Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells

The effect of extracellular calcium ion (Ca2+) concentration on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induction of vitamin D-dependent calcium-binding protein (calbindin-D28K) and its mRNA levels was examined in primary chick kidney cells in vitro. When exposed to normal medium Ca2+ (1.0 mM), 1,25-(OH)2D3 increased calbindin-D28K mRNA, as measured by Northern analysis, by 4-10 fold over basal levels by 12 to 24 h after addition of hormone. In the presence of 0.5 mM Ca2+, 1,25-(OH)2D3 induced calbindin-D28K mRNA by only 2 fold, whereas, when cells were exposed to 2 mM Ca2+, the induction was 10-15 fold. This calcium modulation of 1,25-(OH)2D3 induction was also observed at the level of calbindin-D28K protein concentrations as measured by radioimmunoassay. The alterations in medium Ca2+ were not associated with any change in the rate of total RNA or protein synthesis. These studies suggest that both Ca2+ and 1,25-(OH)2D3 participate in the regulation of calbindin-D28K gene expression in the kidney.     

Age-related alterations in calbindin-D28K induction by 1,25-dihydroxyvitamin D3 in primary cultures of rat renal tubule cells.

In vivo studies have indicated that renal calbindin-D28K protein and mRNA levels decrease in adult and old rats, and this decrease parallels the age-associated decline in serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels. However, diminished renal responsiveness to 1,25-(OH)2D3 with advancing age could also contribute to decreased calbindin-D28K expression. To study renal responsiveness with age, primary cell cultures were established from the kidney cortices of young (1 month old), adult (10-12 months old), and old (20-24 months old) rats. Cells were incubated in medium K-1 containing 2% fetal calf serum. Calbindin-D28K protein levels were determined by Western blot and enzyme-linked immunosorbent assay. In young animals, the levels of calbindin-D28K declined from 12.1 +/- 1.3 micrograms/mg protein in the intact kidney to 1.6 +/- 0.07 micrograms/mg protein in cells that had been cultured for 3 days in the absence of 1,25-(OH)2D3. This sharp decline in calbindin-D28K protein concentration moderated by days 6-8. The continuous presence of 10(-7) M 1,25-(OH)2D3 in the medium did not abolish the decline. The low levels of calbindin-D28K in the cells cultured in the absence of 1,25-(OH)2D3 provided an excellent experimental system in which to compare the response of the cells to 1,25-(OH)2D3 between age groups. In cultured cells treated with 1,25-(OH)2D3 for 72 h, calbindin-D28K induction was greater in cells from adult and old animals compared to cells from young animals. The ratios of calbindin-D28K content (with vitamin D/without vitamin D) were 2.2 +/- 0.2, 4.7 +/- 0.5, and 7.1 +/- 1.5 for young, adult, and old cells, respectively. These studies suggested that the observed in vivo decrease in renal calbindin-D28K with age is primarily due to the lowered circulating 1,25-(OH)2D3.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.     

Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses.

We have examined the 5' flanking region of the mouse calbindin-D28k gene and identified a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-responsive element by deletion mutant analysis of the native promoter as well as by studies with a heterologous thymidine kinase (TK) promoter. The segment between residues -200 and -169 was found to confer a dose-dependent 1,25-(OH)2D3 responsiveness through the TK promoter in Ros 17/2.8 cells as well as in CV-1 cells cotransfected with pAV-hVDR (human vitamin D receptor expression vector). This region contains sequences homologous to the rat osteocalcin vitamin D response element (VDRE). Incubation of this element with nuclear extracts from 1,25-(OH)2D3-treated Ros 17/2.8 cells or from 1,25-(OH)2D3-treated COS cells that had been transfected with pAV-hVDR resulted in a specific protein-DNA interaction. In addition to 1,25-(OH)2D3, sodium butyrate, a differentiating agent, has also been found to modulate expression of calbindin-D28k. Deletion analysis of the mouse calbindin-D28k promoter as well as studies with a heterologous TK promoter resulted in identification of a butyrate-responsive element between -180 and -150 that was found to bind specifically to nuclear factors from butyrate-treated Ros 17/2.8 cells. This butyrate-responsive element may represent a genetic element acted upon by enhancer binding proteins. In summary, the 5' flanking region of the mouse calbindin-D28k gene contains responsive elements that interact with nuclear factors and may mediate, at least in part, the enhanced expression of this gene by 1,25-(OH)2D3 and butyrate.     

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.     

Hormonal regulation of chick kidney inhibitor of adenosine 3',5'-monophosphate-dependent protein kinase

The endogenous inhibitor of cAMP-dependent protein kinase (PKI) in chick kidney is regulated by the vitamin D status of the animal. To determine the specific factors that are involved in the regulation of chick kidney PKI, chicks were raised on a low (0.05%), normal (1%), or high (3%) calcium diet and given vitamin D3 or vehicle three times a week orally. The results from this experimental protocol show that vitamin D3 or one or more of its metabolites and serum calcium levels are both involved in the regulation of chick kidney PKI in vivo. Measurement of PKI activity in primary cultures of chick kidney cells revealed treatment with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3) led to a 90-95% decrease in PKI activity. This effect of 1,25-(OH)2D3 was dose dependent, and neither PTH nor insulin was able to reverse it completely. Treatment with PTH caused 30-60% increase in PKI activity, and cell cultures that were grown in medium containing either 0.5 or 2 mM calcium chloride had similar PKI activities. Taken together, these results indicate that 1,25-(OH)2D3, the most physiologically active form of vitamin D3, is the predominant regulator of PKI, but serum calcium, indirectly through the regulation of PTH secretion, is also involved.     

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10⁻⁷ M, 1,25(OH)₂D₃ for 24 h resulted in a 20–30% decrease in PKI mRNA. 1,25(OH)₂D₃ treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)₂D₃ directly and tissue-specifically downregulates PKI mRNA in the chick kidney.     

The influence of vitamin D metabolites on collagen synthesis by chick cartilage in organ culture

When growth cartilage from rachitic chicks was cultured in the presence of the calcium-regulating hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), collagen resorption was increased and collagen synthesis decreased compared to control cultures containing no hormone. The minimum concentration of the hormone that caused a statistically significant inhibition of collagen synthesis was 10(-8) mol/l. Collagen synthesis by growth cartilage from normal chicks was also reduced by 1,25-(OH)2D3, showing that it was not an abnormal response of vitamin D-depleted tissue. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 also inhibited collagen synthesis by cultures of growth cartilage but only at higher metabolite concentrations. 1,25-Dihydroxyvitamin D3 (10(-7) mol/l) did not significantly inhibit collagen synthesis by cultures of articular fibrocartilage and of sternal cartilage, tissues that do not calcify physiologically. The minimum concentration of 1,25-(OH)2D3 (10(-9) mol/l) necessary to cause decreased collagen synthesis by embryonic chick calvaria was lower than the value obtained with growth cartilage; this suggests that bone cells may be more sensitive to the hormone in this respect than are growth cartilage chondrocytes. These findings provide evidence of a direct role of 1,25-(OH)2D3 in the control of endochondral bone formation which is consistent with its primary role in the maintenance of plasma calcium homeostasis.     

TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.     

25-Hydroxyvitamin D3 metabolism in mitochondria from primary renal cultures

Previous in vitro studies concerning the renal metabolism of 25-hydroxyvitamin D3 (25(OH)D3) to form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25R-dihydroxyvitamin D3 (24,25(OH)2D3) have utilized intact cell systems. In reflecting upon the possible mechanisms by which hormonally induced changes in the production of 1,25(OH)2D3 and 24,25(OH)2D3 may be brought about, we asked whether altered mitochondrial hydroxylase activities can quantitatively account for changes in the total cellular output of these steroids. Our objective was to delineate between extramitochondrial processes (e.g. altered substrate delivery), and those events restricted to the renal mitochondria (altered hydroxylase activities). We have examined the effect of pretreating primary cultures of chick kidney cells with either 1,25(OH)2D3 or parathyroid hormone (PTH) on 25(OH)D3-hydroxylase activities present in subsequently isolated mitochondria. Pretreatment with 10(-7) M 1,25(OH)2D3 reduced 1 alpha-hydroxylase activity in both cells and mitochondria to approximately 60% of control values by 1 h, and to 25-30% by 2 h. The effect of PTH (10 ng/ml) in both mitochondrial and whole cell preparations was an approximate 40% increase in measured 1 alpha-hydroxylase activity. 10 microM forskolin (FSK) elicited an approximate 2-fold increase in 1,25(OH)2D3 production. Reciprocal effects were observed with respect to 24-hydroxylase activity in both whole cell and mitochondrial preparations in response to exogenous 1,25(OH)2D3, PTH, and FSK. The findings demonstrate that these hormones initiate intracellular events which lead directly to altered 25(OH)D3 1 alpha- and 24-hydroxylase activities within the renal mitochondria.     

Prolactin but not growth hormone stimulates 1,25-dihydroxyvitamin D3 production by chick renal preparations in vitro

We studied the effect of PRL from two species (bovine and turkey) and GH from two species (bovine and turkey) on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by two whole cell preparations from vitamin D-deficient chick kidneys (slices and tubules). We observed that 8 ng/ml turkey PRL stimulated 1,25(OH)2D3 production by renal tubules and slices. Ovine PRL had a similar effect on 1,25(OH)2D3 production but at higher concentrations. In contrast, neither bovine GH nor turkey GH stimulated 1,25(OH)2D3 production appreciably at doses up to 1000 ng/ml. The effect of PRL on 1,25(OH)2D3 production by renal tubules required a 3-h preincubation, although its effect on 1,25(OH)2D3 production by renal slices was immediate. We conclude that PRL, but not GH, directly stimulates 1,25(OH)2D3 production by the chick kidney.     

Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin

In order to determine whether cAMP mediates the effects of PTH on the metabolism of 25-hydroxyvitamin D3 (25-OH-D3) on chick kidney cells in primary culture, the effect of forskolin on the production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was assessed. In 4-h incubations with [3H]25-OH-D3 and forskolin, (1-10 microM) [3H]1,25-(OH)2D3 accumulation was increased 50-100%, and that of [3H]24,25-(OH)2D3 was decreased 30-60%. PTH (1-10 ng/ml) brought about identical changes. Similar results were observed when cultures were preincubated with nonradioactive 25-OH-D3 for 4 h in the presence of PTH and forskolin, followed by a 30-min incubation with radioactive substrate. At a low concentration (0.05 microM), forskolin alone had no effect on the metabolism of [3H]25-OH-D3 but markedly enhanced that of PTH. At maximal concentrations of PTH (10 ng/ml) and forskolin (10 microM), the effects of the two on 25-OH-D3 metabolism were not additive. Both PTH and forskolin decreased the further metabolism of [3H]1,25-(OH)2D3, probably by inhibiting its 24-hydroxylation, but there are also cycloheximide-sensitive steps in the metabolism of 1,25-(OH)2D3 that are not affected by PTH and forskolin. In time course experiments, increased [3H]1,25-(OH)2D3 accumulation could be observed before the detection of 24-hydroxylase activity suggesting that the primary effect of PTH and forskolin is on the production of [3H] 1,25-(OH)2D3 rather than its catabolism. Raising the calcium concentration of the medium to 2.5 mM from the normal 1.8 mM or lowering it to 0.5 mM for 24 h in serum-free medium did not alter the response of 25-OH-D3 metabolism to these agents. The results of these studies indicate that the effects of PTH on the metabolism of 25-OH-D3 by chick kidney cells are mediated by cAMP, since they can be enhanced and mimicked by forskolin, that they are exerted at the level of both 1- and 24-hydroxylase activity, and that they are not dependent on the calcium concentration of the medium.