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PubMed1993

An invertebrate calcium-binding protein of the calbindin subfamily: protein structure, genomic organization, and expression pattern of the calbindin-32 gene of Drosophila.

Antisera against vertebrate calcium-binding proteins cross-react with Drosophila nervous and muscle tissue. We have used an antiserum against carp parvalbumin to isolate from a Drosophila head cDNA library immunopositive expression clones. Tissue in situ hybridization identified a clone that labeled specific neurons and muscles similar to the parvalbumin-like immunohistochemical staining pattern. Five independent cDNAs derive from an mRNA whose open reading frame codes for a 310 amino acid polypeptide. Sequence analysis identifies six EF-hand calcium-binding domains and reveals 42% and 37% homology to chicken calretinin and calbindin D-28k, respectively. Since the positions of 9 out of 10 introns within the ORF are conserved from the Drosophila gene to both vertebrate genes, we conclude that we have identified the first invertebrate member of the calbindin sub-family of calcium-binding protein genes of the EF-hand homolog family. The calbindin-32 gene (cbn) maps to 53E on the second chromosome. It is expressed through most of ontogenesis with a selective distribution in the nervous system and in a few small adult thoracic muscles. The cloning of a Drosophila homolog to vertebrate neuronal Ca(2+)-binding proteins opens new routes to study the so far largely elusive function of these brain molecules.

Amino Acid Sequence↗

PubMed2004

Critical role of calbindin-D28k in calcium homeostasis revealed by mice lacking both vitamin D receptor and calbindin-D28k.

Calbindin (CaBP)-D28k and CaBP-D9k are cytosolic vitamin D-dependent calcium-binding proteins long thought to play an important role in transepithelial calcium transport. However, recent genetic studies suggest that CaBP-D28k is not essential for calcium metabolism. Genetic ablation of this gene in mice leads to no calcemic abnormalities. Genetic inactivation of the vitamin D receptor (VDR) gene leads to hypocalcemia, secondary hyperparathyroidism, rickets, and osteomalacia, accompanied by 90% reduction in renal CaBP-D9k expression but little change in CaBP-D28k. To address whether the role of CaBP-D28k in calcium homeostasis is compensated by CaBP-D9k, we generated VDR/CaBP-D28k double knockout (KO) mice, which expressed no CaBP-D28k and only 10% of CaBP-D9k in the kidney. On a regular diet, the double KO mice were more growth-retarded and 42% smaller in body weight than VDRKO mice and died prematurely at 2.5-3 months of age. Compared with VDRKO mice, the double KO mice had higher urinary calcium excretion and developed more severe secondary hyperparathyroidism and rachitic skeletal phenotype, which were manifested by larger parathyroid glands, higher serum parathyroid hormone levels, much lower bone mineral density, and more distorted growth plate with more osteoid formation in the trabecular region. On high calcium, high lactose diet, blood-ionized calcium levels were normalized in both VDRKO and the double KO mice; however, in contrast to VDRKO mice, the skeletal abnormalities were not completely corrected in the double KO mice. These results directly demonstrate that CaBP-D28k plays a critical role in maintaining calcium homeostasis and skeletal mineralization and suggest that its calcemic role can be mostly compensated by CaBP-D9k.

Animals↗

PubMed1999

Dimorphic expression of medial basal hypothalamic-preoptic area calbindin-D(28K) mRNA during perinatal development and adult distribution of calbindin-D(28K) mRNA in Sprague-Dawley rats.

The calcium-binding protein, calbindin (CALB) is: (a) distributed throughout the central nervous system (CNS), (b) abundant in neurons and, (c) thought to act as a buffer by binding intracellular calcium, mediating neurogenesis (cell profileration) and neuronal programmed cell death. Using Northern analysis, CALB mRNA distribution was characterized in 12 different adult brain regions. Additionally, CALB mRNA levels were characterized in the medial basal hypothalamus (MBH) and preoptic area (POA) in perinatal male and female rats, in order to compare this mRNA pattern to the dimorphic MBH-POA CALB protein profile our laboratory previously reported. Three CALB mRNA species were detected (at 1.9, 2.8 and 3.2 kilobase pairs) in all CNS regions. The smallest mRNA transcript (at 1.9 kilobase pairs) was the most abundant of the three CALB mRNAs. To quantify these mRNA signals, CALB mRNA levels were normalized to 18s ribosomal RNA bands. Among the various adult brain sites assayed, the cerebellum expressed the highest CALB mRNA signals. High CALB mRNAs were observed in the MBH-POA, olfactory bulb and hippocampal regions. Moderate CALB mRNA levels were seen in the striatum and frontal cortex while moderate to low CALB mRNA levels were observed in the posterior cortex, entorhinal cortex, midbrain, pons, thalamus and medulla. During perinatal development, MBH-POA CALB mRNA levels were lowest at gestation day (GD) 18, increased four-fold in newborns and remained at moderate levels during early postnatal development. Male CALB mRNA levels were notably greater than female values at GD 18 and in newborns. Whereas, at PND 2, the CALB mRNA levels were approximately equivalent in males and females. These findings suggest that in the adult CNS CALB mRNAs vary among different brain regions. The present male vs. female MBH-POA CALB mRNA levels confirm previously reported dimorphic protein patterns of MBH-POA CALB during perinatal development. Thus, the genesis of sexually dimorphic structures may be influenced by the dimorphic CALB expression in the MBH-POA region.

Animals↗

PubMed1988

Molecular cloning of mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K): expression of calbindin-D28K RNAs in rodent brain and kidney.

We report the isolation of a cloned cDNA for the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K; CaBP28K) by immunological screening of a lambda gt11 bacterial expression library. The library contained cDNAs copied from poly(A)RNA of adult mouse cerebellum. We confirmed the identity of the CaBP28K cDNA by comparing its DNA sequence with that of chick CaBP28K cDNA. In the coding region, 79% of the mouse cDNA sequence was identical to the reported sequence of CaBP28K cDNA derived from chicken intestine. Rat brain and kidney each contain three species of poly(A)RNA that hybridize to CaBP28K cDNA--a major species of 1.9 kb, and rarer components of 2.8 kb and 3.2 kb. All three RNAs appear to be transcribed from a single gene. The ratios of these CaBP28K RNAs were the same in brain and kidney. In the cerebellum, in situ hybridization reveals that CaBP28K RNAs are confined to Purkinje neurons.

Animals↗

PubMed1992

Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta.

The appearance of the calcium-binding proteins (CaBP-D28K and CaBP-D9K) in embryonic mice tissues was determined using a sensitive immunohistochemical assay. CaBP-D28K first appears in myenteric nerve plexuses of the duodenum on day E15, in duodenal villus cells on day E16, in Purkinje cells of the cerebellum on day E19, in cells of the mesonephric duct on day E11 and in the metanephric duct on day E12. CaBP-D9K first appears in enterocytes of the duodenum on day E18, in trophoblastic giant cells (TGC) of the placenta on day E10, and in the metanephric duct on day E15. A differential time of appearance and colocalization of the two CaBPs is demonstrated in the embryonic mouse kidney, suggesting either that vitamin D does not control both CaBPs in the foetus or that the vitamin D control is unequal. The early appearance and location of CaBP-D9K in TGCs may suggest that these cells play an important role in transplacental transfer of calcium.

Animals↗

PubMed1992

Heterogeneous development of calbindin-D28K expression in the striatal matrix.

In the present study, we attempted to trace the development of the striatal matrix by analyzing the ontogenetic expression of calbindin-D28K (calbindin), a calcium binding protein selectivity expressed in medium-sized neurons of the matrix compartment of the mature rat's caudoputamen. The localization of calbindin was documented in a series of developing rat brains, as was the compartmental location of these cells relative to tyrosine hydroxylase (TH)-immunostained dopamine islands, sites of future striosomes. Medium-sized striatal neurons appeared in the striatum at embryonic day (E) 20, and from their first appearance, the calbindin-positive neurons had highly heterogeneous distributions. They first formed a latticework of patches and bands in a ventral region of the caudoputamen. By postnatal day (P) 7, this early calbindin-positive lattice had evolved into a mosaic in which circumscript pockets of low calbindin-like immunoreactivity appeared in more extensive calbindin-rich surrounds. With further development, the mosaic gradually encroached on all but the dorsolateral caudoputamen, a district that is calbindin-poor at adulthood. A special lateral branch of the striatal calbindin system was also identified, distinct from the rest of the calbindin-positive mosaic in several developmental characteristics. In the parts of the caudoputamen where the developing calbindin system and dopamine island system were both present, the dopamine islands invariably lay in calbindin-poor zones. Most dopamine islands, however, only filled parts of the corresponding calbindin-poor zones. Moreover, there were some calbindin-poor zones for which TH-positive dopamine islands could not be detected. Thus during development, calbindin was expressed in the extrastriosomal matrix of the striatum, but the matrix could be divided into calbindin-rich and calbindin-poor zones. In the calbindin-rich regions, there were patches of especially intense calbindin expression and zones of weaker expression. These results suggest that there is neurochemical heterogeneity in the striatal matrix during the prolonged developmental period in which the early calbindin-positive lattice expands to form the calbindin-positive matrix of the mature striatum. Surprisingly, calbindin expression in the matrix, although eventually distributed in strictly complementary fashion to striosomes, does not originate as a system complementary to dopamine islands. The prolonged disparity between the borders of dopamine islands and calbindin-poor zones, and the different spatiotemporal schedules of development of the islands and the calbindin gaps suggest instead that the final match between the borders of striosomes and surrounding matrix results from dynamic processes occurring early in postnatal development. Candidate mechanisms for the gradual adjustment of these borders are proposed.

Aging↗

PubMed2000

Regulation of renal calbindin-D28K.

Calbindin-D28k is an intracellular protein with high affinity for calcium. In the kidney, this protein is exclusively localized in the distal tubule and in the proximal part of the collecting ducts. Functionally, calbindin-D28k is supposed to be involved in the regulation of the reabsorption of calcium and possibly magnesium in the distal nephron though the exact regulatory mechanisms are not yet known. Thus, several theories regarding the functional role of calbindin-D28k have been proposed: The carrier theory describes calbindin-D28k as a transport protein which binds calcium and then transports it from the luminal to the basolateralcell membrane. The buffer theory assumes that calbindin-D28k functions by binding calcium ions to prevent intracellular calcium concentrations from reaching toxic levels. The activator theory describes that calbindin-D28k increases the activity of calcium channels or the enzymatic activity of the Ca++-Mg++-ATPase in the luminal membrane and thereby increases the tubular reabsorption of calcium. The renal calbindin-D28k is dependent upon vitamin D. Pharmacological doses of the active vitamin D metabolite 1,25-(OH)2D increases the concentrations of renal calbindin-D28k, whereas the concentration of calbindin-D28k is low in conditions with reduced levels of circulating 1,25-(OH)2D. Likewise, plasma calcium concentrations, uremia and hypertension affect calbindin-D28k expression. However, several studies have rendered probable the effect of additional factors in the regulation of renal calbindin-D28k. The aim of the present dissertation therefore was to examine the regulation of renal calbindin-D28k in a series of physiological and pathophysiological conditions established in vivo in the rat. A possible correlation between hypertension and calbindin-D28k was examined in three models of experimental hypertension: the genetically defined spontaneous hypertensive rat, the salt-sensitive Dahl rat and the renovascular hypertensive rat. These three models clearly demonstrated three separate patterns in the calcium metabolism, but the studies were unable to support a role for calbindin-D28k in the development of hypertension. In all three models the development of hypertension caused an increased plasma 1,25-(OH)2D. This increase was accompanied by either unaltered or reduced levels of renal calbindin-D28k possibly secondary to a cellular resistance against 1,25-(OH)2D. Magnesium binds to calbindin-D28k with a relatively high affinity. The regulation of urinary magnesium excretion takes place in the distal tubule where calbindin-D28k is found in high concentrations. Therefore, a possible relation between magnesium and calbindin-D28k was examined. The studies demonstrated not previously known connections between magnesium intake, urinary magnesium excretion and renal calbindin-D28k which suggests that this protein is involved in the regulation of magnesium homeostasis by the kidney. Calcitonin increases the reabsorption of calcium in the distal tubule. Therefore, the effect ofcalcitonin on renal calbindin-D28k was examined both by eliminating the endogeneous calcitonin production by a selective thyroidectomy followed by an autotransplantation of the parathyroid glands and further by infusion of calcitonin. These studies demonstrated unchanged concentrations of renal calbindin-D28k. It was concluded that the increased calcium reabsorption induced by calcitonin in the distal tubule is not mediated by calbindin-D28k. Urinary calcium excretion is in part regulated by the action of PTH on calcium reabsorption in the distal nephron. Previous reports of increased expression of renal calbindin-D28k in uremic rats led us to suggest that secondary hyperparathyroidism associated with uremia induced the synthesis of renal calbindin-D28k. Therefore, the effect of PTH was examined in a study comprising selective parathyroidectomy and infusions of PTH, PTHrP, 1,25-(OH)2D and calcium. (ABSTRACT TRUNCATED)

Animals↗

PubMed1992

Transient calbindin-D28k-positive systems in the telencephalon: ganglionic eminence, developing striatum and cerebral cortex.

Calbindin-D28k (calbindin) is a member of the superfamily of calcium-binding proteins implicated in the regulation of intracellular calcium. In the mature brain, calbindin is widely expressed in neurons of the forebrain and the hindbrain, and in the telencephalon calbindin-like immunoreactivity is particularly strongly expressed by medium-sized neurons of the striatum and by certain other neurons in the cortex and subcortex. We have traced the development of calbindin expression in the forebrain of the rat, and report here that in addition to the steady development of these calbindin-positive neuronal systems, transient waves of calbindin expression occur in cells of the ventricular zones of the basal ganglia and cortex and in cells of the telencephalic regions derived from these ventricular zones including radial glia of the developing striatum. In the striatum and its ventricular zone (the ganglionic eminence, or GE) we identified four transient calbindin-positive systems in the perinatal period. First, calbindin-immunoreactive cells began to appear in the GE by embryonic day (E)18, and by E20 an extensive dorsal and lateral part of the GE was marked by dense calbindin-like immunoreactivity in the ventricular zone. This calbindin system peaked at postnatal day (P)0-P3 and disappeared by P15. Its presence suggests that the GE is divisible on a molecular basis into lateral and medial districts that may correspond to derivatives of the lateral and medial ventricular ridges. Second, a system of calbindin-positive processes appeared in the dorsal and lateral caudoputamen with temporal and spatial distributions matching the germinal zone system. Many of these processes could be traced from calbindin-positive cells in the ventricular zone of the GE, including processes stretching across the full width of the dorsal caudoputamen. Double-staining experiments demonstrated that these radial processes were Rat.401-positive, suggesting that they form a subset of radial glia in the developing telencephalon. These findings demonstrate that during development calbindin is expressed in glial as well as neural cells. They further suggest that the radial glia associated with the GE form heterogeneous populations, the transient calbindin-positive radial glia being associated with the lateral ridge of the GE and its derivatives. Third, a scattered population of calbindin-positive cells with morphologies different from the common medium-sized calbindin-immunoreactive neurons of the striatum appeared in the dorsal and lateral striatum from about E20 to P15. Some of these cells were close to the transient calbindin-positive radial processes in the same region, but others were not.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed2000

Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections.

Calbindin-D28k (calbindin) is a calcium-binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline-synthesising neurons [phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR)] was examined (Granata and Chang [1994] Brain Res. 645:265-277). However, detailed information about the distribution of calbindin-IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin-IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)-IR, or PNMT-IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin-IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin-IR neurons were found. Within the ventral pons, a population of calbindin-IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin-IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin-IR neurons was present in the ventral medulla. Seventy-five percent of these calbindin-IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin-IR neurons were a subpopulation of PNMT-IR cells. In the caudal ventral medulla, no TH-IR or PNMT-IR cells were calbindin-IR. In the intermediolateral cell column, close appositions of calbindin-IR terminals on identified sympathetic preganglionic neurons as well as calbindin-IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT-IR neurons in the rostral ventrolateral medulla.

Animals↗

PubMed1995

Retinoic acid regulates the expression of the calcium binding protein, calbindin-D28K.

It is a well established fact that the calcium-binding protein, calbindin-D28k, is influenced by vitamin D in intestine and kidney. However, very little is known concerning the regulation of calbindin-D28k in brain. Although few genes that are regulated by retinoic acid (RA) have been identified in the nervous system, we now report that the human medulloblastoma cell line D283 (which is derived from cerebellum and has a distinctly neuronal phenotype) contains calbindin-D28k endogenously and that calbindin protein and mRNA can be induced 10- to 15-fold in these cells by 10(-7) M RA. These findings are the first evidence of RA-mediated regulation of calbindin. The time course of response, as determined by Northern blot analysis, indicated that the first significant increase in calbindin-D28k mRNA is at 12 h with a plateau of calbindin mRNA induction at 72 h after RA treatment. The induction of calbindin mRNA by RA was preceded by an induction of retinoic acid receptor-alpha mRNA and was accompanied by an induction of retinoid X receptor-alpha mRNA. Calbindin-D28k mRNA levels in D283 medulloblastoma cells as well as the induction of calbindin mRNA by RA were not significantly affected by 1,25-dihydroxyvitamin D3 treatment. Deletion mutant analysis of the native calbindin-D28k promoter and cotransfection of CV-1 or D283 medulloblastoma cells in the presence of retinoic acid receptor-alpha and/or retinoid X receptor-alpha expression vectors as well as results of nuclear transcription assays did not indicate transcriptional regulation of calbindin-D28k by RA. Studies of calbindin-D28k mRNA in control and RA-pretreated D283 medulloblastoma cells at various times (3-24 h) after treatment with 4 micrograms/ml actinomycin D indicated that the half-life of calbindin-D28k mRNA was significantly increased in the presence of RA, suggesting regulation of calbindin-D28k mRNA stability by RA. Thus, calbindin-D28k is one of the few known targets of RA action in cells that express a neuronal phenotype. In addition, our findings present further evidence of an interrelationship between the actions of 1,25-dihydroxyvitamin D3 and the active metabolites of vitamin A.

Animals↗

PubMed1991

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

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

Animals↗

PubMed1996

Effect of vitamin D metabolites and analogs on renal and intestinal calbindin-D in the rat.

The effects on renal and intestinal calbindin-D of vitamin D3 metabolites and synthetic 20-epi-vitamin D3 analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)2D3 0.01, 0.05, 0.1, and 0.4 microg/kg x d, 24,25-(OH)2D3 0.1, 1 and 10 microg/kg x d, and 25-(OH)D3 10 and 400 microg/kg x d. The dosage of the synthetic analogs were MC903 0. 1, 10, and 100 microg/kg x d, EB1213 0.1 and 10 microg/kg x d, KH1060 0.1 and 0.4 microg/kg x d, and GS1725 0.01 and 0.1 microg/kg x d. Two control groups had either vehicle alone or no treatment. N = 8 in each group. 1,25-(OH)2D3 increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)2D3 increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24, 25-(OH)2D3 that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D3 did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)2D3 increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity.

Administration, Cutaneous↗

PubMed2005

Interaction of calbindin D28k and inositol monophosphatase in human postmortem cortex: possible implications for bipolar disorder.

OBJECTIVES: Therapeutically relevant concentrations of lithium (Li) exert an uncompetitive inhibition on inositol monophosphatase (IMPase). It has recently been shown that calbindin D28k (calbindin) activates IMPase. Purified calbindin attaches to a specific amino acid sequence on purified IMPase enhancing its activity by several hundred fold. We studied whether calbindin activates IMPase in postmortem human brain crude homogenate, whether differences in calbindin levels between lymphocytes and brain may be responsible for our previous finding of reduced IMPase activity in lymphocytes but not brain of bipolar patients, and whether calbindin protein levels are altered in postmortem brain from bipolar patients versus control subjects and schizophrenic and major depressive patients. METHODS: IMPase activity in human postmortem brain specimens with or without 10 microM human recombinant calbindin was quantified spectrophotometrically in an enzyme-linked immunosorbent assay (ELISA) reader. Calbindin protein levels in postmortem brain were determined using Western blot analysis. RESULTS: Supplementation of human recombinant calbindin to postmortem human brain crude homogenate enhanced IMPase activity by 3.5-fold. No difference in postmortem temporal cortex calbindin protein levels was found between bipolar patients versus comparison groups. Two-fold higher calbindin protein levels were found in Li-treated bipolar patients compared with other bipolar patients. Subchronic Li treatment in mice did not affect brain calbindin protein levels significantly. Chronic Li treatment reduced calbindin protein levels in the frontal cortex but not in the hippocampus. CONCLUSIONS: Calbindin is a physiological activator of IMPase in human brain. Protein levels of calbindin are not altered in postmortem temporal cortex of bipolar patients.

Animals↗

PubMed2003

Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k.

UNLABELLED: This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation. INTRODUCTION: Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells. MATERIALS AND METHODS: Apoptosis was induced by addition of dexamethasone (dex; 10-6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by 32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot. RESULTS: The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 microM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway. CONCLUSION: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.

Animals↗

PubMed1991

Modulation of quail intestinal and egg shell gland calbindin (Mr 28,000) gene expression by vitamin D3, 1,25-dihydroxyvitamin D3 and egg laying.

The effects of vitamin D3 sources, egg production and egg cycle on the genomic expression of calbindin (Mr 28,000) in the intestine and egg shell gland (ESG) of quail were characterized by Northern blot and solution hybridization, using synthetic oligonucleotide probe. In vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-fed quail, onset of egg production induced duodenal and ESG calbindin mRNA and calbindin synthesis. Duodenal calbindin mRNA was slightly higher during the period of shell calcification as compared with the period during which shells were not formed (ESG inactivity). ESG calbindin mRNA was markedly higher during the period of shell calcification than of ESG inactivity. Increasing dietary intake of [3H]1 alpha-hydroxyvitamin D3 increased the duodenal, but not ESG, content of 1,25-(OH)2D3 and calbindin. Duodenal calbindin and its mRNA were absent in vitamin D-deficient quail and were not affected by egg laying. ESG calbindin in the vitamin D-deficient quail was not affected by egg laying, but calbindin mRNA increased in the vitamin D-deficient birds during shell calcification. The results suggest that: (a) intestinal calbindin mRNA and calbindin are induced and/or regulated, either directly or indirectly, by 1,25-(OH)2D3; (b) intestinal calbindin and its mRNA are further induced at the onset of egg laying by an additional stimulator besides 1,25-(OH)2D3; (c) 1,25-(OH)2D3 is required for the expression of the latter stimulator; (d) ESG calbindin mRNA and calbindin are induced in egg-laying birds by a stimulator associated with the egg cycle; and (e) the induction of ESG calbindin mRNA does not need vitamin D metabolites, but 1,25-(OH)2D3 is required for the translation of the mRNA.

Animals↗

PubMed1997

Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat beta cell line (RIN 1046-38).

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.

Animals↗

PubMed1988

Analysis of rat vitamin D-dependent calbindin-D28k gene expression.

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.

Aging↗

PubMed1996

The role of gonadal hormones in gene expression of calbindin (Mr 28,000) in the laying hen.

Acute and chronic changes in calbindin (Mr 28,000) mRNA and calbindin concentrations were determined to assess the roles of steroid hormones in calbindin mRNA and calbindin synthesis in the eggshell gland (ESG). The results support an earlier suggestion that calbindin gene expression in the ESG is associated with Ca2+ flux through the ESG. The evidence includes wide oscillation of the mRNA during the diurnal egg cycle, in close temporal association with egg shell calcification. Progesterone (single im injection of 1 mg/kg body weight, BW) prolonged the period of egg formation and reduced the rate of Ca2+ transport and the concentration of calbindin mRNA in the ESG. Dexamethasone (single im injection of 5 mg/kg BW) prolonged the period of egg formation, increased shell Ca2+, and reduced calbindin mRNA in the ESG and intestine. Testosterone (single im injection of 2 mg/kg BW) did not affect calbindin mRNA synthesis in the ESG. The effects of estradiol on the synthesis of calbindin mRNA in the ESG of sexually immature or laying birds were minor, while it affected plasma Ca in the same birds. The antiestrogen Tamoxifen (60 mg/kg BW, given orally) reduced plasma Ca, but did not affect the synthesis of calbindin mRNA in the ESG. The antiprogesterone RU-38486 (20 mg/kg BW, orally) increased shell Ca2+ but had no effect on plasma Ca or the synthesis of calbindin mRNA. It appears that estrogens alone cannot account for the markedly elevated synthesis of calbindin mRNA in the ESG of the laying bird. The hypothesis that the regulatory mechanism for the synthesis of calbindin mRNA in the ESG may involve a stimulator(s), associated with the onset of production, and an oscillating depressor(s) is supported and both appear to be closely linked to the reproductive cycle. The specific in vivo effect of progesterone on calbindin mRNA in the ESG, together with its already known changes during the ovulatory cycle in birds, supports the idea that it is a depressor.

Animals↗