To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

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CaBP-9k may be involved in the active calcium absorption and embryo implantation. Although we generated CaBP-9k KO mice to explore its function, no distinct phenotypes were observed in these KO mice. It can be hypothesized that TRPV5 and 6 and plasma membrane calcium ATPase 1b may play a role in the regulation of calcium transport to compensate CaBP-9k deficiency in its KO model. Introduction: Active calcium transport in the duodenum and kidney is carried in three steps: calcium entry through epithelial Ca 2+ channels (TRPV5 and TRPV6), buffering and/or transport by calbindin-D9k (CaBP9k) and -D28k (CaBP-28k), and extrusion through the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger 1. Although the molecular mechanism of calcium absorption has been studied using knockouts (KOs) of the vitamin D receptor and CaBP-28k in animals, the process is not fully understood. Materials and Methods:We generated CaBP-9k KO mice and assessed the phenotypic characterization and the molecular regulation of active calcium transporting genes when the mice were fed different calcium diets during growth. Results: General phenotypes showed no distinct abnormalities. Thus, the active calcium transport of CaBP9k-null mice proceeded normally in this study. Therefore, the compensatory molecular regulation of this mechanism was elucidated. Duodenal TRPV6 and CaBP-9k mRNA of wildtype (WT) mice increased gradually during preweaning. CaBP-9k is supposed to be an important factor in active calcium transport, but its role is probably compensated for by other calcium transporter genes (i.e., intestinal TRPV6 and PMCA1b) during preweaning and renal calcium transporters in adult mice. Conclusions: Depletion of the CaBP-9k gene in a KO mouse model had little phenotypic effect, suggesting that its depletion may be compensated for by calcium transporter genes in the intestine of young mice and in the kidney of adult mice.

Maintenance of uterine calcium balance is crucial for physiological functioning, including smooth muscle contraction and embryo implantation. Although calbindins were previously thought to act as important uterine calciumprocessing genes for female reproductive function, they were not enough to attest the roles of calcium ions in the reproductive organs. Previously, we reported that rat transient receptor potential cation channel, subfamily V, member 6 (TRPV6) was expressed and regulated by hormones in the uterus. In the present study, we observed uterine TRPV6 expression in a mouse model to clarify the mutual roles of these two calcium-processing genes in female reproductive organs. We investigated uterine TRPV6 mRNA expression during the estrous cycle and pregnancy, as well as its regulation by the steroid hormones estrogen (E2) and progesterone (P4) in mice. Uterine TRPV6 mRNA levels increased at estrus and fluctuated in the uterus, placenta, and fetal membrane during pregnancy. Uterine TRPV6 mRNA increased in mid-and late pregnancy, and its expression was strongly induced in midpregnancy in the labyrinth and spongy zones of the placenta, and in the fetal membrane. E2 (17␤-estradiol) was found to regulate uterine TRPV6 expression in the luminal and glandular epitheliums. In addition, we determined that ER␣ tightly regulated uterine TRPV6 transcription. Together, these results suggest that for uterine function in normal pregnancy, TRPV6 is regulated by E2 via an ER␣-dependent pathway.estrogen receptor-␣; uterus IN MAMMALS, CALCIUM IONS may be involved in control of uterine muscle contraction and embryo implantation. The balance between contraction and relaxation is extremely important throughout pregnancy and during labor. However, regulation of uterine calcium is not yet fully understood. A model for calcium ions in intestinal cells suggests that calcium flows into the cytoplasm via channel proteins, i.e., calcium transporters. These transporters transfer calcium using calcium-binding proteins (Calbindin-D9k or -28k), which are extruded from the cell membrane by a plasma membrane Ca 2ϩ ATPase (28, 31). Transient receptor potential vanilloid type 5 (TRPV5) and 6 (TRPV6) are found in the apical membranes of intestinal and renal epithelial cells and have been proposed as mediators for calcium uptake during trans-cellular transport (7). TRPV6 is also known as epithelial calcium channel 2 (EcaC2) and calcium transporter 1 (CaT1); TRPV5 is also called EcaC1 and CaT2. These closely related proteins are expressed primarily in cells of the duodenum and kidney that are involved in calcium absorption or reabsorption (25). TRPV6 was first cloned from rat duodenum and was detected subsequently in both human and mouse duodena (12,22). The genes encoding these proteins are located closely together on the same chromosome and their genomic structures are similar (12,19,20). TRPV6 is expressed in the duodenum, jejunum, ileum, kidney, and exocrine tissues such as the pancreas, prostate, mammary, and sweat glands (7,12,29,32). I...

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Dexamethasone (Dex) is a member of the glucocorticoids (GCs), and is broadly used as an anti-inflammatory medication. Continuous administration with GCs induces adverse effects and suffering in humans (i.e., osteoporosis) due to negative calcium balance derived from low re- and absorption in the duodenum and kidney. A cytosolic calcium-binding protein, calbindin-D9k (CaBP-9k), is dominantly expressed in the renal and intestinal tissues involved in calcium re- and absorption and plays an active role in calcium transport. In the present study, we employed adrenalectomized (ADX) and sham-treated (Sham) male mice to examine the effect of Dex on CaBP-9k gene expression in the duodenum and kidney. Dex significantly reduced the levels of duodenal CaBP-9k mRNA and protein, and it restored ADX-induced decrease in renal CaBP-9k protein compared with the level of Sham control. Dex treatment increased calcium and phosphate levels in the sera of both Sham and ADX mice. In a time course experiment, Dex significantly decreased duodenal CaBP-9k at the transcriptional and translational levels at 3 days, whereas it temporarily increased CaBP-9k mRNA and protein levels at 12 and 24 h. Altered CaBP-9k expression by Dex was completely reversed by mifepristone, an antagonist for the GC receptor (GR). In addition, duodenal CaBP-9k and GR were colocalized on the enterocyte (duodenocyte), supporting a role for GR in regulating CaBP-9k. In ovariectomized (OVX) and ADX female mice daily treated with Dex for 3 days, duodenal CaBP-9k was expressed at the same level as in male mice. Also, no cross-activity of progesterone and Dex on their receptors was observed. Taken together, these results indicate that mouse CaBP-9k gene may be regulated by Dex in a tissue-specific manner, and reduced duodenal CaBP-9k via the GR pathway may take part in negative calcium absorption of GC-induced osteoporosis, whereas renal CaBP-9k may not be involved in the regulation of calcium homeostasis.

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