The selenoprotein thioredoxin reductase is expressed in peripheral blood monocytes and THP1 human myeloid leukemia cells--regulation by 1,25-dihydroxyvitamin D3 and selenite

1,25(OH)2 Vitamin D3 (1,25(OH)2D3) and adhesion propagate monocyte differentiation. We identified the selenoprotein thioredoxin reductase (TrxR) as a new molecular target for 1,25(OH)2D3 in monocytes during this process. In THP1 monocytic leukemia cells 1,25(OH)2D3 stimulated TrxR mRNA levels 2-4-fold by 4-8 h and enhanced TrxR activity (60%) (as measured by the dithionitrobenzole-assay) after 24 h, which declined below baseline after 96 h. The addition of 100 nM selenite enhanced (approx. 50%) basal and stimulated enzyme activity in THP1 cells. The relative stimulation by 1,25(OH)2D3 was very similar but peak levels were sustained in THP1 cells up to 48 h. Human peripheral blood monocytes (PBM) of different donors showed very low basal TrxR steady state mRNA levels which were markedly enhanced (as analyzed by Northern blotting) after 4 h of adherence to culture dishes. 1,25(OH)2D3 (100 nM) further stimulated TrxR mRNA expression (4 h, 3-fold). TrxR enzyme activity mirrored the mRNA changes. Basal activity was stimulated approx. 25% by adhesion in culture alone and was further stimulated (approximately 15%) by 1,25(OH)2D3 after 4 h. By 24 h similar results were achieved but the effect of 1,25(OH)2D3 could be seen in the presence of 100 nM selenium only. The expression of TrxR and its regulation by 1,25(OH)2D3 and selenite in monocytes might be important for their induction of differentiation and maintenance of function.     

1,25-Dihydroxycholecalciferol effects on plasma calcitonin levels and calcitonin mRNA in normal or partially vitamin D-depleted rats

The physiological effect of 1,25-(OH)2D3 on the regulation of calcitonin (CT) secretion was studied by measuring plasma CT levels and CT mRNAs extracted from thyroid glands of normal (D+) or partially vitamin D-depleted rats (D-). In both groups, acute 1,25-(OH)2D3 administration of 0.1 microgram/kg b.w. yielded an early drop in plasma calcium concentrations (around 0.6-1 mg/dl) with a maximum decrease 15 min after treatment. In spite of this hypocalcemia, a significant rise in plasma CT levels was observed within 5 min in D+ animals and within 30 min in D- animals after injection of the vitamin D metabolite. Nevertheless, the increased CT secretion was not associated with a marked and sustained rise in CT mRNA levels measured by dot-blot hybridization or CT mRNA activity evaluated by translation assay. By contrast to the observations made previously using supra-physiological doses of the vitamin D metabolites, no clear-cut effect on CT mRNA levels was found with lower doses. If we hypothesized that 1,25-(OH)2D3 plays a physiological role in CT secretion, our results suggest that this rapid control could be exerted at a post-translational level may be via an increase in the cytoplasmic ionized calcium concentration of C-cells.     

Regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D3 in human osteosarcoma cells

Synthesis of type I and III collagens has been examined in MG-63 human osteosarcoma cells after treatment with the steroid hormone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Analysis of total [3H]proline-labeled proteins and pepsin-derived collagens revealed that 1,25-(OH)2D3 selectively stimulated synthesis of alpha 1I and alpha 2I components of type I collagen after 6-12 h. Consistent with previous reports (Franceschi, R. T., Linson, C. J., Peter, T. C., and Romano, P. R. (1987) J. Biol. Chem. 262, 4165-4171), parallel increases in fibronectin synthesis were also observed. Hormonal effects were maximal (2- to 2.5-fold versus controls) after 24 h and persisted for at least 48 h. In contrast, synthesis of the alpha 1III component of type III collagen was not appreciably affected by hormone treatment. Of several vitamin D metabolites (1,25-(OH)2D3, 25-dihydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) tested for activity in stimulating type I collagen synthesis, 1,25-(OH)2D3 was found to be the most active. Analysis of collagen mRNA abundance by Northern blot hybridization indicated that both types I and III procollagen mRNAs were increased 4-fold after a 24-h exposure to 1,25-(OH)2D3. Pro alpha 1I mRNA remained elevated through the 48-h time point while pro alpha 2I and pro alpha 1III mRNAs returned to control values. These results indicate that the regulation of collagen synthesis by 1,25-(OH)2D3 is complex and may involve changes in translational efficiency as well as mRNA abundance. 1,25-(OH)2D3 also caused at least a 20-fold increase in levels of the bone-specific calcium-binding protein, osteocalcin. These results are consistent with the hypothesis that 1,25-(OH)2D3 is stimulating partial differentiation to the osteoblast phenotype in MG-63 cells.     

1,25-Dihydroxyvitamin D3 has opposite effects on the expression of parathyroid secretory protein and parathyroid hormone genes

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Differential effects of 1,25-dihydroxyvitamin D3 upon intestinal vitamin D3-dependent calbindin (a 28,000-dalton calcium binding protein) and its mRNA in D-replete and D-deficient chickens

The effect of vitamin D3 status upon the responsiveness of chick intestinal epithelium to exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was studied. Intestinal calbindin [A recent consensus decision was made to redesignate the vitamin D-dependent calcium binding protein as "calbindin-D28K" (R.H. Wasserman (1985) in Vitamin D: Chemical, Biochemical, and Clinical Update (Norman, A.W., Schaefer, K., Grigoleit, H.-G., and Herrath, D.V., Eds.), pp. 321-322, de Gruyter, Berlin/New York).] protein and intestinal calbindin mRNA were quantitated in birds which had been raised on a vitamin D3-deplete (-D) or on a vitamin D3-replete (+D) diet. 1,25(OH)2D3 stimulated intestinal calbindin mRNA levels in -D chickens in a proportional dose-dependent manner, when measured at both 12 and 48 h after administration of the hormone. A first increase was observed with 1,25(OH)2D3 concentrations between 0.065 and 0.65 nmol. The maximal stimulation achieved by 1,25(OH)2D3 (6.5-18 nmol) in -D tissue was approximately 10-fold over the calbindin mRNA levels present in vehicle-treated birds. The increase of calbindin mRNA in -D birds was associated with a similar dose-dependent increase in calbindin protein in 1,25(OH)2D3-treated -D birds after 12 or 48 h. In +D intestine, while exogenous 1,25(OH)2D3 also increased calbindin mRNA levels in a dose-dependent fashion, the maximal stimulation observed after 5 h (1.2- to 2-fold) was clearly less than that observed in -D intestine. In contrast to -D birds, intestinal calbindin levels in +D birds were decreased by administration of exogenous 1,25(OH)2D3. Administration of 32.5 to 65 nmol 1,25(OH)2D3 resulted in an approximately 1.8-fold repression compared to vehicle-treated birds. This differential responsiveness between +D and -D intestines with respect to 1,25(OH)2D3 was not explained either by differences in the uptake in the chromatin fractions of these tissues or by metabolism of radiolabeled 1,25(OH)2D3. Dietary withdrawal of vitamin D3 led to a gradual decline in ambient intestinal calbindin levels, while intestinal sensitivity to 1,25(OH)2D3 was restored. These findings suggest that vitamin D3 status regulates intestinal responsiveness to the seco-steroid 1,25(OH)2D3.     

24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs

A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D(3) toxicosis.     

Production and metabolic clearance rates of 1,25-dihydroxyvitamin D3 during maturation in rats: studies using a rapid, primed-infusion technique

I have adapted the primed-infusion technique for the rapid estimation of the metabolic clearance rate (MCR) and production rate (PR) of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in chronically catheterized conscious rats. Following a bolus injection, [3H]-1,25(OH)2D3 was infused iv at a constant rate for 7 h. Steady-state [3H]-1,25(OH)2D3 levels in plasma were achieved within 3 h. HPLC-purification of plasma [3H]-1,25(OH)2D3 was necessary. This rapid, primed-infusion technique thus eliminates the need for protracted infusions to achieve steady-state plasma [3H]-1,25(OH)2D3 levels. The MCR averaged 0.201 +/- 0.003 ml.min-1.kg-1 in fed male rats weighing 200-300 g. This MCR is approximately 50% lower than that seen in other species. Overnight fasting was without effect on the MCR. The MCR increased in direct proportion to body weight in maturing rats (6-16 weeks old) weighing 150-450 g. Thus, the MCR can be normalized per kg across this age range. However, 1,25(OH)2D3 production and plasma levels both decreased by 65-67% as the rats matured. The failure of a 3-fold decrease in plasma 1,25(OH)2D3 levels to affect the MCR suggests that 1,25(OH)2D3 stimulates its own catabolism only at markedly elevated concentrations.     

25-Hydroxyvitamin D3 loading test in primary hyperparathyroidism, hypoparathyroidism and pseudohypoparathyroidism

Plasma 1,25-dihydroxyvitamin D (1,25-(OH)2D) level, which is considered to be an indicator of parathyroid function, is possibly modified by the level of vitamin D. In the present study, we have investigated parathyroid function in terms of enhancement of the plasma levels of 1,25-(OH)2D after oral administration of 100 micrograms of 25-hydroxyvitamin D3 (25OHD3) in 9 cases of primary hyperparathyroidism (1 degree HPT), 7 cases of hypoparathyroidism (HP), 2 cases of pseudohypoparathyroidism (PHP) and 6 normal subjects. The plasma levels of 25-hydroxyvitamin D (25OHD) increased and reached a peak at 6-12 hours after the administration of 25OHD3. The plasma levels of 1,25-(OH)2D slightly increased but remained within the normal range after 25OHD3 administration in 3 of the normal subjects whose basal levels were rather low, but the increase in plasma 1,25-(OH)2D in control subjects was not statistically significant. In cases of 1 degrees HPT, the plasma 1,25-(OH)2D level rose significantly in all cases (P less than 0.05), although the pattern of the increase was not uniform. These increases were remarkable in the patients whose basal levels were low. On the other hand, an increase in the level was rarely observed in any of the cases of HP and in one of the cases of PHP. In another case, normocalcemic PHP, the plasma 1,25-(OH)2D level rose.(ABSTRACT TRUNCATED AT 250 WORDS)