Dibutyryl cAMP enhances the effect of 1,25-dihydroxyvitamin D3 on a human promyelocytic leukemia cell, HL-60, at both the receptor and the postreceptor steps.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces differentiation of a human promyelocytic leukemia cell line, HL-60, into monocytes/macrophages, and 25-hydroxyvitamin D3- and 1,25-(OH)2D3-24-hydroxylase activities in HL-60 mitochondria via a steroid-hormone receptor mechanism. Dibutyryl cyclic adenosine monophosphate (dbcAMP), a granulocyte inducer, significantly augmented the differentiation-inducing effect of 1,25-(OH)2D3 along the monocyte/macrophage pathway. Furthermore, dbcAMP significantly potentiated the effect of 1,25-(OH)2D3 on HL-60 cells to hydroxylate 1,25-(OH)2[26,27-3H]D3 to form 1,24,25-(OH)3[26,27-3H]D3. DbcAMP seemed to augment the effect of 1,25-(OH)2D3 in part through upregulation of the 1,25-(OH)2D3 receptor, because 10(-7) M dbcAMP increased 1,25-(OH)2D3 receptor levels approximately 2.3-fold, which was similar to a 1.9-fold augmentation by the same concentrations of dbcAMP of 1,25-(OH)2D3-induced cell characteristics to hydroxylate C-24 of 1,25-(OH)2[26,27-3H]D3. However, dbcAMP is also known to enhance HL-60 cell differentiation caused by other differentiation inducers. We have established another HL-60 clone which acquires resistance to 1,25-(OH)2D3 in the induction of cell differentiation by a defect at the postreceptor step, as reflected by resistance to other differentiation inducers, such as retinoic acid and dimethyl sulfoxide. Even in this resistant clone, dbcAMP significantly enhanced the differentiation-inducing effect of 1,25-(OH)2D3. Of interest, this clone showed resistance to dbcAMP in the induction of cell differentiation. Furthermore, we have demonstrated that intracellular cAMP levels were significantly lower in uremic serum-treated cells than in cells treated with normal human serum and that a significant positive correlation was found between intracellular cAMP levels and 1,25-(OH)2D3-induced cell differentiation. These data indicated that the intracellular cAMP level is one of the major determinants of 1,25-(OH)2D3-induced HL-60 cell differentiation and that dbcAMP could enhance the effects of 1,25-(OH)2D3 on HL-60 cells not only by increasing 1,25-(OH)2D3 receptor levels but also at the postreceptor step.     

Calcium-transport in quiescent and 1,25-dihydroxycholecalciferol-stimulated human osteosarcoma cells. Role in 24 hydroxylase enhancement

The present study evaluates in osteosarcoma cells, the effects of a calcium channel inhibitor nicardipine in 24-hydroxylase activity and 45Ca desaturation curve in presence of 1,25-dihydroxycholecalciferol (1,25(OH)2D3). This sterol induced an increase in 24-OHase activity and 45Ca fluxes. Nicardipine reversed the effect of 1,25(OH)2D3 on 45Ca fluxes but reinforced the enhancement of the 24-OHase activity. The fact that the effects of 1,25(OH)2D3 were reduced by cycloheximide support the hypothesis of a de novo protein synthesis. Our study has allowed us to dissociate the effects of 1,25(OH)2D3 on 24-OHase enhancement from those on Ca2+ transport.     

In vivo activation of the intracrine vitamin D pathway in innate immune cells and mammary tissue during a bacterial infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D(3) 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D(3) to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Subsequently, synthesis of 1,25(OH)(2)D(3) by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14(+) cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)(2)D(3), was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)(2)D(3) production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)(2)D(3) for local regulation of vitamin D responsive genes.     

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.     

Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.     

Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3-induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.     

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.     

Effect of 1,25-dihydroxyvitamin D3 on human cancer cells in vitro

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.     

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.     

Expression of mRNAs for type-I collagen, bone sialoprotein, osteocalcin, and osteopontin at different stages of osteoblastic differentiation and their regulation by 1,25 dihydroxyvitamin D3

We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.     

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.     

25-hydroxy-vitamin d metabolism in human colon cancer cells during tumor progression

RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1alpha-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH)3-D3 and 1,25-(OH)2- 24-oxo-D3. Likewise, 25-(OH)-D3 is metabolized into 24,25-(OH)2D3, 23,25-(OH)2D3, and 23,25-(OH)2-24-oxo-D3. Poorly-differentiated cancers expressed low levels of 1alpha-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1alpha,25-(OH)2-D3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1alpha,25-(OH)2-D3 particularly at late stages of colon cancer progression.     

Induction of Cholinergic Expression in Developing Spinal Cord Cultures

The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (1 mM) produced a 2-3-fold stimulation of ChAT activity in developing cultures whereas 8-bromo cGMP had no effect. A phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, also increased (2-fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2-16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP-mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1-day exposure to dbcAMP. 125I-Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7-9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of monocyte chemotaxis by 1 alpha,25-dihydroxyvitamin D3. Stimulation of defective migration of AIDS patients

Preincubation for 20 h with 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) markedly augmented the chemotactic responsiveness of human blood monocytes to the classical chemoattractant, FMLP. A modest enhancement of monocyte spontaneous locomotion in the absence of chemoattractants was also observed. Maximal increase of monocyte migration was observed after pretreatment with 10(-9) M 1,25(OH)2D3 and was detectable at FMLP concentrations ranging from 10(-10) to 10(-7) M. Pretreatment with 1,25(OH)2D3 augmented the number of monocyte high affinity FMLP receptors (1500 +/- 220 and 3800 +/- 300 sites per cell for untreated and 1,25(OH)2D3-pretreated cells, respectively) with no significant changes in Kd values (2 +/- 0.5 nM and 4 +/- 1 nM, for untreated and 1,25(OH)2D3-pretreated monocytes). Enhanced chemotaxis was not restricted to FMLP, because 1,25(OH)2D3-treated monocytes showed enhanced migration also in response to activated C components and chemotactic cytokines. In agreement with previous observations, monocytes from AIDS patients showed defective migration capacity. In vitro exposure to 1,25(OH)2D3 stimulated monocyte migration in all 10 patients examined with considerable quantitative differences among individuals. Regulating the responsiveness of mature monocytes to chemo-attractants, 1,25(OH)2D3, produced systemically or in situ by immunocompetent cells, could play a role in the regulation of the recruitment of monocytes at sites of inflammation, cell-mediated immunity, or bone resorption. The potential of 1,25(OH)2D3 as a restorative agent under conditions of defective phagocyte recruitment deserves further exploration.     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

1,25-Dihydroxyvitamin D3 regulation of phorbol ester receptors in HL-60 leukemia cells

In this study the relationship between cell binding of phorbol 12,13-dibutyrate (PDBu) and induction of differentiation by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was examined. Binding of [3H]PDBu increased within 12 h of 1,25-(OH)2D3 treatment, and a 60-130% increase in [3H]PDBu receptor levels was observed within 24 h. By 48 h, however, [3H]PDBu binding was not different from control. Scatchard analysis of [3H]PDBu binding showed no statistical differences in Kd value (Kd approximately equal to 30 nM) between 1,25-(OH)2D3-treated and control cells 22 h post-treatment; however, a 2-fold increase in Bmax was observed in treated (338 +/- 24 pmol/10(9) cells) compared to control cultures (170 +/- 14 pmol/10(9) cells). Stimulation of [3H]PDBu binding was dependent on 1,25-(OH)2D3 concentrations over a range of 1-100 nM. Homogenates from 1,25-(OH)2D3-treated HL-60 cells also demonstrated an increase (70%) in [3H]PDBu binding to the Ca2+/phospholipid-dependent enzyme protein kinase C as assessed by incubation of cell homogenates with [3H]PDBu in the presence of saturating phosphatidylserine and calcium concentrations. This suggests that the increase in [3H]PDBu binding cannot be entirely explained by modulation of the latter two agents. Cycloheximide (5 microM), an inhibitor of protein synthesis, ablated the 1,25-(OH)2D3-stimulated increase in [3H]PDBu binding to intact HL-60 cells. These data demonstrate that an increase in [3H]PDBu binding occurs early in the course of 1,25-(OH)2D3-induced differentiation, results from an increased number of [3H]PDBu-binding site, and is dependent on protein synthesis.