Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1-36) [hPTHrP-(1-36)] versus hPTH-(1-34) on serum calcium,plasma 1,25-dihydroxyvitamin D concentrations,and fractional calcium excretion in healthy human volunteers

PTH and PTH-related protein (PTHrP) cause primary hyperparathyroidism and humoral hypercalcemia of malignancy (HHM), respectively. These syndromes are similar in several important ways, but differ in several characteristic, yet unexplained, ways. Two of the unresolved questions in HHM and hyperparathyroidism involve renal physiology. 1) Why does renal proximal tubular production of 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] differ between the two syndromes? 2) Do distal tubular calcium responses to PTH and PTHrP differ in the two syndromes? To address these questions, we compared the two peptides, human PTH-(1-34) and PTHrP-(1-36), in a direct, head to head study using a continuous, steady state infusion of each peptide at the same dose in normal human volunteers for 46 h. We had previously described such methods as applied to PTHrP, but a direct multiday comparison of PTHrP to PTH has not previously been reported. In two groups (seven subjects each) of healthy young (25- to 35-yr-old) normal volunteers, PTH and PTHrP infused at 8 pmol/kg.h displayed similar calcemic effects, although PTH was slightly more potent in this regard. Both peptides also displayed similar phosphaturic effects. In addition, both peptides had similar effects on renal tubular calcium handling, yielding fractional calcium excretion values of approximately 3.5%, some 50% below the values (6.5%) observed in subjects rendered similarly hypercalcemic by the infusion of calcium. In contrast to these several quantitatively similar effects of PTH and PTHrP, PTH tended to be selectively more effective than PTHrP in stimulating renal production of 1,25-(OH)(2)D. These studies indicate that renal tubular calcium reabsorption is likely to contribute to hypercalcemia in patients with HHM. In addition, PTH may be selectively more effective than PTHrP in stimulating 1,25-(OH)(2)D production, in contrast to its phosphaturic, calcemic effects and its effects to stimulate nephrogenous cAMP excretion and renal tubular calcium reabsorption.     

Glucocorticoid and 1,25-dihydroxyvitamin D modulate the degree of adenosine 3',5'-monophosphate-dependent protein kinase isoenzyme I and II activation by parathyroid hormone in rat osteosarcoma cells

Glucocorticoid increases and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases PTH activation of adenylate cyclase and cAMP-dependent protein kinase in rat osteosarcoma cells (ROS 17/2.8). Since selective cAMP-dependent protein kinase isoenzyme activation may account for specific physiological hormonal responses, we investigated steroid effects on activation of isoenzymes I and II in response to PTH using a new ion exchange separation procedure. Pretreatment of cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM) or 1,25-(OH)2D3 altered the degree of cAMP-dependent protein kinase isoenzyme activation by PTH in accordance with their modulation of intracellular cAMP accumulation, but did not alter the amount of each isoenzyme present or the order in which isoenzymes I and II were activated. In all treatment groups isoenzyme I was preferentially activated by low doses of PTH, while high concentrations activated both isoenzymes, as predicted by the relative affinities of each isoenzyme for cAMP. Glucocorticoid reduced the concentration of bovine PTH-(1-34) required for maximal activation of isoenzyme I from 1 to 0.05 ng/ml and that required for activation of isoenzyme II from 10 to 1 ng/ml. This effect was abolished by simultaneous treatment of cells with 1,25-(OH)2D3. At doses of PTH that caused partial activation (0.05-0.1 ng/ml for isoenzyme I; 1 ng/ml for isoenzyme II), 1,25-(OH)2D3 treatment attenuated this activation. In all groups both isoenzymes were fully activated by 100 ng/ml PTH. Control experiments demonstrated that isoenzyme activation is not a result of cell disruption over the range of PTH doses that regulation by steroid hormone was observed. These results extend our studies on modulation of the cAMP pathway by steroid hormones and make it feasible to correlate selective isoenzyme activation with specific responses to PTH.     

Mutual up-regulation of thyroid hormone and parathyroid hormone receptors in rat osteoblastic osteosarcoma 17/2.8 cells

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.     

Glucocorticoids and 1,25-dihydroxyvitamin D3 regulate parathyroid hormone stimulation of adenosine 3',5'-monophosphate-dependent protein kinase in rat osteosarcoma cells

Glucocorticoids increase and 1,25-dihydoxyvitamin D3 [1,25-(OH)2D3] decreases the activity of PTH-responsive adenylate cyclase, altering intracellular cAMP in a rat osteoblast-like cell line (ROS 17/2.8). This study was undertaken to measure the subsequent activation of the cAMP-dependent protein kinase (PKA). Pretreatment of ROS cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM), shifted the dose-response curve for PKA activation by PTH upward compared to the control value. Basal PKA activity was enhanced 50% by TRM, and the PTH concentration required for maximal activation of PKA decreased from 1.0 to 0.05 ng/ml. At the lowest effective PTH concentration (0.05 ng/ml) the mean PKA activity ratio increased to 0.73 in TRM-treated cells compared with 0.45 in untreated cells. Pretreatment with 1,25-(OH)2D3 had opposite effects, shifting the dose-response curve for PKA activation by PTH downward and to the right, decreasing the basal activity ratio from 0.26 to 0.16, and increasing the PTH concentration required for maximal activation to 10 ng/ml. 1,25-(OH)2D3-treated cells stimulated with 0.5-1 ng/ml PTH consistently had lower PKA activity ratios than untreated cells. Simultaneous treatment with 1,25-(OH)2D3 reversed the effect of TRM. There were no differences in total PKA activity (2.57 +/- 0.09 pmol 32P/min.micrograms protein) between treatment groups, suggesting that TRM and 1,25-(OH)2D3 do not alter the cellular PKA concentration. In control experiments exogenous PKA was added to sonication buffer of PTH-stimulated cells to verify that the TRM and 1,25-(OH)2D3 shifts in PKA activation at low PTH doses occur before sonication. cAMP-dependent protein kinase activation was also studied by measuring the progressive occupation of regulatory subunit-binding sites by hormonally stimulated endogenous cAMP. [3H] cAMP binding was expressed as the percent change in bound [3H]cAMP per microgram protein compared to that in unstimulated cells not steroid treated. [3H]cAMP binding to all cytosol fractions decreased as PTH increased over the concentration range predicted by our PKA activation experiments. TRM treatment shifted the curve for [3H]cAMP binding to regulatory subunit downward and to the left, and 1,25-(OH)2D3 treatment shifted it upward and to the right. In cells treated with both TRM and 1,25-(OH)2D3, the curve was similar to control curve. Sonicating unstimulated cells in buffer containing comparable concentrations of added cAMP did not alter [3H]cAMP binding. These and the previous controls suggest that changes in PKA activation at low doses of PKA reflect cellular events occurring before cell disruption.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered calbindin mRNA expression and calcium regulating hormones in rat diabetic pregnancy

Offspring of rats with diabetes mellitus are at risk of reduced calcium and bone mineral content. Altered expression of the maternal calcium binding proteins, calbindin-D(9K) and calbindin-D(28K), which are involved in renal and placental calcium transport, may underlie these problems.We have investigated the effect of diabetes on circulating concentrations of regulatory hormones with respect to calbindin-D mRNA concentrations. Three rat groups were studied; control (CP), streptozotocin-induced diabetic (DP), and insulin-treated diabetic (DPI) pregnant rats. Calbindin-D(9K) and calbindin-D(28K) mRNA abundance in placenta and maternal kidney were measured at days 7, 15, 18 and 21 of gestation, together with serum or plasma concentrations of 1,25 dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)), parathyroid hormone (PTH), PTH-related protein (PTHrP), calcitonin, oestradiol and IGF-I. An increase in placental calbindin-D(9K) mRNA abundance between days 18 and 21 in CP and DPI rats was severely blunted in the DP rats. In contrast, renal calbindin-D(28K) mRNA abundance was greater at days 7, 15 and 18 in DP compared with CP rats, as was calbindin-D(9K) at day 18. Calcitonin concentrations showed no differences between the groups, and both PTH and IGF-I were reduced over the first half of gestation, unlike the calbindins. In contrast, the concentrations of PTHrP and 1,25(OH)(2)D(3) were reduced at term in the DP group compared with the other two groups. Plasma oestradiol concentrations were lower in DP than in CP rats at days 7, 15 and 18, and most striking was the absence in DP rats of the peak of oestradiol seen at day 18 in CP rats. Despite the similarity between changes in placental calbindin mRNA and 1,25(OH)(2)D(3), previous work has shown placental calbindin-D(9K) regulation to be vitamin-D-independent. These studies produce suggestive evidence, therefore, that PTHrP and oestradiol may be involved in the altered calbindin-D expression by kidney and placenta in rat diabetic pregnancy.     

Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.     

Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.     

Parathyroid hormone (PTH)-related protein, PTH, and 1,25-dihydroxyvitamin D in dogs with cancer-associated hypercalcemia.

Circulating N-terminal PTH-related protein (PTHrP), N-terminal PTH, and 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations were measured in normal dogs and dogs with cancer-associated hypercalcemia (CAH), parathyroid adenomas, and miscellaneous tumors. PTHrP was undetectable (less than 1.8 pM) in normal dogs and increased in dogs with CAH due to adenocarcinomas derived from apocrine glands of the anal sac (44.9 +/- 27 pM), lymphoma (8.3 +/- 4.4 pM), and miscellaneous carcinomas (13.3 +/- 11.4 pM). The PTHrP concentration decreased in dogs with lymphoma and anal sac adenocarcinomas after successful treatment of CAH. The PTHrP concentration had a significant linear correlation with total serum calcium in dogs with anal sac adenocarcinomas and hypercalcemia, but not in dogs with lymphoma and hypercalcemia. Serum N-terminal PTH concentrations were usually in the normal range (12-34 pg/ml) for all groups of dogs except dogs with parathyroid adenomas (83 +/- 38 pg/ml). The serum PTH concentration increased after successful treatment of CAH. Serum 1,25-(OH)2D concentrations were decreased, normal, or increased in dogs with CAH, and 1,25-(OH)2D levels decreased after treatment of CAH. In summary, circulating concentrations of PTHrP are consistently increased in dogs with CAH, and PTHrP appears to play an important role in the induction of hypercalcemia.     

Conflicting actions of parathyroid hormone-related protein and serum calcium as regulators of 25-hydroxyvitamin D(3)-1 alpha-hydroxylase expression in a nude rat model of humoral hypercalcemia of malignancy.

In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.     

Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D(3). I. Calcium transport,receptor kinetics,and signal transduction in intestine of male chickens

To study the physiological relevance of membrane-initiated steroid signaling, we investigated the correlation of age in male chickens with the magnitude of responses to 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)(2)D(3) (130 pM) responsiveness as a function of age, showed a decreased intestinal Ca(2+) transport. Western analyses of isolated basal lateral membranes indicated a decreased expression of the membrane-associated rapid response binding protein with increasing age. Saturation analyses of [(3)H]1,25-(OH)(2)D(3) binding to basal lateral membranes, revealed an allosteric interaction identified as cooperative binding. A significant increase in K(d) was observed with increasing age, indicating decreasing affinity. Determinations of the number of binding sites yielded a binding capacity of 190-250 fmol/mg protein during growth and maturation, whereas in adulthood (58 wk) saturable binding was no longer observed. Data obtained in parallel analyses of binding of [(3)H]1,25-(OH)(2)D(3) to nuclear fraction vitamin D receptor, in contrast, indicated an absence of cooperative binding and an absence of significant changes in K(d) or binding capacity with age. Membrane-initiated signal transduction by 1,25-(OH)(2)D(3) was assessed by determination of protein kinase C and A activities. Stimulation of protein kinase C activity in response to 1,25-(OH)(2)D(3) decreased with age, whereas no age-correlated changes in steroid-stimulated protein kinase A activities were observed. Thus, in conclusion, our experiments demonstrate that there is a decrease in responsiveness to exogenous 1,25-(OH)(2)D(3) as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1,25-(OH)(2)D(3), a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.     

Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D3 secretion by rat proximal tubules.

PTH stimulates mammalian renal proximal tubule cell synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a Ca-dependent process. In the present study regulation of 1,25-(OH)2D3 secretion by PTH, phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the Ca ionophore A23187, and calcitonin was evaluated in perifused rat proximal tubule cells isolated by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low Ca diet secreted 1,25-(OH)2D3 at a rate 2.5 times that of tubule cells from rats fed a normal Ca diet. Perifusion of tubules with human PTH-(1-34) (10(-7) M) induced an immediate and sustained increase in 1,25-(OH)2D3 secretion. Perifusion with either A23187 or 12-O-tetradecanoylphorbol 13-acetate caused transient increases in hormone secretion, while both agents perifused simultaneously resulted in a sustained increase in 1,25-(OH)2D3 secretion. Perifusion of tubule cells with the protein kinase-C (PKC) inhibitor staurosporine blocked the PTH-induced increase in 1,25-(OH)2D3 secretion. Calcitonin had no effect on 1,25-(OH)2D3 secretion rates. The results of the present studies show that an activator of PKC increases 1,25-(OH)2D3 secretion by mammalian proximal tubule cells and suggest that the phospholipase-C/PKC signalling system may mediate PTH stimulation of 1,25-(OH)2D3 secretion.     

Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells

Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).     

Combined effects of dexamethasone and 1,25-dihydroxyvitamin D3 on parathyroid hormone secretion in cultured bovine parathyroid cells

The present studies investigate the effects of glucocorticoids on the function of the parathyroid glands using primary cultures of bovine parathyroid cells. Treatment of parathyroid cell cultures with dexamethasone for 48 h caused a dose-dependent stimulation of PTH secretion. The minimal concentration of dexamethasone required for a significant stimulation of PTH secretion was 0.1 nM. The stimulatory effect of dexamethasone on the secretion of PTH was found within 12 h of treatment with 100 nM dexamethasone. The steroids deoxycorticosterone and cortexolone, which do not have glucocorticoid activity were without effect of PTH secretion. Since glucocorticoids may modulate the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in other tissues, additional studies were performed to evaluate the interactions of glucocorticoids and 1,25-(OH)2D3. Addition of 1,25-(OH)2D3 to parathyroid cell cultures for 48 h significantly suppressed PTH secretion. In the presence of dexamethasone, however, 1,25-(OH)2D3 also significantly decreased PTH secretion, although it did not reduce PTH secretion to control levels. The treatment of parathyroid cell cultures with 100 nM dexamethasone did not affect the parathyroid cell content of 1,25-(OH)2D3 receptors. In summary, these studies indicate that glucocorticoids significantly increase the secretion of PTH in vitro. This stimulatory effect can be inhibited by 1,25-(OH)2D3. The parathyroid gland is an additional site of physiological antagonism of glucocorticoids and 1,25-(OH)2D3.     

Regulation of vitamin D-1alpha-hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney

We investigated the effects of dexamethasone on vitamin D-1alpha-hydroxylase and -24-hydroxylase expression and on vitamin D receptor (VDR) content in the kidneys of mice fed either a normal (NCD) diet or a calcium- and vitamin D-deficient (LCD) diet for 2 weeks. For the last 5 days mice received either vehicle or dexamethasone (2 mg/kg per day s.c.). Dexamethasone significantly increased plasma calcium concentrations without changing plasma concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in both NCD and LCD groups. Northern blot and enzyme activity analyses in NCD mice revealed that dexamethasone increased renal VDR mRNA expression modestly and greatly increased 24-hydroxylase mRNA abundance and enzyme activity, but did not affect 1alpha-hydroxylase mRNA abundance and enzyme activity. In mice fed an LCD diet, dexamethasone increased renal VDR mRNA expression 1.5-fold, decreased 1alpha-hydroxylase mRNA abundance (52%) and activity (34%), and markedly increased 24-hydroxylase mRNA abundance (16-fold) and enzyme activity (9-fold). Dexamethasone treatment did not alter functional VDR number (B(max) 125-141 fmol/mg protein) or ligand affinity (K(d) 0.13-0.10 nM) in LCD mice. Subcutaneous injections of 1,25(OH)(2)D(3) (0.24 nmol/kg per day for 5 days) into NCD mice strongly increased renal 24-hydroxylase mRNA abundance and enzyme activity, while there was no effect of dexamethasone on renal 24-hydroxylase expression in these mice. This may be due to overwhelming induction of 24-hydroxylase by 1,25(OH)(2)D(3). These findings suggest that glucocorticoid-induced osteoporosis is caused by direct action of the steroids on bone, and the regulatory effect of glucocorticoids on renal 25-hydroxyvitamin D(3) metabolism may be less implicated in the initiation and progression of the disease.