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Parathyroid hormone (PTH) or activators of protein kinase A (PKA) up-regulate the vitamin D receptor (VDR) and augment the induction by 1,25-dihydroxyvitamin D(3) of the expression of target genes (24-hydroxylase and osteopontin) in osteoblastic cells. To understand regulatory mechanisms involved, we asked whether the inducible cAMP early repressor (ICER), which serves as a dominant negative regulator of cAMP-induced transcription in other endocrine systems, may similarly play a role in modulation of vitamin D hormone action. In this study we demonstrate that PTH or 8-bromo-cAMP rapidly induces ICER mRNA and protein in osteoblastic cells. In UMR 106 osteoblastic cells transfected with an expression vector containing the ICER II-gamma coding sequence, cAMP or PTH enhancement of 1,25-dihydroxyvitamin D(3)-induced osteopontin and 24-hydroxylase mRNA and transcription is inhibited. The vitamin D response element is sufficient for the PKA enhancement of VDR-mediated transcription and is also sufficient to observe the inhibitory effect of ICER. Our data indicate that the mechanism of the inhibitory effect of ICER involves an inhibition of PKA-induced VDR transcription, and this inhibition may be mediated in part by binding of ICER to a cAMP response element-like sequence in the VDR promoter. This study provides evidence for the first time that ICER has a key regulatory role in the PKA enhancement of VDR transcription and therefore in the cross-talk between the PKA signaling pathway and the vitamin D endocrine system.

This article describes studies on the glucose-induced responses of intracellular Ca2+ concentration ([Ca2+]i), insulin release, and redistribution of calbindin-D28k, a calcium-binding regulatory protein, in beta-cells of pancreatic islets of calbindin-D28k knockout (KO) and wild-type mice (C57BL6) as well as in betaHC-13 control cells and betaHC-13 CaBP40 cells (beta-cell line overexpressing calbindin-D28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca2+]i (mean increase in [Ca2+]i, i.e., delta[Ca2+]i, was 296 nM) compared with wild-type mice (delta[Ca2+]i = 97 nM). betaHC-13 CaBP40 cells showed little change in [Ca2+]i upon elevation of glucose from 5.5 to 32.7 mM, whereas betaHC-13 control cells exhibited significant increases in [Ca2+]i, (delta[Ca2+]i = 510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in betaHC-13 control cells, whereas in betaHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D28k in beta-cells affect both [Ca2+]i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This co-localization may be an important regulatory function concerning Ca2+ influx via L-type VDCC and exocytosis of insulin granules.

When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)(2)D(3)-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)(2)D(3)-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)(2)D(3) alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)(2)D(3) or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

We present the case of a patient with acute pulmonary embolism and a large right atrial and a smaller pulmonary artery thrombus. Thrombi were successfully treated with intravenous heparin. Echocardiography was used to document the morphological evolution of thrombi and to evaluate the medical treatment.

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.

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., (Copyright 1999 Elsevier Science B.V.)

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

We report the case of a patient who was admitted to the hospital with acute pulmonary embolism 2 weeks after a complicated pelvis fracture. Echocardiography revealed a large, long, and mobile thrombus in the right atrium. The patient was scheduled to undergo urgent surgical thrombectomy. Preoperative echocardiography did not detect any thrombi in the right heart and pulmonary artery. The obvious embolism of this large thrombus in the pulmonary circulation was silent as the patient remained asymptomatic and hemodynamically stable. We discuss the contribution of echocardiography to the appropriate therapeutic management of right atrial thrombi and particularly to the cancellation of urgent operative thrombectomy.

Mutations in the presenilin 1 (PS-1) gene account for many cases of early-onset autosomal dominant inherited forms of Alzheimer's disease. Recent findings suggest that PS-1 mutations may sensitize neurons to apoptosis induced by trophic factor withdrawal and exposure to amyloid beta-peptide (Abeta). We now report that overexpression of the calcium-binding protein calbindin D28k prevents apoptosis in cultured neural cells expressing mutant PS-1 (L286V and M146V missense mutations). Elevations of the intracellular Ca2+ concentration and generation of reactive oxygen species induced by Abeta, and potentiated by mutant PS-1, were suppressed in calbindin-overexpressing cells. Impairment of mitochondrial function by Abeta (which preceded apoptosis) was exacerbated by PS-1 mutations and was largely prevented by calbindin. These findings suggest that PS-1 mutations render neurons vulnerable to apoptosis by a mechanism involving destabilization of cellular calcium homeostasis, which leads to oxidative stress and mitochondrial dysfunction.

The influence of kainic acid (KA), which induces acute seizures, on expression of mRNA for the calcium-binding protein, calbindin-D28k, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and early-response genes [c-fos, zif268 (NGFI-A), nur77 (NGFI-B)] was examined in rat hippocampus by Northern blot analysis. A significant increase (3.2-fold) in BDNF mRNA was observed 1 h after KA injection (12 mg/kg i.p.) and peak expression (9.4-fold) occurred 3 h after KA. The induction of BDNF mRNA was preceded by the induction of c-fos, mRNA (30 min after KA) and was followed by the induction of calbindin-D28k mRNA (3.5-fold 3 h after KA; a maximal response was at 3-6 h after KA). Region-specific changes, analyzed by immunocytochemistry and in situ hybridization, indicated that the most dramatic increases in calbindin protein and mRNA after KA treatment were in the dentate gyrus. Although calbindin-D28k and BDNF mRNAs were induced, a 3.4-3.8-fold decrease in NT-3 mRNA was observed by Northern analysis 3-24 h after KA treatment. Calbindin-D28k gene expression was also examined in rats with a chronic epileptic state characterized by recurrent seizures established with an episode of electrical stimulation-induced status epilepticus (SE). When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed. Our findings suggest that the induction of calbindin-D28k mRNA (which may be interrelated to the induction of BDNF mRNA) is an early response which may not be related to enhanced neuronal activity or seizures per se, but rather to maintaining neuronal viability.

Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.

This study was undertaken to determine whether adaptations to short-term exercise training after myocardial infarction, could affect the response of heart rate, blood pressure and double product at submaximal workload, and the behavior of electrocardiographic ST segment depression. We studied 60 patients (group A) who underwent a modest level exercise training for 3 months and 40 patients (group B) who did not participate in this program. All these subjects were involved in the trial 1 week after discharge from the hospital. Submaximal treadmill stress test was performed after the 3-month period. The stress test duration was longer (p < .01), heart rate (p < .001), systolic blood pressure (p < .01) and double product (p < .01) at submaximal workload were lower and the onset of ST depression of 1 mm was delayed (p < .01) in group A. In conclusion, postinfarction short-term exercise training increases the exercise tolerance, decreases the heart rate, systolic blood pressure, and double product response to exercise and improves the ischemic threshold.

Changes in gene expression after kindled seizures were examined using microdissection of discrete brain areas and Northern and slot blot analyses. Experimental animals were kindled with either of two protocols: (1) a paradigm in which 50 Hz/10 s stimulus trains were delivered every 30 min through hippocampal electrodes (12 stimulations every other day for 4 days) and (2) a traditional approach in which 50 Hz/10 s stimulus trains were given to the hippocampus three times daily for 16 days. Rats were sacrificed 24 h or 30 days after the last kindled seizure. We first examined the possibility that kindling may affect transcription of mRNA for neurotransmitter receptors. We found significant decreases (22-58%) in AMPA/kainate activated glutamate receptor mRNAs (GluR1, -2, -3 mRNAs) in hippocampus, amygdala/entorhinal cortex and in frontoparietal cortex 24 h but not 30 days after rapidly kindled seizures. However, changes in GABA receptor alpha 1, alpha 2, alpha 4 or beta 1 mRNAs were not observed in any brain region 30 days after traditional kindling or 24 h after rapidly kindled seizures. In addition, we tested whether changes in the expression of proenkephalin could be detected after kindling. We found significant increases (1.7-10 fold) in proenkephalin mRNA in the frontoparietal cortex, hippocampus and in the amygdala/entorhinal cortex 24 h but not 30 days after rapidly kindled seizures. Our findings suggest that changes in glutamate receptor and proenkephalin gene expression are robust, acute sequelae to kindled seizures and may be involved in kindling.

The distal tubule is a major site of hormone regulated Ca2+ reabsorption. We have previously shown that in the rabbit, vitamin D depletion decreases both the Ca2+ uptake by the luminal membrane of nephron distal segments and the ATP-dependent Ca2+ transport by the basolateral membrane of these same segments. We also reported that in in vitro experiments, the vitamin D-dependent calcium binding protein (CaBP) 28K enhances Ca2+ uptake by the high-affinity Ca2+ transport system of the distal luminal membranes. The purpose of the present study was to investigate the role of CaBP 9K in Ca2+ transport by the two polar membranes of the proximal and distal nephron segments. Preloading the luminal membranes of the two series of tubules with 10 microM CaBP 9K did not modify 0.5 mM Ca2+ uptake by these membranes. In contrast, preincubation of distal tubule basolateral membranes with 10 microM CaBP 9K strongly enhanced ATP-dependent Ca2+ transport by these membranes. A similar although more modest effect was observed in proximal basolateral membranes. This effect was present only in calmodulin depleted membranes. Calmodulin also increased the ATP-dependent Ca2+ uptake by the depleted membranes. The two actions were not additive. A dose-response curve showed that the maximal CaBP 9K effect was obtained at 10(-5) M. CaBP 9K increased the Vmax of ATP-dependent uptake from 0.43 +/- 0.03 to 0.79 +/- 0.06 nmol/mg/10 sec (P < 0.005), leaving the Km unchanged. It is concluded that CaBP 9K activates the Ca2+ pump in the basolateral membrane of both the proximal and distal tubule.(ABSTRACT TRUNCATED AT 250 WORDS)

Several clearance and micropuncture studies suggest that vitamin D deficiency decreases calcium (Ca2+) reabsorption by the nephron. In confirmation of these observations, we reported recently that in vitamin D depleted rabbits, Ca2+ transport by both the luminal and the basolateral membranes of the distal tubule was significantly diminished. It is also in the distal tubule that vitamin D stimulates the synthesis of two calcium binding proteins (CaBP) with molecular weights of 28K and 9K. The purpose of this study was to investigate the role of the CaBP 28K in the regulation of Ca2+ transport by the two polar membranes of the distal nephron of the rabbit. Results were compared to those obtained with the corresponding membranes of the proximal tubule. The basolateral and luminal membranes were purified from proximal and distal tubule suspensions separately. Ca2+ uptake was measured by the rapid filtration technique. Preloading the luminal membrane vesicles from proximal and distal tubules with 3 microM CaBP 28K strongly enhanced the initial Ca2+ uptake by the two membranes. When the vesicles, preloaded with CaBP 28K and incubated with 0.5 mM Ca2+, were abruptly diluted in a medium containing 10 microM A 23187 and 2 mM EGTA, the release of Ca2+ was slower than with control vesicles, suggesting that the excess of transported Ca2+ was bound inside the vesicles. The effect of CaBP 28K was dose-dependent with a half-maximal effect at 10(-6) M and a maximal effect at 5 x 10(-6) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Emerging data indicate that neurotrophic factors and cytokines utilize similar signal transduction mechanisms. Although neurotrophic factors can protect CNS neurons against a variety of insults, the role of cytokines in the injury response is unclear. We now report that TNF beta and TNF alpha (1-100 ng/ml) can protect cultured embryonic rat hippocampal, septal, and cortical neurons against glucose deprivation-induced injury and excitatory amino acid toxicity. The elevation of intracellular calcium concentration ([Ca2+]i) induced by glucose deprivation, glutamate, NMDA, or AMPA was attenuated in neurons pretreated with TNF beta. The mechanism whereby TNFs stabilize [Ca2+]i may involve regulation of the expression of proteins involved in maintaining [Ca2+]i homeostasis, since both TNF beta and TNF alpha caused a 4- to 8-fold increase in the number of neurons expressing the calcium-binding protein calbindin-D28k. These data suggest a neuroprotective role for TNFs in the brain's response to injury.

1. 1,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in addition to regulating calcium homeostasis, also has antiproliferative and prodifferentiating effects. 2. Most studies concerning the therapeutic potential of analogs of 1,25(OH)2D3, which are antiproliferative and prodifferentiating but do not cause hypercalcemia, have been done using leukemic cells. 3. Recent evidence from both in vivo and in vitro studies has indicated that 1,25(OH)2D3 or analogs of 1,25(OH)2D3 can inhibit the growth of breast cancer cells, thus suggesting the therapeutic potential of analogs of 1,25(OH)2D3 in the treatment of breast cancer.

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III) to that of calbindin-D28k in 40 human fetal and postnatal cerebella ranging from 12 weeks gestation to adulthood. In the external granule layer of the developing cerebellar cortex, beta III staining was present in the premigratory (postmitotic) zone of horizontal neurons but was absent in "epithelioid" cells of the subpial proliferative mitotic zone. In the molecular layer, intense beta III staining was associated with parallel fibers, stellate/basket neurons and migrating fusiform granule neurons. beta III staining was also present in internal granule neurons. In contrast, beta III was not detectable in fetal and neonatal Purkinje neurons and Golgi II neurons, but was evident in these neurons from juvenile and adult cerebella. Calbindin-D28k staining was present in Purkinje neurons also delineating their somatic spines ("pseudopodia"), lateralizing and apical dendrites (including dendritic spines), subpopulations of small to intermediate-sized Golgi II neurons in the internal granule layer ("synarmotic cells" of Landau), large to medium-sized subcortical Golgi II neurons and neurons of cerebellar roof nuclei, at various gestational stages and postnatally. It was absent in the external granule layer, parallel fibers, stellate/basket and internal granule neurons. Variable degrees of beta III and calbindin-D28k staining were detected in subpopulations of immature neuroepithelial cells of the ventricular matrix at the roof of the fourth ventricle. Glial (including Bergmann glia) and mesenchymal cells were not stained for either antigenic determinants. The differential expression of calbindin-D28k and beta III defines distinct populations of neurons in the developing human cerebellar cortex and supports the ontogenetic concept of Ramon y Cajal.

Calbindin-D28k appears in the metanephric kidney during embryogenesis. We studied the temporal appearance and spatial distribution of calbindin-D28k mRNA in the developing kidneys of 12-day fetal through 21-day postnatal mice by in situ hybridization. 35S-UTP-labeled antisense (cRNA) probe to calbindin-D28k mRNA hybridized to the ureteric buds of 12-day embryos, whereas adjacent metanephrogenic tissue was unlabeled. By embryonic day 13, Y-shaped bodies of "advancing" ureteric buds were labeled intensely. In 16-day embryos, ampullae of ureteric buds were located immediately beneath the renal capsule and labeled strongly, in contrast to metanephric tubules and S-shaped bodies. The former were unlabeled and the latter were labeled only at points of contact with the ampullae. Subsequently, the ampullae of the metanephric ureteric buds hybridized with the cRNA probe, and from the 18th embryonic to the 21st postnatal day, this labeling was intense. The cRNA probe did not hybridize with the renal vesicles, proximal tubules, or tubular segments of Henle's loop derived from nephrogenic blastema, but it did label distal nephron segments. By the 21st postnatal day, collecting ducts and ureter no longer were labeled. In conclusion, calbindin-D28k mRNA is present in the developing mouse kidney, and its distribution during nephrogenesis is identical to that of calbindin-D28k per se. Collectively, these findings show that the calbindin-D28k gene is transcribed and its message is translated by the cells of the ureteric bud during the initial stage of renal morphogenesis.

Apoptosis, or programmed cell death, plays an essential role in specific cell deletion during normal embryonic and adult development in vertebrate and invertebrate species. Recent evidence suggests that signal transduction pathways governing cellular proliferation and cell cycle progression also mediate the physiological response to changes in the extracellular environment that trigger the anti-proliferative state characteristic of apoptosis.

We studied the effects of dietary calcium on kidney, femur, testis, liver, heart and brain concentrations of lead, magnesium, iron, copper, calcium and zinc in rats exposed to lead for 1 y. Renal levels of the 28,000 Da, vitamin D-dependent, calcium-binding protein calbindin-D28K were also measured. Seventy-two weanling male Sprague-Dawley rats were randomly assigned to one of nine treatment groups. Rats were fed diets containing 0.1, 0.5 or 2.5% Ca for 52 wk and were simultaneously given either 0, 50 or 100 mg lead/L in their drinking water. Rats fed the 0.1% Ca diet had organ lead concentrations that were two- to 20-fold greater than the corresponding animals fed 0.5% Ca. Rats fed diets containing 2.5% Ca had the lowest organ lead concentrations. Despite substantial effects of diet Ca on organ lead concentrations, Ca did not significantly influence concentrations of most other divalent metals studied with the exception of kidney calcium and magnesium, testis iron, plasma ionic calcium and magnesium, and several femur metals. Kidney calcium concentrations were lower in rats fed 2.5% Ca diets than in those fed 0.1 or 0.5% Ca diets. For rats not given lead, renal calbindin concentrations were highest in rats fed 0.1% Ca, and lowest in rats fed 2.5% Ca. Lead inhibited an increase in renal calbindin in the rats fed 0.1% Ca, but paradoxically increased renal calbindin levels in animals fed 2.5% Ca.

Brain levels of the calcium binding protein Calbindin-D28K (CaBP28K) and CaBP28K mRNA were measured for various animal models of neurodegenerative diseases (MPTP-treated C57BL/6J mice and Sprague-Dawley rats receiving striatal/intraperitoneal kainic acid or quinolinic acid into the nucleus basalis magnocellularis). Brain areas were tested (radioimmunoassay, Western blot, slot blot, and Northern blot) for levels of CaBP28K and CaBP28K mRNA. The various models did not exhibit any changes in protein or mRNA levels from the controls, suggesting that CaBP28K-containing neurons were not lost after exposure to these neurotoxins. Immunocytochemical characterization of the substantia nigra of the MPTP-treated mice revealed that there was significant dopaminergic cell loss in this brain area after MPTP treatment. The majority of dopaminergic neurons that degenerated did not contain CaBP28K. The small percentage of surviving neurons were CaBP28K-positive. These results suggest that the presence of CaBP28K may protect neurons from calcium-mediated neurotoxicity.

Quantitative autoradiographic analysis of [3H] 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding in vitamin D deficient mice provided evidence for high levels of specific binding in choroid plexus and, to a lesser extent, ventral hippocampus. Sucrose gradient analysis yielded a 3-4S peak of specific [3H]1,25(OH)2D3 binding in bovine choroid plexus, but not amygdala or hippocampus. Scatchard analysis of [3H]1,25(OH)2D3 binding in bovine choroid plexus yielded KD = 0.23 +/- 0.06 nM and Nmax = 43.5 +/- 0 fmol/g tissue (n = 5). This result indicates the presence of significant receptor-like [3H]1,25(OH)2D3 binding sites in the choroid plexus and, thus, suggests roles for this hormone in regulating the entry of calcium into the brain and/or in the central regulation of calcium homeostasis.

Studies from our laboratory concerning regulation of calbindin include regulation by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], receptor regulation as a possible mechanism for modulating calbindin's response to hormone, tissue specific regulation and regulation by factors other than 1,25(OH)2D3. With regard to receptor regulation, we found that the induction of calbindin mRNA in intestine and kidney by 1,25(OH)2D3 is not accompanied by a corresponding alteration in vitamin D receptor (VDR) mRNA in the vitamin D-deficient, low calcium rat. However, in the vitamin D-replete rat, administration of 1,25(OH)2D3 results in an induction of both calbindin and VDR mRNA in these tissues. These results suggest the presence of an inhibitor of 1,25(OH)2D3-mediated receptor up-regulation in the vitamin D-deficient, low calcium animal. Glucocorticoids can also regulate calbindin gene expression. Dexamethasone treatment (50 micrograms.100 g body weight-1.d-1 for 4 d) results in a 75% decrease in rat intestinal calbindin-D9k mRNA. This decrease may be related to the inhibition of intestinal calcium absorption previously observed after glucocorticoid administration. Kidney calbindin-D28k mRNA is unaffected by glucocorticoid treatment, indicating tissue specificity of the glucocorticoid response. To evaluate more precisely the means whereby 1,25(OH)2D3 and other modulators can influence calbindin gene expression, we isolated the chromosomal gene for calbindin-D28k by screening a mouse genomic library in cosmid. Ros 17/2.8 cells were transfected with recombinant plasmids in which the mouse calbindin promoter is fused to the reporter gene encoding chloramphenicol acetyltransferase. Deletion studies have enabled us to identify sequence elements in the mouse calbindin-D28k gene that confer basal activation and a hormone inducible response.

Purified porcine erythrocyte membrane Ca(2+)-ATPase and 3':5'-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D28k (CaBP-D28k). The concentration of CaBP-D28k required for half-maximal activation (K0.5 act.) of the Ca(2+)-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition of either excess CaM or CaBP-D28k. 3':5'-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K0.5 act. = 90 nM) or calmodulin (K0.5 act. = 1.2 nM). CaBP-D28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.

In this study the ontogenesis of rat intestinal vitamin D receptor (VDR) gene expression was examined. When Northern and slot blot analyses were used to examine the expression of intestinal VDR mRNA in 15-, 18-, 22-, and 28-day-old rats, induction of VDR mRNA was not observed until 22 days postpartum. Since little is known, particularly in the neonate, concerning the in vivo regulation of VDR gene expression, we examined the possibility that glucocorticoids and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] could affect the developmental expression of the intestinal VDR gene. To examine the effect of glucocorticoids, rat pups received three sequential injections (one per day) of hydrocortisone (5, 2.5, and 2.5 mg/100 g BW). Hydrocortisone administration before day 14 or on days 19-21 was not effective in inducing VDR mRNA. However, a significant 3.8-fold increase in intestinal VDR mRNA was observed in rats injected with hydrocortisone from days 15-17. The hydrocortisone effective period coincides with the glucocorticoid-sensitive period of rat intestinal development. It should be noted, however, that the up-regulation of VDR was accompanied by an increase in actin mRNA, suggesting that the effect is not specific for VDR. Similarly, when rats were bilaterally adrenalectomized on day 17 (killed on day 22), a 4-fold decrease in VDR mRNA was observed, accompanied by a decrease in actin mRNA. However, when rats were injected with 1,25-(OH)2D3 (25 ng/day.100 g BW) from days 15-17, levels of intestinal VDR mRNA were significantly increased by 1.5-fold, and this change was specific for VDR mRNA. In summary, our results indicate that hydrocortisone and 1,25-(OH)2D3 can precociously induce intestinal VDR mRNA, suggesting the involvement of glucocorticoids and 1,25-(OH)2D3 in the regulation of VDR gene expression in the developing rat intestine. However, our results also indicate that the effect of glucocorticoids (unlike the effect of 1,25-(OH)2D3) is not specific for VDR mRNA, but may reflect general effects of glucocorticoids on intestinal maturation.

Previous studies have been directed at the elucidation of neuron-specific gene expression in the mammalian central nervous system. In particular, we have identified a series of marker molecules that are expressed in cerebellar Purkinje cells with varying degrees of specificity. Here, we show by light microscopic immunocytochemistry and Northern transfer and hybridization that two of these markers, namely, L7 and PEP19, are expressed in the retina of mouse and rabbit, while a third marker, cerebellin, is absent. Light and electron microscopic immunocytochemistry proves that L7-like immunoreactivity is restricted to rod bipolar cells, while PEP 19-like immunoreactivity is distributed in both rod and cone bipolars. PEP19 is also expressed by subsets of amacrine and ganglion cells. The density of PEP19-positive bipolar cells is greater than that of L7-positive bipolar cells, although the density of each is approximately equal in central and peripheral portions of the retina. An antiserum to a fourth Purkinje cell marker, vitamin D-dependent calcium-binding protein-28 kD (CaBP), reveals primarily axonless horizontal cells, but also subsets of rod bipolar, amacrine, and, in the mouse but not in the rabbit, ganglion cells. The processes of immunoreactive cell bodies form discrete bands in the internal plexiform layer, and mixtures of the antisera help distinguish their identity. Thus, these Purkinje cell markers can be used at the electron microscopic level to unravel the extremely complex neuropil of this retinal layer. Furthermore, knowledge of the retinal distribution of this panel of molecules is of general value for future studies of retinal neuronal typology and can serve to map the densities of subsets of bipolar cells throughout the retina. The expression of L7 and PEP19 in bipolar cells and in Purkinje cells suggests a biochemical relationship between these two spatially distant neuronal populations.

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.

In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.

Calbindin-D28k is a highly abundant protein found in neurons in selected brain regions, including cells in sensory systems of the brainstem. Because of its capacity to bind cytosolic Ca++, calbindin-D28k is thought to contribute to the regulation of compartmental Ca++ concentrations in neurons. In this study of the rat spinal trigeminal nucleus, calbindin-D28k was localized with immunoperoxidase and immunogold methods. Results showed that immunoreactive calbindin-D28k neurons were widely distributed to all regions of the nucleus, but were particularly numerous in the substantia gelatinosa. Some trigemino-thalamic neurons that were identified by retrograde labeling of a conjugated wheat-germ agglutinin with horseradish peroxidase also contained calbindin-D28k immunoreactivity. Most of the calbindin-D28k labeling was found in cell bodies and dendrites. Axon terminals were rarely stained. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also heavily associated with euchromatin within nuclei. These findings show that immunoreactive calbindin-D28k is localized to both interneurons and projecting neurons of the spinal trigeminal nucleus. Many of these cells are likely to receive glutamatergic afferent inputs, which may act in part by increasing Ca++ flux into the neurons. Calbindin-D28k has a high capacity for buffering Ca++ and under some conditions may protect neurons against glutamate-induced excitotoxicity. We speculate that calbindin-D28k may function to regulate calcium concentrations in spinal trigeminal neurons.

A calcium binding protein of Mr 29,000 which cross-reacts with antibodies raised against chick calbindin-D28k was previously reported to be present in rat cerebellum [Pochet, R., Parmentier, M., Lawson, D. E. M., & Pasteels, J. L. (1985) Brain Res. 345, 251-254]. It was suggested that the Mr 29,000 protein represents another form of calbindin-D28k. In our laboratory we were able to identify Mr 28,000 and 29,000 proteins in rat, human, and chick cerebellum by their ability to bind 45Ca in a 45Ca blot assay. Two calcium binding proteins of Mr 27,680 and 29,450 were isolated from rat cerebelli by the use of gel permeation chromatography and preparative gel electrophoresis. After reverse-phase high-performance liquid chromatography (HPLC) the proteins were sequenced. Sequence analysis by tandem mass spectrometry indicated only 52% identity between the rat cerebellar Mr 28,000 and 29,000 proteins. Thus they are not different forms of the same protein, as previously suggested. Eighty-nine percent identity was observed between the rate cerebellar Mr 29,000 protein and chick calretinin [Rogers, J. H. (1987) J. Cell Biol. 105, 1343-1353]. The difference in identity between the rat cerebellar Mr 29,000 protein and chick calretinin may be due to species differences, and thus this protein is most likely rat calretinin. However, barely detectable levels of mRNA and weak immunoreactivity have been reported for calretinin in cerebellum. Since we found the Mr 29,000 calcium binding protein to be an abundant protein in rat cerebellum, these results suggest either posttranscriptional regulation of calretinin in cerebellum or species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal systems for calcium homeostasis are crucial for neuronal development and function and may also contribute to selective neuronal vulnerability in adverse conditions such as exposure to excitatory amino acids or anoxia, and in neurodegenerative diseases. Previous work demonstrated the presence and differential distribution of calcium-binding proteins in the CNS. We now report that a subpopulation of neurons in dissociated cell cultures of embryonic rat hippocampus expresses calbindin-D28k (Mr 28,000 calcium-binding protein) immunoreactivity and that these neurons are relatively resistant to neurotoxicity induced by either glutamate or calcium ionophore. Direct comparisons of dynamic aspects of intracellular calcium levels and calbindin-D28k immunoreactivity in the same neurons revealed that calbindin-D28k-positive neurons were better able to reduce free intracellular calcium levels than calbindin-D28k-negative neurons. These findings indicate that the differential expression of calbindin-D28k in hippocampal neurons occurs early in development and may be one determinant of selective neuronal vulnerability to excitotoxic insults.

Untreated diabetic (BB) rats exhibited compensatory intestinal growth which was associated with hyperplasia and was accompanied by an increase in unoccupied vitamin D receptors. Although vitamin D receptors were increased, low circulating 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] prevented amplification of the action of 1,25(OH)2D3, as evidenced by reductions in calbindin D-9K and alkaline phosphatase activity in the BB rat intestine compared to control. In the kidney, a lesser degree of compensatory growth was observed which was not associated with hyperplasia, and no significant effect of diabetes on vitamin D receptors or calbindin D-28K was observed. These studies suggest tissue-specific changes in 1,25(OH)2D3 metabolism during spontaneous diabetes which may be related to the hyperplasia which occurs during compensatory tissue growth.

Calbindin-D28k (CaBP28k) protein and gene expression were examined in the mouse cerebellum during development and aging utilizing slot and Northern blot hybridization analyses for mRNA levels, Western blot analysis and radioimmunoassay (RIA) for protein levels, and by in situ studies using immunocytochemistry and hybridization cytochemistry on prepared tissue sections. Samples were obtained and analyzed from C57BL/6J mice aged day of birth and postnatal weeks 1, 2, 4, 8, and 120. A specific cDNA and antibody for CaBP28k were utilized in these studies. Analysis of mRNA levels showed a steady rise in CaBP28k mRNA from birth to a peak at postnatal week (3.4-fold increase) and then a decline to steady-state levels at postnatal weeks 4 and 8 (47% reduction of peak level) followed by a reduction of CaBP28k mRNA to birth levels at postnatal week 120. The specificity of the changes observed was tested by reprobing blots with beta-actin cDNA. Analysis of CaBP28k protein levels by both Western blot and RIA showed a similar pattern. In situ analysis of CaBP28k mRNA levels, based on hybridization signal (silver grains per cell), demonstrated a rise in cellular CaBP28k mRNA levels which peaked at postnatal week 2 (416.9 +/- 52.1) and then declined to steady-state levels by postnatal weeks 4 and 8 (267.4 +/- 35.8). Cellular CaBP28k mRNA levels exhibited a dramatic reduction in the aged cerebellum (postnatal week 120; 78.3 +/- 16.0). The levels of cellular CaBP28k mRNA corresponded to the intensity of immunoreactive CaBP28k localized by immunocytochemistry. The results are consistent with the hypothesis that CaBP28k may play a critical role in Purkinje cell maturation and maintenance.(ABSTRACT TRUNCATED AT 250 WORDS)

The time and place of initial contacts between afferent axons and their target cells are not known for most regions of the mammalian CNS. To address this issue, we have selectively visualized afferent climbing fiber axons together with their synaptic targets, Purkinje cells, in postnatal mouse cerebellum. Climbing fibers were orthogradely labeled by injection of rhodamine isothiocyanate into their brainstem source, the inferior olivary nucleus. Purkinje cells were localized with an antibody to a calcium-binding protein, calbindin D-28k (CaBP), in the same section or in adjacent sections. A novel view of the olivocerebellar projection and the morphology of climbing fiber arbors prior to the well-known "nest" stage has emerged from this analysis. At birth, climbing fibers project into the zone of Purkinje cells, before these cells have aligned into a monolayer. During this phase, climbing fibers have simple morphologies consisting of relatively unbranched terminal arbors and small tapered growing tips. Purkinje cells are arranged 3-6 cells deep and have tufted dendrites and relatively smooth somata. By postnatal days 3-4, climbing fibers branch over several adjacent Purkinje cell perikarya, which are still organized in a band several cells thick. From postnatal days 5-7, when climbing fibers subsequently make focused nests on individual cells, Purkinje somata are smoother and form a more distinct monolayer. Up to this time, however, climbing fibers continue to associate with Purkinje perikarya, even though Purkinje cell dendrites have emerged and branched extensively. By postnatal days 8-10, climbing fiber terminals climb onto the trunk of the relatively mature Purkinje dendritic tree. At birth, mossy fibers originating from the pontine nuclei resemble immature climbing fibers in that they also have a simple unbranched morphology and growing tips, but project only so far as the internal granule cell layer. Occasional individual fibers reach into the Purkinje zone both at postnatal day 0 and postnatal day 4, confirming that the fibers formerly described as "combination fibers" (Mason and Gregory, S4. J. Neurosci, 4:1715-1735) can be mossy in origin. These data demonstrate that climbing fibers project among Purkinje cells earlier than suspected, before these afferents begin to arborize and form pericellular nests. Our observations are not in accord with the view derived from autoradiographic tracing studies that as in other cortical areas, climbing afferents wait in the vicinity of Purkinje cells in the early neonatal period, then advance onto these cells in synchrony with Purkinje cell alignment into a monolayer and dendritic maturation.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene expression for calbindin-D28k, the 28,000 relative molecular mass vitamin D-dependent calcium-binding protein, was measured in cells of the murine nephron by in situ hybridization on tissue sections (hybridization cytochemistry). Radiolabeled (35S-UTP), single-stranded RNA complementary to calbindin-D28k-mRNA (probe RNA) was prepared from linearized cDNA template and used for the hybridizations. Autoradiography was carried out and cellular levels of hybridization signal (silver grains) were quantified. After correction for background the concentration of silver grains was more than 350% greater in the distal tubule than in either the proximal tubule or the glomerulus. The relative cellular level of mRNA in the cytoplasm, as reflected in silver grains/cell, of the distal tubules with probe RNA was 3.4 times greater than that with control RNA. Cells of the distal tubule were the only apparent sites of specific hybridization with probe RNA. The presence of calbindin-D28k-mRNA in the distal tubule corresponded to the localization of calbindin-D28k by immunocytochemistry.