Synopsis The biologically active metabolite of vitamin D, 1,25(OH)2D3, affects mineral homeostasis and has numerous other diverse physiological functions including effects on growth of cancer cells and protection against certain immune disorders. This chapter reviews the role of vitamin D hydroxylases in providing a tightly regulated supply of 1,25(OH)2D3. The role of extrarenal 1α(OH)ase in placenta and macrophages is also discussed as well as regulation of the hydroxylases and vitamin D hydroxylases in aging and chronic kidney disease. Understanding specific factors involved in regulating the hydroxylases may lead to the design of drugs that can selectively modulate the hydroxylases. The ability to alter levels of these enzymes would have therapeutic potential for the treatment of various diseases including bone loss disorders and certain immune diseases.
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.
Vitamin D maintains calcium homeostasis and is required for bone development and maintenance. Recent evidence has indicated an interrelationship between vitamin D and health beyond bone, including effects on cell proliferation and on the immune system. New developments in our lab related to the function and regulation of target proteins have provided novel insights into the mechanisms of vitamin D action. Studies in our lab have shown that the calcium-binding protein, calbindin, which has been reported to be a facilitator of calcium diffusion, also has an important role in protecting against apoptotic cell death in different tissues including protection against cytokine destruction of osteoblastic and pancreatic beta cells. These findings have important implications for the therapeutic intervention of many disorders including diabetes and osteoporosis. Recent studies in our laboratory of intestinal calcium absorption using calbindin-D(9k) null mutant mice as well as mice lacking the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin-D(9k) and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)(2)D(3) in intestine and kidney is 25(OH)D(3) 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)(2)D(3). In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP beta, SWI/SNF (complexes that remodel chromatin using the energy of ATP hydrolysis) and the methyltransferases, CARM1 and G9a. Evidence is also presented for C/EBP beta as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)(2)D(3) and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)(2)D(3) mediates its multiple biological effects.
Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6−/−) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. At the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6−/− mice receiving a normal (~ 1%) or low (~ 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or ageing mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6+/+ and Trpv6−/− mice (−35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6−/− mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.
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