Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides.

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ±â€¯3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ±â€¯6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis.

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Vitamin D target proteins: function and regulation.

Recent findings have indicated that calbindin-D(28k), the first known target of vitamin D action, is present in osteoblasts and protects against TNF and glucocorticoid induced apoptosis of osteoblastic cells. Cytokine mediated destruction of pancreatic beta cells, a cause of insulin dependent diabetes, is also inhibited by calbindin-D(28k). In calbindin-D(28k) transfected pancreatic beta cells free radical formation by cytokines is inhibited by calbindin. Thus, besides its role as a facilitator of calcium diffusion, calbindin has a major role in protecting against cellular degeneration in different cell types. Besides calbindin, the other known pronounced effect of 1,25(OH)(2)D(3) in intestine and kidney is increased synthesis of 25(OH)D(3) 24-hydroxylase (24(OH)ase) which is involved in the catabolism of 1,25(OH)(2)D(3). We have noted that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)(2)D(3). These studies establish C/EBPbeta as a novel 1,25(OH)(2)D(3) target gene and indicate a role for C/EBPbeta in 24(OH)ase transcription. These studies extend our previous studies related to factors that affect vitamin D receptor (VDR) mediated 24(OH)ase transcription (YY1, TFIIB, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment.

1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function- and cell-specific additive effects.

Vitamin D3 derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)2D3 and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)2D3 10(-10) and 10(-9) M were synergistic with ATRA 10(-8) M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)2D3 and ATRA individually induced an accumulation of MCF-7 cells in the G1 phase of the cell cycle and an associated increase in p21WAFI/CiP1, p27KiP1 and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)2D3 but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)2D3, a noncalcemic analogue 1,25(OH)2-16-ene-23-yne-D3, ATRA, AGN195183, an RARalpha-specific agonist, and AGN190168 (tazarotene), an RARbeta/gamma-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)2-16-ene-23-yne-D3 combined with ATRA or with tazarotene at 10(-9) M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)2D3, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.

Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis.

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.

Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells.

In this study, the interrelationship between signal transduction pathways and 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] action was examined in UMR106 osteoblastic cells. Treatment of these cells with 8-bromo-cAMP (1 mM) resulted in an upregulation of the vitamin D receptor (VDR) and an augmentation in the induction by 1,25(OH)2D3 of 25(OH)D3 24-hydroxylase [24(OH)ase] and osteopontin (OPN) mRNAs as well as gene transcription. Transfection with constructs containing the vitamin D response element devoid of other promoter regulatory elements did not alter the cAMP-mediated potentiation, suggesting that cAMP-enhanced transcription is due, at least in part, to upregulation of VDR. Treatment with phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA) 100 nM], an activator of protein kinase C, significantly enhanced 1,25(OH)2D3-induced OPN mRNA and transcription but had no effect on VDR or on 24(OH)ase mRNA or transcription. Studies using OPN promoter constructs indicate that TPA-enhanced OPN transcription is mediated by an effect on the OPN promoter separate from an effect on VDR. Thus interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, changes in cellular phosphorylation may play a significant role in determining the effectiveness of 1,25(OH)2D3 on transcriptional control in cells expressing skeletal phenotypic properties.

YY1 represses vitamin D receptor-mediated 25-hydroxyvitamin D(3)24-hydroxylase transcription: relief of repression by CREB-binding protein.

Ying Yang transcription factor (YY1) can repress or activate transcription. 25-Hydroxyvitamin D(3)-24-hydroxylase [24(OH)ase], an enzyme involved in the catabolism of 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], is up-regulated at the transcriptional level by 1,25-(OH)(2)D(3) to self-induce its deactivation. Here we report that YY1 can repress 1,25-(OH)(2)D(3)-induced 24(OH)ase transcription in CV1 cells transfected with vitamin D receptor (VDR) expression vector or in LLCPK(1) cells that contain VDR endogenously. With increasing amounts of YY1 DNA transfected (500 ng to 2 microg), ligand-dependent VDR activation of 24(OH)ase transcription was steadily repressed (maximum repression was 10-fold). Thus, YY1 may be a key modulator preventing activation at times that do not require the enzyme to be expressed. Relief of YY1 repression was observed in the presence of TFIIB or CBP (CREB binding protein) suggesting that YY1 may exert repression, in part, by sequestering TFIIB/CBP. Glutathione-S-transferase (GST) pull-down assays identified regions in the N and C termini of CBP that can bind YY1. In addition, the N-terminal region of CBP that interacts with YY1 can inhibit YY1 from binding to TFIIB. Thus, CBP may alleviate YY1-mediated repression, in part, by preventing YY1 from binding to TFIIB, which is required for VDR-mediated transcription. In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. In addition, our findings demonstrate a role for CBP in relief of repression of VDR-mediated transcription.

Acute hypoxia-induced alterations of calbindin-D28k immunoreactivity in cerebellar Purkinje cells of the guinea pig fetus at term.

Purkinje cells (PCs) are vulnerable to hypoxic/ischemic insults and rich in calcium and calcium-buffering/sequestering systems, including calcium-binding proteins (CaBPs). Calbindin-D28k is an EF-hand CaBP, which is highly expressed in PCs where it acts primarily as a cellular Ca++ buffer. Elevation of [Ca++] in the cytosol and nuclei of PCs is pivotal in hypoxic/ischemic cell death. We hypothesize that hypoxia results in decreased concentration, or availability of calbindin-D28k in PCs, thereby decreasing their buffering capacity and resulting in increase of intracellular and intranuclear [Ca++]. Cerebellar tissues from normoxic fetuses were compared to fetuses obtained from term pregnant guinea pigs exposed to hypoxia [7% FiO2] for 60 min. The pregnant guinea pigs were either killed upon delivery immediately following hypoxia (Hx0h) or were subsequently allowed to recover for 24 h (Hx24h) or 72 h (Hx72h). Fetal brain hypoxia was documented biochemically by a decrease in brain tissue levels of ATP and phosphocreatine. Compared to normoxic fetuses, there is a predominantly somatodendritic loss or decrease of calbindin-D28k immunohistochemical staining in PCs of Hx0h (p < 0.005), Hx24h (p < 0.05), and Hx72h (p < 0.005) fetuses. Hypoxia-induced alterations of calbindin-D28k immunoreactivity are qualitatively similar at all time points and include a distinctive intranuclear localization in subpopulations of PCs. A similar trend is demonstrated by immunoblotting. Subpopulations of TUNEL+/calbindin-D28k- PCs lacking morphologic features of apoptosis or necrosis are demonstrated in Hx24h and Hx72h fetuses. The present study demonstrates an abrogating effect of perinatal hypoxia on calbindin-D28k immunoreactivity in cerebellar PCs. The perturbation of this Ca++ buffer protein in hypoxia-induced neuronal injury may herald delayed cell death or degeneration.

Myocardial contusion presented as acute myocardial infarction after chest trauma.

A 46-year-old male patient developed an acute myocardial infarction and congestive heart failure following blunt chest trauma. Electrocardiogram (ECG) revealed acute anterior myocardial infarction. Echocardiography showed akinesis of interventricular septum, dyskinesis in apical anterior wall, and severe impairment of left ventricular overall systolic function. Coronary angiography revealed normal coronary arteries. The patient followed a low-intensity physical medicine rehabilitation program. Follow-up was without new complications or deterioration of congestive heart failure. Five months later the patient presented with fulminant acute pulmonary edema and cardiogenic shock. Cardiopulmonary resuscitation was unsuccessful.

The role of calbindin and 1,25dihydroxyvitamin D3 in the kidney.

The identification of a putative apical Ca++ channel in 1,25dihydroxyvitamin D3 responsive epithelia (proximal intestine and the distal nephron) as well as recent studies using calbindin-D28k knock-out mice indicating the first direct in-vivo evidence for a role for this calcium-binding protein in renal calcium absorption suggest mechanisms, which had remained incomplete, related to the control of renal calcium absorption.

Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity.

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death.

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.

Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP).

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.

Concentration- and cell type-specific effects of calbindin D28k on vulnerability of hippocampal neurons to seizure-induced injury.

The calcium-binding protein calbindin D28k (CB) is expressed in limited subpopulations of neurons in the brain. In the hippocampus, CB is expressed in all dentate granule cells and a subpopulation of CA1 pyramidal neurons, but is absent from CA3 neurons. This pattern of CB expression is inversely correlated with neuronal vulnerability to seizure-induced damage suggesting the possibility that expression of CB confers resistance to excitotoxicity. While data from cell culture studies support an excitoprotective role for calbindin, it is not known whether CB is a key determinant of neuronal vulnerability in vivo. We therefore examined the pattern of damage to hippocampal neurons following intrahippocampal injection of the seizure-inducing excitotoxin kainate in CB homozygous (CB-/-) and CB heterozygous (CB+/-) knockout mice in comparison with wild-type mice (CB+/+). Whereas the extent of damage to CA1 neurons was similar in CB-/- and CB+/+ mice, damage to CA1 neurons was significantly reduced in CB+/- mice. Dentate granule neurons were not damaged following kainate-induced seizures in CB+/+, CB+/- or CB-/- mice. These findings suggest that CB can modify vulnerability of hippocampal CA1 neurons to seizure-induced injury, and that either CB is not a critical determinant of resistance of dentate granule neurons, or compensatory changes occur and lack of CB is not the only difference between CB-/- and CB+/+ mice.

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines.

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Effect of calbindin D 28K on sodium transport by the luminal membrane of the rabbit nephron.

We previously reported that in the rabbit, the vitamin D-dependent calcium binding protein 28K (CaBP 28K) increases calcium (Ca2+) transport in the distal tubule by opening a high affinity Ca2+ channel in the luminal membrane. Since Na+ and Ca2+ transports are interdependent in this membrane, we questioned whether the calbindin has any influence on Na+ transport. Luminal membranes from rabbit proximal and distal tubules were purified and 22Na uptake by the membrane vesicles was measured using the rapid filtration technique. The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes pH 7.4, with either 3 microM CaBP or the carrier. Incubation medium contained 1 mM 22NaCl, 278 mM mannitol, and 20 mM Tris-Hepes pH 7.4. The presence of 3 microM CaBP 28K in the distal luminal membrane vesicles increased the 0.5 mM Ca2+ uptake from 0.91 +/- 0.21 to 1.84 +/- 0.33 pmol/microg/10 s (P < 0.01) and decreased 1 mM Na+ uptake from 0.62 +/- 0.15 to 0.27 +/- 0.08 pmol/microg/10 s (P < 0.05). A similar decrease of Na+ uptake was observed in proximal luminal membrane experiments. The effect on Na+ uptake by the distal membrane was dose-dependent with a IC50 of 4.5 microM. Addition of 2 mM Ca2+ to the incubation medium decreased 1 mM Na + uptake from 0.62 +/- 0.15 to 0.49 +/- 0.12 pmol/microg/10 s (P < 0.05), but did not influence the effect of CaBP 28K on Na+ uptake. Experiments performed in the presence and absence of ethyl isopropyl amiloride (EIPA) suggest that the effect of calbindin involves the Na+/H+ exchanger activity.

Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury.

Tumor necrosis factor alpha (TNFalpha) is widely expressed in both neurons and glia and has been shown to be upregulated after traumatic brain injury (TBI). TNFalpha receptor activation results in activation of the transcription factor nuclear factor kappaB (NF-kappaB), which may serve an antiapoptotic role via the induction of target genes manganese superoxide dismutase (MnSOD) and/or calbindin. In the present study, we used a controlled cortical impact model of TBI with pertinent lines of transgenic mice to combine both morphological characterization and molecular analysis to elucidate the role of TNFalpha after TBI. Measurements of both the lesion volume and the blood-brain barrier breach indicated exacerbations in mice rendered genetically deficient in both the p55 and p75 TNFalpha receptors (TNFR-KO) compared with wild-type animals. Additionally, animals genetically altered to overexpress MnSOD showed a significant decrease in lesion volume compared with that of control littermates, whereas no alterations were observed in mice lacking the calcium-binding protein calbindin D28k. Analysis of NF-kappaB activation and relative levels of MnSOD revealed delayed responses in the injured cortex of TNFR-KO animals compared with wild-type animals, implying that endogenous TNFalpha may be neuroprotective after TBI.

Expression of 25(OH)D3 24-hydroxylase in distal nephron: coordinate regulation by 1,25(OH)2D3 and cAMP or PTH.

Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1-34); 10 nM] was found to potentiate the effect of 1, 25(OH)2D3 on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1, 25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1, 25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3 in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.

Expression of calbindin-D28k in C6 glial cells stabilizes intracellular calcium levels and protects against apoptosis induced by calcium ionophore and amyloid beta-peptide.

The calcium binding protein, calbindin-D28k is normally present in neurons. Recently we reported that brain injury and tumor necrosis factors (TNFs) induce calbindin-D28k in astrocytes. TNF-treated calbindin expressing astrocytes were resistant to acidosis and calcium ionophore toxicity, suggesting that calbindin may have a cytoprotective role in astrocytes in the injured brain (M.P. Mattson, B. Cheng, S.A. Baldwin, V.L. Smith-Swintosky, J. Keller, J. Geddes, Scheff, J.W., Christakos, S., Brain injury and tumor necrosis factors induce calbindin-D28k in astrocytes: evidence for a cytoprotective response, J. Neurosci. Res., 42 (1995) 257). In order to obtain direct evidence for a role of calbindin, using the eukaryotic expression vector pREP4, rat calbindin-D28k was stably expressed in C6 rat astocytoma glial cells. Cytotoxicity in response to calcium ionophore or amyloid beta-peptide (which accumulates in the brain in Alzheimer's disease and has been reported to be neurotoxic) was measured by MTT reduction in vector transfected cells and in calbindin transfected clones. Stably expressed calbindin resulted in increased cell survival in the presence of calcium ionophore (1-10 microM) or amyloid beta-peptide (10-100 microM). In addition, the calcium ionophore or amyloid beta-peptide mediated rise in intracellular calcium in vector transfected cells was significantly attenuated in calbindin expressing cells. Apoptotic cell death was detected by the Hoechst method in vector transfected C6 glial cells treated with calcium ionophore or beta-amyloid (34-36% apoptotic cells/culture). The number of apoptotic nuclei was significantly attenuated in similarly treated calbindin-D28k transfected clones (10-13% apoptotic cells/culture; p<0.01). Our results support the involvement of calcium fluxes in apoptosis and suggest that calbindin-D28k, by buffering calcium, can suppress death in apoptosis susceptible cells in the central nervous system.