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.     

Peptide binding proclivities of calcium loaded calbindin-D28k

Calbindin-D28k is known to function as a calcium-buffering protein in the cell. Moreover, recent evidence shows that it also plays a role as a sensor. Using circular dichroism and NMR, we show that calbindin-D28k undergoes significant conformational changes upon binding calcium, whereas only minor changes occur when binding target peptides in its Ca(2+)-loaded state. NMR experiments also identify residues that undergo chemical shift changes as a result of peptide binding. The subsequent use of computational protein-protein docking protocols produce a model describing the interaction interface between calbindin-D28k and its target peptides.     

In vitro enzyme activation with calbindin-D28k, the vitamin D-dependent 28 kDa calcium binding protein.

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.     

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen.

The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line

The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen

Summary The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca²⁺ pump was not detectable in the infundibulum or the magnum. Calbindin-D_28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca²⁺ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D_28k differed, co-localizing with the Ca²⁺ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca²⁺ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

Expression of calbindin-D28k in developing and growing chick testes.

Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Expression of calbindin-D 28k in developing and growing chick testes

Summary Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca²⁺-dependent fashion. Molecular docking and conformational sampling studies of the Ca²⁺-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.

Structured summary of protein interactions

Calbindin-D28K and Caspase-3bind by isothermal titration calorimetry(View interaction)     

Inhibition of bioenergetics alters intracellular calcium,membrane composition,and fluidity in a neuronal cell line

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca²⁺-pump activity measured by⁴⁵Ca²⁺ uptake was >92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (³H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca²⁺ from the incubation medium ablated the CN/2-DG induced release of³H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (_c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca²⁺-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca²⁺ as a result of Ca²⁺-pump inhibition and thereby activate Ca²⁺-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.     

Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca(2+)](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca(2+)](i)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in [Ca(2+)](i)followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak [Ca(2+)](i)levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of [Ca(2+)](i), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria. In studies of delayed, Ca(2+)]-dependent cell death, CaBP transfected cells exhibited enhanced survival 24h after a 1-h exposure to 200 microM NMDA. However, necrotic cell death observed after the first 6h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca(2+)-buffering effect of CaBP which serves to limit Ca(2+)entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis.     

Calcium movements and intracellular calcium distribution in neoplastic GH3 cells

Experiments were carried out to investigate the nature of the calcium homeostatic mechanisms in neoplastic GH3 rat pituitary cells. GH3 cells grown and maintained in Ham's F10 culture medium contained 35 nmoles calcium/mg cell protein. When stimulated by thyrotropin releasing hormone (TRH) or elevated K+ concentrations, only the latter caused cell calcium levels to rise although both resulted in hormone release. When exposed to EGTA, the GH3 cells lost calcium. When the temperature was lowered to 4 degrees C, the cells gained calcium and when rewarmed were able to extrude the previously accumulated calcium. The increased cell calcium following cold exposure could be blocked by prior treatment with rotenone. If rotenone was added subsequent to the cold exposure, it did not block the extrusion seen upon rewarming. In the absence of glucose in the medium, the GH3 cells took up more calcium upon exposure to 4 degrees C, and upon rewarming the cells could not return to their previous low levels. There are thus significant differences in calcium homeostasis between the neoplastic GH3 cells and their normal pituitary counterparts. When intracellular calcium was localized with the potassium pyroantimonate technique, there was calcium found in/on mitochondria, membrane bound vesicles and plasma membrane. Nuclear staining was sparse, and nucleolar staining was virtually absent. Upon stimulation with TRH, there was a decrease in mitochondrial calcium along with increases in both plasma membrane and nucleolar calcium levels. Since total calcium is unchanged, this indicates a significant calcium redistribution in response to TRH. The increased nucleolar calcium may reflect a calcium dependent increase in mRNA synthesis as has been reported. Since TRH presumably acts at a surface receptor, the increased plasma membrane calcium might be functionally related to receptor activation.     

Heterogeneity in calbindin-D28k expression in oxytocin-containing magnocellular neurons of the rat hypothalamus

We have used a double-labeling immunofluorescence method to examine whether oxytocin-containing magnocellular neurons possess a calcium-binding protein, calbindin-D28k, in the hypothalamus of the rat. In the supraoptic nucleus, most oxytocin-immunoreactive cells were also stained for calbindin-D28k. However, in the magnocellular part of the paraventricular nucleus nearly all oxytocin-labeled cells were devoid of calbindin-D28k. In the anterior commissural nucleus, approximately one-third of oxytocin-stained cells were also calbindin-D28k-immunoreactive, but the other cells were negative for calbindin-D28k. This study indicates that there may be distinct chemical features between oxytocin-containing magnocellular neurons of the supraoptic nucleus compared to those of the paraventricular nucleus.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis by disruption of intracellular calcium homeostasis in human neuronal cell line SHSY5Y

The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P?<?0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P?<?0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.