Calbindin-D9k is a Novel Risk Gene for Neurodegenerative Disease.

BACKGROUND/AIMS: Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-D9k (CaBP-9k) acts as calcium modulators and sensors in various tissues. However, the neurobiological functions of CaBP-9k are unknown. METHODS: We used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice. RESULTS: We found that the brains of CaBP-9k KO mice have increased APP/ß-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice. CONCLUSION: Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.

Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D9k.

Calbindin D9k is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I. There is a significant discrepancy between simulated and experimental order parameters for site I residues in the case of no ion bound in site I. However, it was found in the simulations that a Na+ ion can bind in site I, and the resulting order parameters determined from the simulations are in excellent agreement with experiment. Comparisons are made to X-ray structures of other S100 family members in which Na+ ions were observed or suggested to be bound in site I. © 2019 Wiley Periodicals, Inc.

The Protective Role of Calbindin-D9k on Endoplasmic Reticulum Stress-Induced Beta Cell Death.

Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.

Impact of subchronic exposure to triclosan and/or fluoride on estrogenic activity in immature female rats: The expression pattern of calbindin-D9k and estrogen receptor α genes.

This study explored the influence of triclosan (TCS) in the absence and presence of sodium fluoride (NaF) on estrogenic activity and thyroid function of adolescent female rats. The results indicated that the individual exposure to TCS evoked a significant decline in T3 and T4 but the levels of estradiol, FSH, and LH were significantly elevated beside marked up regulation of calbindin-D9k and estrogen α mRNA expression. On the other hand, the single exposure to NaF causes insignificant changes in thyroid hormones, but evoked a trend toward an increase in both estradiol and LH levels. No significant differences in the TSH level were recorded among the experimental groups. The joint exposure to TCS and NaF induced a significant improvement in thyroid and reproductive hormone levels. Overall, these findings revealed that exposure to TCS resulted in significant endocrine and reproductive alterations in immature female rats, while TCS + NaF coexposure resulted in lessening most effects.

Intestinal Regulation of Calcium: Vitamin D and Bone Physiology.

The principal function of vitamin D in the maintenance of calcium homeostasis is to increase intestinal calcium absorption. This conclusion was made from studies in vitamin D receptor (VDR) null mice which showed that rickets and osteomalacia were prevented when VDR null mice were fed a rescue diet that included high calcium, indicating that the skeletal abnormalities of the VDR null mice are primarily the result of impaired intestinal calcium absorption. Although vitamin D is critical for controlling intestinal calcium absorption, the mechanisms involved have remained incomplete. This chapter reviews studies, including studies in genetically modified mice, that have provided new insight and have challenged the traditional model of VDR-mediated calcium absorption.

Trans- and paracellular calcium transport along the small and large intestine in horses.

Intestinal calcium absorption plays a key role in the maintenance of calcium homeostasis and may either occur by paracellular or transcellular mechanisms. The horse has some unique peculiarities in calcium homeostasis compared to other species including a high absorptive capacity for calcium in the intestine, high plasma calcium concentrations, high renal excretion, and low plasma concentrations of vitamin D metabolites. So far, knowledge about the underlying mechanisms and the regulation of intestinal calcium absorption is still limited concerning this species. Several studies have documented that intestinal calcium transport in horses is not as dependent on vitamin D as in other species. However, published data on other potential regulatory mechanisms are still lacking. In the present study, paracellular and transcellular transport mechanisms for intestinal calcium transport along the intestinal axis were identified in horses using the Ussing chamber technique. Furthermore, the expression of respective transport proteins including transient receptor potential vanilloid member 6, calbindin-D9k and calcium ATPase type 1 in line with the determined calcium flux rates was documented. In respect to regulation of transepithelial calcium transport, novel regulatory proteins for maintaining calcium homeostasis such as B-box and SPRY-domain containing protein and calmodulin were investigated for the first time in equine intestinal tissues in this study. This provides the basis for a new approach for a better understanding of equine calcium homeostasis regulation.

The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics.

Calbindin-D28k (CBD-28k) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg · kg(-1) · day(-1)) or furosemide (FSM; 30 mg · kg(-1) · day(-1)) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides.

An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.

Distribution of and steroid hormone effects on calbindin-D9k in the immature rat brain.

Calbindin-D9k (CaBP-9k), one of the major calcium-binding and calcium-buffering proteins, is important in the physiological functioning of organs. The neuroanatomical localization of CaBP-9k in the rodent brain has not been reported; thus, this study investigated the neuroanatomical distribution of CaBP-9k and the regulation of CaBP-9k expression on steroid hormones in the immature rat brain. To confirm the influence of steroid hormones on CaBP-9k expression, immature female rats were injected for 5 days with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182, 780 and RU 486). The localization and expression of the CaBP-9k protein in brain regions were identified by immunofluorescence and western blot assays, respectively. We observed that CaBP-9k expression was especially strong in hypothalamus, cerebellum, and brain stem. In addition, CaBP-9k was colocalized with mature-, GABAergic, dopaminergic, and oxytocinergic neurons. We also observed that the CaBP-9k protein level was significantly increased by P4 and reversed by antagonist RU 486 treatment in immature rat brain. In summary, CaBP-9k positive cells have a wide distribution in the immature rat brain, and CaBP-9k expression is regulated by P4. We suggest that CaBP-9k expression regulated by steroid hormone may serve as an important regulator of cytosolic calcium concentration in the brain.

Effects of estrogen on esophageal function through regulation of Ca2+-related proteins.

BACKGROUND: The calcium ion is important for physiological functions in all tissues and organs and essential to many vital functions, including hormone secretion and muscle contraction. The intracellular concentration of calcium is regulated by calcium related proteins such as CaBP-9k, PMCA1, and NCX1. In this study, we investigated the relationship between calcium regulation and esophageal functions such as mucin secretion and smooth muscle contraction. METHODS: To evaluate the influence of sex steroid hormones, immature rats were treated for 3 days with estradiol (E2), progesterone (P4), and their antagonists (ICI 182,780, and RU486). Esophageal function, transcription level, and localization of CaBP-9k, PMCA1, NCX1, ERα, and MUC2 were examined in the esophagus. RESULTS: Transcriptional level of Cabp-9k and Muc2 was increased by E2, but not by P4. CaBP-9k, PMCA1, and MUC2 were mainly localized in the mucosal layer. Acidic mucosubstances in the esophagus were increased by E2 and recovered by ICI treatment. Unlike the expression of Cabp-9k, mRNA levels of Pmca1, Ncx1, and Erα were only decreased in response to E2, and recovered by ICI co-treatment group. The contraction of the esophagus and mRNA level of Mylk were reduced by E2. Overall, E2 upregulated mucus secretion, but downregulated muscle contraction in the esophagus through regulation of the expression of calcium related genes and the resultant intracellular calcium level. CONCLUSIONS: The regulation of E2 in the function of esophagus may be applied to treat esophageal diseases such as reflux esophagitis, achalasia, and esophageal cancer.

Induction of the Estrogenic Marker Calbindn-D9k by Octamethylcyclotetrasiloxane.

Interrupting the hormonal balance of an organism by interfering with hormones and their target receptors gives rise to various problems such as developmental disorders. Collectively, these reagents are known as endocrine disruptors (EDs). Cyclic volatile methyl siloxanes (cVMSs) are a group of silicone polymers that including octamethylcyclotetrasiloxane (D4). In the present study, we examined the estrogenicity of D4 through in vitro and in vivo assays that employed calcium-binding protein 9K (calbindin-D9k; CaBP-9K) as a biomarker. For in vitro investigation, GH3 rat pituitary cells were exposed to vehicle, 17ß-estradiol (E2), or D4 with/without ICI 182 780 (ICI). CaBP-9K and progesterone receptor (PR) both were up-regulated by E2 and D4 which were completely blocked by ICI. Transcription of estrogen receptor α (ER α) was decreased by E2 and D4 but increased by ICI. D4 was also administered to immature female rats for an uterotrophic (UT) assay and detection of CaBP-9K. Ethinyl estradiol (EE) or D4 was administered subcutaneously with or without ICI. Although uterine weight was not significant altered by D4, an effect thought to be due to cytochrome P450 (CYP), it induced CaBP-9K and PR gene expression. Based on these results we reveal that D4 has estrogenic potential proven under in vitro and in vivo experimental conditions.

Biomarker genes for detecting estrogenic activity of endocrine disruptors via estrogen receptors.

Endocrine disruptors (EDs) are compounds used in various industrial products, drugs, and cosmetics. They can be found in the environment and disturb the endocrine and reproductive systems, resulting in adverse effects to humans and wildlife such as birth defects and developmental disorders. Since several EDs have a structure similar to that of endogenous steroid hormones such as estrogens, they intend to have an affinity for steroid hormone receptors and alter hormone-mediated metabolism by binding to these receptors. EDs are therefore a global concern and assays should be developed to efficiently determine whether these compounds are detrimental to biological systems. Diverse experimental methods may help determine the endocrine disrupting potential of EDs and evaluate the adverse effects of a single and/or combination of these reagents. Currently, biomarkers have been employed to objectively measure EDs potency and understand the underlying mechanisms. Further studies are required to develop ideal screening methods and biomarkers to determine EDs potency at environmentally relevant concentrations. In this review, we describe the biomarkers for estrogenicity of EDs identified both in vitro and in vivo, and introduce a biomarker, cabindin-D(9k) (CaBP-9k), that may be used to assess estrogenic activity of EDs.