderly. We studied the effect of age, body mass index, age at menopause, current use of thiazides, loop diuretics, and estrogens, smoking, and disability on SOS and BUA. Comparisons were made between ultrasound measurements and bone mineral density (BMD) measurements of the lumbar spine and proximal femur using DXA. We found a significant decline with age in SOS and BUA in men (-0.4 and -0.1 %/year, respectively) and women (-1.3 and -0.4%/year, respectively), which persisted after adjustment for body mass index. Age at menopause was not associated with SOS or BUA. Pack-years of smoking was negatively related to SOS in both sexes and to BUA in men. Severe disability was associated with lower SOS and BUA in men, but not in women. Despite the small number of exposed persons, current corticosteroid use was associated with lower BUA in men. The other risk factors examined did not affect the ultrasound measurements. We observed modest correlations between SOS or BUA, on the one hand, and BMD of the lumbar spine, on the other hand (r = 0.32-0.42). Similar correlations were found between ultrasound measurements and BMD measurements of the proximal femur. We conclude that in persons 55 years or over (1) there is a significant age-related decline of BUA and SOS, which is about three times higher in women compared with men, and (2) ultrasound measurements are not able to predict low BMD in hip or spine.

Parathyroid hormone sensitizes long bones to the stimulation of bone resorption by 1,25-dihydroxyvitamin D3.

In response to hypocalcemia the serum PTH level increases rapidly followed by a PTH-induced rise in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] production. Therefore, bone is first exposed to increased PTH levels before increased 1,25-(OH)2D3 levels. In the present study the effect of pretreatment with PTH on 1,25-(OH)2D3-induced bone resorption was examined. Bone resorption was measured as release of prelabeled 45Ca during culture from 17-day-old fetal mice radii/ulnae and metatarsals. Radii/ulnae and metatarsals are characterized by differences in development. In radii/ulnae mature osteoclasts are present, whereas in metatarsals only different stages of preosteoclasts can be found. Preincubation for 24 h but not 4 h with PTH increases the stimulation of bone resorption by 1,25-(OH)2D3 in fetal radii/ulnae but not in metatarsals. Coincubation of PTH and 1,25-(OH)2D3 did not result in a significant change in bone resorption compared to 1,25-(OH)2D3 alone. The observed difference in the effect of pretreatment with PTH between radii/ulnae and metatarsals indicates that PTH does not stimulate the development of early osteoclast precursors but that a certain level of differentiation of the osteoclast precursor is required. Pretreatment with prostaglandin E2 resulted in an effect similar to that of PTH. Inhibition of prostaglandin synthesis by indomethacin prevented the potentiation of 1,25-(OH)2D3-induced bone resorption by pretreatment with PTH. Thus, the present study demonstrates that PTH sensitizes responses to 1,25-(OH)2D3. PTH must be present before 1,25-(OH)2D3 to observe a potentiation of 1,25-(OH)2D3-induced bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive axial and peripheral assessment of bone mineral content: a comparison between osteoporotic women and normal subjects.

We compared different methods of bone densitometry in women with spinal osteoporosis and normal subjects to assess their discriminatory capability. The methods used included: quantitative computed tomography of the spine (QCT) specified as to trabecular (QCTtrab) and cortical bone (QCTcort), dual-photon absorptiometry of the spine (DPAspine), single-photon absorptiometry of the distal and proximal forearm (SPAdist and SPAprox), and quantitative roentgen microdensitometry of the phalanx (QMD). A total of 25 postmenopausal osteoporotic women and 24 healthy comparison subjects matched for age and years since menopause were studied. In the osteoporotic group an average decrement of the axial bone mineral density of -50% (p less than 0.001) and -20% (p less than 0.001) were observed for QCTtrab and QCTcort, respectively. For DPAspine, SPAdist, SPAprox, and QMD the difference between normal and osteoporotic subjects was -20% (p less than 0.001), -12% (p less than 0.05), -7% (NS), and -6% (NS), respectively. With the peripheral measurements (SPA and QMD), alone or in combination, no adequate discrimination between women with or without vertebral compression fractures could be obtained. Although QCTtrab showed the highest diagnostic sensitivity (81%), it appears not to be superior to DPAspine. Combinations of the various axial and peripheral measurements did not result in an essentially better sensitivity. In normal women as well as in osteoporotic individuals the trabecular and cortical QCT measurements showed two opposite trends, suggesting an increase in cortical and a decrease in trabecular density from L1 to L3.

Vertebral deformities and functional impairment in men and women.

The objective of this study was to assess the prevalence and health effects of vertebral deformities in men and women. The study was carried out as part of the cross-sectional baseline phase of The Rotterdam Study, a prospective population-based cohort study of residents aged 55 years or over of a district of Rotterdam, The Netherlands. The prevalence of vertebral deformities according to a modification of the Eastell method and concomitant functional impairment were assessed in a random sample of 750 men and 750 women. The prevalence of moderate (grade I) vertebral deformities was 8 and 7% in men and women, respectively. For severe deformities (grade II), these percentages were 4 and 8%. In men, the prevalence of both moderate and severe deformities increased with age. In women, however, the prevalence of moderate vertebral deformities remained constant, opposite to a marked increase in severe deformities. Moderate vertebral deformity was significantly associated with impaired rising in men only. Severe vertebral deformity was associated with a significantly increased risk of general disability and the use of a walking aid in both men and women, impaired bending in men, and impaired rising in women. It is concluded that (1) vertebral deformities are only slightly less common in men than in women from the general population and (2) severe progression with age occurs in women only and (3) severe vertebral deformity is, particularly in men, related to functional impairment.

Course of bone mass during and after hormonal replacement therapy with and without addition of nandrolone decanoate.

A total of 33 women with postmenopausal osteoporosis were matched pairwise by age, years since menopause, and body mass index and randomized to receive either cyclic estrogen-progestagen replacement treatment (group 1) or the same treatment plus nandrolone decanoate (ND; group 2). Both groups were treated during 3 years and subsequently followed for another year off treatment. A year after cessation of the treatment the distal forearm bone mineral content in group 2 was significantly higher than that in group 1. Bone mass measurements in the axial skeleton already showed a significant difference in favor of group 2 after 3 years treatment, which persisted during the year off treatment. The decline in lumbar bone mineral mass and density in the 1 year off treatment was similar in both groups. Correction for body mass did not change these results. Bone turnover parameters did not show significant differences between the two groups after cessation of treatment. A higher muscle mass, induced by ND, could partly explain the differences between the groups since even 1 year after treatment was stopped an increased serum creatinine level was still observed in group 2.

Effects of 24R,25-dihydroxyvitamin D3 in combination with 1 alpha-hydroxyvitamin D3 in predialysis renal insufficiency: biochemistry and histomorphometry of cancellous bone.

The effect of combined administration of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and 1 alpha-hydroxyvitamin D3 (1 alpha-(OH)D3) was studied in 24 non-dialyzed patients with chronic renal insufficiency (CRI), matched pairwise as to age, sex, and creatinine clearance (Cr.cl). Low Ca intake had been supplemented beforehand. Then, 1 alpha-(OH)D3 (mean dose 0.55 micrograms daily) was given orally to all patients for 3 months (T0 to T3). Subsequently, patients were assigned randomly to 6 months further treatment either with 1 alpha-(OH)D3 alone (Group A) or with 1 alpha-(OH)D3 plus a high dosage of 24,25-(OH)2D3 (50 micrograms orally, twice weekly) (Group B). Histomorphometry was performed at T0, T3, and T9. In both groups iPTH was equally suppressed, into the lower normal range. Whereas in Group A, serum Ca rose steadily and Cr.cl declined, in Group B both parameters levelled off between T6 and T9. At T9, in Group A the elevated resorption and osteoid indices had normalized markedly, but osteoblasts (Ob.Pm) and mineralizing boundaries (M.Bd) were depressed considerably between T3 and T9. In contrast, in Group B, preservation of Ob.Pm and improved mineralizing activity were observed (M.Bd at T9 > T3 > T0). Resorption indices hardly changed. In the patients with high Ob.Pm at T0, cancellous bone area increased significantly. This was not observed in Group A. Thus, in Group B, osteoblast recruitment appeared maintained and M.Bd appeared normalized. Decline of remodeling toward an adynamic state with an increased risk of hypercalcemia appeared prevented.

Regulation of osteocalcin production and bone resorption by 1,25-dihydroxyvitamin D3 in mouse long bones: interaction with the bone-derived growth factors TGF-beta and IGF-I.

Bone cells produce multiple growth factors that have effects on bone metabolism and can be incorporated into the bone matrix. Interplay between these bone-derived growth factors and calciotropic hormones has been demonstrated in cultured bone cells. The present study was designed to extend these observations by examining the interactions between either transforming growth factor-beta (TGF-beta) or insulin-like growth factor-I (IGF-I) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a mouse long bone culture model with respect to osteocalcin production and bone resorption. In contrast to the stimulation in rat and human, in the fetal mouse long bone cultures, 1,25(OH)2D3 caused a dose-dependent inhibition of osteocalcin production. Both the osteocalcin content in the culture medium and in the extracts of the long bones was reduced by 1,25(OH)2D3. This effect was not specific for fetal bone because 1,25(OH)2D3 also reduced osteocalcin production by the neonatal mouse osteoblast cell line MC3T3. TGF-beta inhibited whereas IGF-I dose-dependently increased osteocalcin production in mouse long bones. The combination of TGF-beta and 1,25(OH)2D3 did not result in a significantly different effect compared with each of these compounds alone. The IGF-I effect was completely blocked by 1,25(OH)2D3. In the same long bones as used for the osteocalcin measurements, we performed bone resorption analyses. Opposite to its effect on osteocalcin, 1,25(OH)2D3 dose-dependently stimulated bone resorption. TGF-beta reduced and IGF-I did not change basal (i.e., in the absence of hormones) bone resorption. Our results show that 1,25(OH)2D3-enhanced bone resorption is dose-dependently inhibited by TGF-beta and IGF-I. Regression analysis demonstrated a significant negative correlation between 1,25(OH)2D3-induced bone resorption and osteocalcin production. The specificity for their effect on 1,25(OH)2D3-stimulated bone resorption was assessed by testing the effects of TGF-beta and IGF-I in combination with parathyroid hormone (PTH). Like 1,25(OH)2D3, PTH dose-dependently stimulates bone resorption. However, PTH-stimulated bone resorption was not affected by TGF-beta. Like 1,25(OH)2D3-stimulated bone resorption, IGF-I inhibited the PTH effect but at a 10-fold higher concentration compared with 1,25(OH)2D3. In conclusion, the present study demonstrates growth factor-specific interactions with 1,25(OH)2D3 in the control of osteocalcin production and bone. With respect to bone resorption, these interactions are also hormone specific. The present data thereby support and extend the previous observations that interactions between 1,25(OH)2D3 and bone-derived growth factors play an important role in the control of bone metabolism. These data together with the fact that TGF-beta and IGF-I are present in the bone matrix and potentially can be released during bone resorption support the concept that growth factors may control the effects of calciotropic hormones in bone in a localized and possibly temporal manner. Finally, in contrast to human and rat, in mice 1,25(OH)2D3 reduces osteocalcin production and this reduction is paralleled by stimulation of bone resorption by 1,25(OH)2D3. These data thereby show a dissociation between osteocalcin production and bone resorption.

A large-scale population-based study of the association of vitamin D receptor gene polymorphisms with bone mineral density.

Conflicting results have been reported on the association between restriction fragment length polymorphisms (RFLPs) at the vitamin D receptor (VDR) gene locus (i.e., for BsmI, ApaI, and TaqI) and bone mineral density (BMD). We analyzed this association in a large population-based sample (n = 1782) of men and women aged 55-80 years using a novel direct haplotyping polymerase chain reaction (PCR) test to monitor the three polymorphic sites simultaneously. The direct haplotyping test we developed demonstrated a larger degree of genetic polymorphism at the VDR gene locus than described until now. None of the individual RFLPs were associated with BMD at the proximal femur. By analyzing allele dose effects, we identified a VDR haplotype allele weakly associated with low BMD. This allele, as one representative of the group of b alleles, is different from the BsmI allele previously reported by other groups to be associated with low BMD. This suggests allelic heterogeneity at the VDR locus in relation to BMD. Our results indicate at most a small effect of the VDR genotype on BMD in this elderly population. Since anonymous polymorphisms were analyzed, alternative explanations for our results include linkage to another nearby bone-metabolism related gene.

Evidence for involvement of 17beta-estradiol in intestinal calcium absorption independent of 1,25-dihydroxyvitamin D3 level in the Rat.

The sex steroid 17beta-estradiol (17beta-E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3-month-old Brown Norway rats was designed to investigate the role of 17beta-E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham-operated, ovariectomized (OVX), and OVX supplemented with either a 0.025-mg or 0.05-mg 17beta-E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17beta-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17beta-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17beta-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17beta-E2-supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18. 1 +/- 2.1 and 16.4 +/- 2.2 pmol/l compared with 143.5 +/- 29 pmol/l for the OVX group), the OVX-induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17beta-E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17beta-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17beta-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17beta-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17beta-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption. (J Bone Miner Res 1999;14:57-64)

Role of calcium and cAMP in heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in an osteoblast cell line.

To understand further the mechanism of action of parathyroid hormone (PTH) in the stimulation of the number of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding sites in UMR 106-01 cells we studied the role of cAMP and calcium. In addition to PTH other agents known to act via the cAMP signal pathway, prostaglandin E2, forskolin and dibutyryl cAMP, caused an increase in 1,25(OH)2D3 binding. Addition of the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl)adenine resulted in a marked decrease of PTH-stimulated cAMP production but this was not followed by a reduction of 1,25(OH)2D3 receptor up-regulation by PTH. Increasing the intracellular calcium concentration by Bay K 8644 and A23817 independent of an activation of the cAMP signal pathway did not result in an increased 1,25(OH)2D3 binding. The calcium channel blockers nitrendipine and verapamil and chelating extracellular calcium with EGTA all reduced cAMP-mediated stimulation of 1,25(OH)2D3 binding. This reduction was not due to a reduce cAMP production as verapamil even potentiated PTH- and forskolin-stimulated cAMP production in a dose-dependent manner. The present study provides evidence for an interrelated action of calcium and cAMP in the heterologous up-regulation of the 1,25(OH)2D3 receptor. The current data show an interaction between the cAMP and calcium signal pathway at (1) the level of cAMP generation/degradation, and (2) a level located distal in the cascade leading to 1,25(OH)2D3 receptor up-regulation.