2D3-induced Increase Research Articles

Transcriptional regulation and renal localization of 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression: effects of the Hyp mutation and 1,25-dihydroxyvitamin D3.

X-Linked hypophosphatemic (HYP) mice respond to low phosphate (Pi) intake with a fall in the serum concentration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and an increase in the renal activity of 1,25-(OH)2D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway that degrades 1,25-(OH)2D3 to its final inactivation product. In contrast, normal mice respond to a low Pi diet with an adaptive increase in serum 1,25-(OH)2D3 levels and no change in renal 24-hydroxylase. The low Pi response in Hyp mice involves a 3-fold increase in renal 24-hydroxylase maximum velocity and a corresponding increase in 24-hydroxylase immunoreactive protein and messenger RNA (mRNA). To determine the mechanism for the increase in 24-hydroxylase mRNA in the mutant strain, we examined the effect of actinomycin D on renal 24-hydroxylase mRNA abundance and measured renal 24-hydroxylase gene transcription by nuclear run-off assay in Hyp mice fed control and low Pi diets for 4 h. Vehicle and 1,25-(OH)2D3-treated normal mice were also studied 2-4 h post-treatment. Actinomycin D abrogated the increase in renal 24-hydroxylase mRNA elicited by a low Pi diet in Hyp mice and by 1,25-(OH)2D3 in both normal and Hyp mice. 24-Hydroxylase gene transcription, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was increased 2-fold by feeding the low Pi diet to Hyp mice (n = 4; P < 0.05) and 5.4-fold by 1,25-(OH)2D3 administration to normal mice (n = 3; P < 0.01). In situ hybridization localized 24-hydroxylase transcripts to the proximal tubule of normal and mutant mice fed control and low Pi diets and showed that the 1,25(OH)2D3-induced increase in 24-hydroxylase mRNA occurred in the same nephron segment. The present study demonstrates that 1) transcriptional activation can account for the increase in renal 24-hydroxylase mRNA in Pi-deprived Hyp mice and for 24-hydroxylase mRNA induction by 1,25-(OH)2D3; and 2) the renal proximal tubule is the primary site of increased expression of 24-hydroxylase mRNA.

1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose-response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0.5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67.4 +/- 8.8 fmol/mg protein in vehicle-treated cells to 96.7 +/- 12.4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1.08 +/- 0.10 nmol/l for 1,25-(OH)2D3-treated and 0.97 +/- 0.11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 micrograms/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.

Gramicidin D Is a Potent Insulin Secretagogue; Dependence on Extracellular Calcium Influx

Gramicidin D, a sodium ionophore, was identified as a potent insulin secretagogue in the mouse β-cell line, βTC3. Gramicidin D stimulated insulin secretion by 3.2-fold relative to control cells incubated with vehicle alone. Using ion-specific fluorescent probes, gramicidin D (1 μM) increased the intracellular concentrations of Na+ ([Na+]i) and Ca2+ ([Ca2+]i). By contrast, no changes in pHi were detected in cells exposed to ionophore. The increase in [Ca2+]i was biphasic and characterized by an initial peak at 1-2 minutes followed by a sustained second phase, The addition of EGTA (2 mM) to the extracellular medium abolished gramicidin D-induced increase in [Ca2+]i and insulin secretion. These parameters were also profoundly inhibited by the L-type Ca2+-channel inhibitor, verapamil (20 μM). These findings suggest that insulin secretion induced from βTC3 cells by gramicidin D is mediated via the promotion of Ca2+-influx.

Effect of 1,25-dihydroxyvitamin D3 and diltiazem on tissue calcium in uremic rat.

The concentration of calcium was measured in the aorta, heart, and kidney of uremic rats treated with 100 ng/kg/day 1,25-dihydroxyvitamin D3 (1,25 D3) or 60 mg/kg/day diltiazem for 12 weeks. The concentration of calcium was increased in the aorta, heart, and kidney of uremic rats, and was further increased by administration of 1,25 D3. The 1,25 D3-induced increase in calcium in the aorta was inhibited by diltiazem, but this effect was not accompanied by a decrease in serum calcium x phosphate products. Diltiazem had no effect on the 1,25 D3-induced increase of calcium in the heart and kidney. Thus, in uremia 1,25 D3 may promote the calcification of the aorta; calcium antagonists may protect against calcification without a reduction in serum calcium x phosphate products.

1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i.

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.

Transforming growth factor β-induced dissociation between vitamin D receptor level and 1,25-dihydroxyvitamin D 3 action in osteoblast-like cells

In the present study the interaction between a locally produced factor in bone, transforming growth factor β (TGFβ) and a systemic regulator of bone metabolism, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) was investigated. In rat (UMR 106, ROS 17/2.8) and human (MG-63) osteoblastic cell lines and in isolated fetal rat osteoblasts TGFβ caused a comparable increase in vitamin D receptor (VDR) level. A maximum was observed after 6 h at 1 ng/ml TGFβ. Scatchard analysis revealed that up-regulation of VDR is due to an increase in receptor number and not to a change in affinity. This was supported by Northern blot analysis which showed a dose- and time-dependent increase in VDR mRNA by TGFβ. To assess the significance of the TGFβ-induced increase in VDR level for l,25-(OH) 2D 3 effects cells were preincubated with TGF for 4 h (causing a 2–3-fold increase of the VDR level) and subsequently incubated with l,25-(OH) 2D 3 for 4 h and 24 h. TGFβ preincubation potently inhibited subsequent l,25-(OH) 2D 3 stimulation of osteocalcin production in both ROS 17/2.8 and MG-63 cells on protein as well as mRNA level. A similar inhibition by TGFβ was observed on the 1,25-(OH) 2D 3-induced increase in osteopontin mRNA. The current study demonstrates dissociation between regulation of VDR level and modulation of two l,25-(OH) 2D 3 biological responses by TGFβ in osteoblast-like cell lines of different origin. This dissociation shows that, besides interaction at VDR level also at other levels in the cell interaction(s) exist between TGFβ and 1,25-(OH) 2D 3. Besides, these data emphasize the potential importance of the interplay of locally produced factors and systemic calciotrophic hormones in the regulation of bone metabolism.

1 alpha,25-(OH)2 vitamin D3 enhances expression of the genes encoding Ca(2+)-binding proteins MRP-8 and MRP-14

Two closely related Ca(2+)-binding proteins, migration inhibitory factor-related protein (MRP)-8 and MRP-14, are synthesized under specific conditions of myeloid cell differentiation. Because 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces myeloid cell differentiation and expression of other S-100 class calcium-binding proteins, we examined the effects of 1,25-(OH)2D3 on MRP mRNA levels in human U-937 histiocytic lymphoma cells. 1,25-(OH)2D3 increased MRP-8 and MRP-14 mRNA levels in a time- and dose-dependent manner. MRP mRNA levels were maximal at 24 h and remained elevated for at least 96 h after exposure of the cells to 1,25-(OH)2D3. MRP-8 mRNA accumulation required 100- to 1,000-fold higher concentrations of 25-(OH)D3, which binds to the 1,25-(OH)2D3 intracellular receptor with 100- to 1,000-fold lower affinity. Other differentiating agents, dimethyl sulfoxide, retinoic acid, and dexamethasone, also increased levels of MRP-8 and MRP-14 mRNA. Phorbol myristate acetate enhanced MRP-14 mRNA levels to a greater extent than MRP-8 mRNA levels, suggesting differential regulation of MRP gene expression by protein kinase C. The 1,25-(OH)2D3-induced relative increase in MRP mRNA levels was not changed by a 1,000-fold reduction in extracellular [Ca2+]. Thus 1,25-(OH)2D3 is potentially a physiological modulator of MRP gene expression. Expression of the MRP-8 and MRP-14 genes may be important for differentiation of myeloid cells.

1,25-Dihydroxyvitamin D 3 stimulates both alkaline phosphatase gene transcription and mRNA stability in human bone cells

We have previously reported that 1,25(OH) 2D 3 stimulated the cellular alkaline phosphatase (ALP) activity and increased the steady-state level of ALP mRNA in a human osteosarcoma cell line (TE-85), under serum-free conditions. To define the molecular mechanism by which 1,25(OH) 2D 3 acts to stimulate ALP activity, the time courses of the increases in ALP activity and in the steady-state ALP mRNA level in response to 1,25(OH) 2D 3 were evaluated. 1,25(OH) 2D 3 progressively increased the steady-state level of ALP mRNA from 5 to 24 h of treatment, at which time a plateau was observed. In contrast, no significant increase in ALP-specific activity was detected until after 10 h of treatment, at which time the activity increased linearly with time up to 72 h. These time courses are consistent with the premise that the increased ALP activity was the result of increased gene expression. Nuclear runoff analysis indicated that the transcription rate of the ALP gene was more than five-fold higher in the 1,25(OH) 2D 3-treated cells than in the control cells. In addition, it was found that 1,25(OH) 2D 3 treatment increased ALP mRNA stability. The 1,25(OH) 2D 3-induced increase in ALP mRNA stability was not due to an interaction of the 1,25(OH) 2D 3—receptor complex with the ALP mRNA, since the removal of 1,25(OH) 2D 3 did not abolish its stabilizing effect. In the presence of cycloheximide, the stabilizing effect of 1,25(OH) 2D 3 was abolished, suggesting that a 1,25(OH) 2D 3-inducible protein factor was involved. Based on these findings, we have proposed a model in which 1,25(OH) 2D 3 stimulated ALP activity in human bone cells through mechanisms involving both (1) increased transcription of the ALP gene and (2) increased stability of ALP mRNA, an effect which requires the de novo synthesis of a protein, a putative ALP mRNA “stabilizing factor.”

1,25-Dihydroxyvitamin D 3 increases the toxicity of hydrogen peroxide: the role of calcium and heat shock

1,25-Dihydroxyvitamin D 3 [1,25-(OH) 2D 3] increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We therefore examined whether 1,25-(OH) 2D 3 would also modulate the susceptibility to H 2O 2-induced oxidative stress. Prior incubation for 24 h with 1,25-(OH) 2D 3 (25 pM or higher) produced unexpected increased H 2O 2 toxicity. Since cellular Ca 2+ may be a mediator of cell injury, we investigated the effects of altering extracellular Ca 2+ ([Ca 2+ e) on 1,25-(OH) 2D 3-enhanced H 2O 2 toxicity, as well as the effects of 1,25-(OH) 2D 3 and H 2O 2 on cytosolic-free Ca 2+ concentration ([Ca 2+] f). Basal [Ca 2+] f in medium containing 1.5 mM Ca 2+ as determined by fura-2 fluorescence was higher in 1,25-(OH) 2D 3-pretreated cells than control cells (137 versus 112 nM, p < 0.005). H 2O 2 induced a rapid increase in [Ca 2+] f (to > 300 nM) in both 1,25-(OH) 2D 3-treated and control cells, which was prevented by a reduction in [Ca 2+] e to less than basal [Ca 2+] f. The 1,25-(OH) 2D 3-induced increase in H 2O 2 toxicity was also prevented by preincubation with 1,25-(OH) 2D 3 in Ca 2+-free medium or by exposing the cells to H 2O 2 in the presence of EGTA. Preexposure of cells to 45°C for 20 min, 4 h earlier, partially prevented the toxic effects of H 2O 2 particularly in 1,25-(OH) 2D 3-treated cells, even in the presence of physiological levels of [Ca 2+] e. Thus, 1,25-(OH) 2D 3 potentiates H 2O 2-induced injury probably by increasing cellular Ca 2+ stores. The protective effects of heat shock are probably exerted at a site distal to the toxic effects of Ca 2+. The 1,25-(OH) 2D 3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca 2+-associated enhanced susceptibility of oxidative injury due to 1,25-(OH) 2D 3.

Major pathway for putrescine synthesis induced by 1α,25-dihydroxyvitamin D3 in chick duodenum

We have reported that a single injection of 1α,25-dihydroxyvitamin D3 into vitamin D-deficient chicks produces a marked accumulation of putrescine in the duodenum by an interconversion pathway. In the present study, we examined the effect of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine, a specific irreversible inhibitor of polyamine oxidase, on the duodenal putrescine synthesis induced by 1α,25-dihydroxyvitamin D3. Addition of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to an assay mixture completely inhibited the activity of duodenal polyamine oxidase in vitro. Prior administration of N1,N2-bis(2,3-butadienyl)-1,4-butanediamine to chicks completely blocked the 1α,25-dihydroxyvitamin D3-induced increase in duodenal accumulation of putrescine in vivo. The increase of the duodenal accumulation of putrescine by 1α,25-dihydroxyvitamin D3 in vitamin D-deficient chicks coincided quantitatively with the amount of N1-acetylspermidine synthesized from spermidine after the injection of the vitamin into the chicks pretreated with the inhibitor of polyamine oxidase. These results clearly indicate that spermidine N1-acetyltransferase plays a preferential role in the increase in duodenal putrescine synthesis by 1α,25-dihydroxyvitamin D3. The rapidly proliferating and maturing epithelium of small intestines will provide a good model for investigating the role of the interconversion of polyamine metabolism in cell growth and differentiation.

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.

Bisphosphonates inhibit 1,25-dihydroxyvitamin D3-induced increase of osteocalcin in plasma of ratsin vivo and in culture medium of rat calvariain vitro

In order to test whether bisphosphonates, which are potent inhibitors of osteoclastic bone resorption, may also act upon osteoblasts, we studied the effect of dichloromethylenebisphosphonate (Cl2MBP) and 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) on in vivo levels and in vitro release of osteocalcin, a bone-specific protein produced by osteoblasts. In rats, 161 mumol/kg of Cl2MBP or 1.61 mumol/kg AHBuBP strongly inhibited the increase of plasma osteocalcin induced by 1,25(OH)2D3. The inhibition was measurable within 24 hours after the administration of bisphosphonate and was independent of any change in bone resorption. The effect upon osteocalcin release was also present in calvaria cultures. 250 microM Cl2MBP strongly inhibited the osteocalcin release induced by 10(-8) M 1,25(OH)2D3. In the presence of 1,25(OH)2D3, protein synthesis and DNA synthesis were also decreased, whereas in the absence of 1,25(OH)2D3, protein synthesis was increased. Thus, bisphosphonates affect the production of a bone-specific protein by osteoblasts in addition to their inhibitory action on osteoclasts.

Effect of 1,25-dihydroxyvitamin D3 on cytosolic calcium in dispersed parathyroid cells

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cytosolic calcium ([Ca]i) of dispersed bovine parathyroid cells, using the fluorescent dye indo-1. The addition of 10(-8) M 1,25-(OH)2D3 caused an increase in [Ca]i by 23.4 +/- 2.7% over a 10 minute period. There was a significant increase in [Ca]i within two minutes of the addition of 1,25-(OH)2D3. 1,25-(OH)2D3 increased [Ca]i in a dose-dependent manner and this occurred with as little as 10(-10) M. Neither 10(-7) M 25-(OH)D3 nor 10(-7) M 24, 25-(OH)2D3 caused a significant increase in [Ca]i. Chelation of extracellular calcium with EGTA blocked the 1,25-(OH)2D3-induced increase in [Ca]i, suggesting that the increase was mainly from extracellular calcium. Neither 10(-5) M verapamil nor 10(-4) M diltiazem blocked the 1,25-(OH)2D3-induced increase in [Ca]i. The present data suggest that 1,25-(OH)2D3 might modify membrane permeability to calcium independent of voltage-dependent calcium channels sensitive to verapamil or diltiazem. The rapid effect of 1,25-(OH)2D3 raises the possibility that its mechanism is independent of genome activation, perhaps attributable to direct interaction with components of the parathyroid cell plasma membrane.

Actin polymerization. The mechanism of action of cytochalasin D.

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.

Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1 alpha,25-dihydroxyvitamin D3-induced putrescine synthesis.

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

The mechanisms of increase in bone resorption induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] and bacterial lipopolysaccharides (LPS) were compared in an in vitro dead bone assay and a living bone assay. 1 alpha,25(OH)2D3 at concentrations of 0.05-5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release 45Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05-5 micrograms/ml similarly enhanced the release of 45Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1 alpha,25(OH)2D3 and LPS dose-dependently stimulated the release of 45Ca from fetal mouse calvaria prelabeled with 45Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1 alpha,25(OH)2D3 or 5 micrograms/ml of LPS. Indomethacin (10(-5) M) completely inhibited the LPD-induced increase of osteoclasts, but not the 1 alpha,25(OH)2D3-induced increase. These results suggest that 1 alpha,25(OH)2D3 and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1 alpha,25(OH)2D3 induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.

Cytochalasin D-induced increase in actin synthesis and content in a variety of cell types

Treatment of a variety of mesenchymal cells (normal and transformed rat fibroblasts, bovine aortic endothelial cells, rabbit smooth muscle cells), exhibiting different cytoskeletal organizations and derived from several species, with doses of cytochalasin D (CD, 2–6 μM for 20 h) sufficient to induce cytoskeletal rearrangement and altered cellular morphology results in an increase in the relative content and rate of synthesis of actin. These data extend our previous findings for HEp-2 cells to other cell types and provide further evidence for our hypothesis that the CD-induced cytoskeletal reorganization triggers stimulation of actin synthesis and the resulting increase in actin content.

Effect of 1,25(OH)2D3 on the relative contributions of skeletal 45Ca and intestinal 40Ca to blood calcium in normal and thyroparathyroidectomized dogs

The effects of gradually increasing doses of 1,25(OH)2D3 on plasma calcium and 45Ca radioactivity were studied in young dogs that had been extensively prelabelled with 45Ca. The effects of orally and intravenously administered 1,25(OH)2D3 were evaluated in normal and thyroparathyroidectomized dogs fed a normal diet. In normal dogs when 1,25(OH)2D3 increased the plasma calcium within the normal range (2.9-3.1 mmol/L) there was no significant increase in plasma 45Ca. In thyroparathyroidectomized dogs, oral or intravenous 1,25(OH)2D3 increased the low blood calcium to a normal level (1.8-2.9 mmol/L) without significantly increasing plasma 45Ca. In normal and thyroparathyroidectomized dogs, any 1,25(OH)2D3-induced increase in plasma calcium above the normal range was associated with a significant increase in 45Ca, indicating mobilization of bone calcium. Intravenous administration of 1,25(OH)2D3 in the normal or thyroparathyroidectomized dogs had a much larger effect than oral doses in mobilizing bone 45Ca when inducing a similar level of hypercalcemia. The major physiological effect of 1,25(OH)2D3 in the low or normal range of plasma calcium is on intestinal absorption of calcium without a significant effect on mobilizing bone calcium. The pharmacological effect of 1,25(OH)2D3 in vivo is to mobilize bone calcium as well as dietary calcium into blood.

Rat duodenal calcium-binding protein messenger rna: Induction by 1,25-dihydroxyvitamin D 3

To extend our previous observations on the regulation of CaBP biosynthesis by 1,25-dihydroxyvitamin D 3, we have studied the specific mRNA encoding this protein in vitamin D-deficient and in vitamin D-repleted rats as well as the rate of its induction after a single injection of 1,25(OH) 2D 3 to vitamin D-deficient animals. The CaBP-mRNA was quantified by translation in a cell-free reticulocyte lysate system. CaBP-mRNA activity and cytoplasmic CaBP (measured by radioimmunoassay), dramatically decreased in rats previously fed a vitamin D-free diet for 5 weeks but neither parameter was zero. In vitamin D-deficient rats, a single injection of 1,25(OH) 2D 3 led to an increase in CaBP-mRNA activity within 2 h. This CaBP-mRNA activity peaked at about 4–6 h and thereafter declined to low value by 48 h. and the changes in mRNA activity always preceded the changes in cytosolic CaBP concentration. These results indicate that the induction of CaBP biosynthesis results from a 1,25(OH) 2D 3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH- 2D 3. This study also shows that the production of many other proteins seem to be under the control of vitamin D 3

Cell-free translation analysis of the vitamin D-dependent calcium binding protein mRNA activity present in total RNA and polysomal extracts from chick intestine

To further elucidate the mechanism(s) involved in the induction of a vitamin D-dependent intestinal calcium binding protein (CaBP), by the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], we have examined by cell-free translation CaBP-mRNA activity present in total cellular RNA extracts or polysomes obtained from chick small intestine. CaBP-mRNA activity was examined by immunochemical analysis of polypeptides completed upon addition of purified intestinal RNA or polysomes to a lysate system. Intestinal polysomes isolated from vitamin D-deficient chicks treated with vitamin D 3, 1,25(OH) 2D 3, or 1α-hydroxyvitamin D 3 were found to complete the synthesis of an immunoprecipitable protein which comigrated with authentic chick intestinal CaBP on SDS polyacrylamide gels. This product was found to represent up to 0.5–1.0% of the total polypeptides directed by polysomes isolated from treated birds but could not be detected among the products directed by intestinal polysomes obtained from vitamin D-deficient birds. A detectable level of polysomal CaBP-mRNA activity was present within 3 h and a maximal activity achieved at 9 h after administration of 1,25(OH) 2D 3. Increases in the levels of cytoplasmic CaBP (measured by radioimmunoassay) were first detected at 6 h, which was significantly later than the polysomal CaBP-mRNA activity. CaBP-mRNA activity in total cellular RNA isolated with guanidinium thiocyanate was also found to be dependent on treatment with vitamin D 3 or 1,25(OH) 2D 3 but represented only 0.1–0.2% of the total mRNA activity. Surprisingly, total RNA and poly(A) RNA were found to direct the synthesis and release of two additional polypeptides that were immunoprecipitated even with an affinity-purified antibody to CaBP. One, an approximately 11,000-dalton polypeptide, was clearly vitamin D dependent; the other, an approximately 38,000-dalton polypeptide, was not. The time course for appearance of CaBP-mRNA activity in total cell RNA extracts following 1,25(OH) 2D 3 treatment was found to be nearly identical to that observed in polysome preparations. These results indicate that induction of CaBP-biosynthesis results, at least in part, from a 1,25(OH) 2D 3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH) 2D 3.