Puneet Dhawan

New insights into the mechanisms of vitamin D action.

The biologically active metabolite of vitamin D, 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) is a secosteroid whose genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific interaction of 1,25(OH)2D3 with the vitamin D receptor (VDR) which heterodimerizes with the retinoid X receptor (RXR). After interaction with the vitamin D response element (VDRE) in the promoter of target genes, transcription proceeds through the interaction of VDR with coactivators and with the transcription machinery. The identification of the steps involved in this process has been a major focus of recent research in the field. However, the functional significance of target proteins as well as the functional significance of proteins involved in the transport and metabolism of vitamin D is also of major importance. Within the past few years much new information has been obtained from studies using knockout and transgenic mice. New insight has been obtained using this technology related to the physiological significance of the vitamin D binding protein (DBP), used to transport vitamin D metabolites, as well as the physiological significance of target proteins including 25‐hydroxyvitamin D3 24‐hydroxylase (24(OH)ase), 25‐hydroxyvitamin D3‐1α‐hydroxylase (1α‐(OH)ase), VDR, and osteopontin. The crystal structure of the DBP and the ligand binding domain of the VDR have recently been reported, explaining, in part, the unique properties of these proteins. In addition novel 1,25(OH)2D3 target genes have been identified including the epithelial calcium channel, present in the proximal intestine and in the distal nephron. Thus in recent years a number of exciting discoveries have been made that have enhanced our understanding of mechanisms involved in the pleiotropic actions of 1,25(OH)2D3.

Vitamin D: Metabolism, Molecular Mechanism of Action, and Pleiotropic Effects

1,25-Dihydroxvitamin D3 [1,25(OH)2D3] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)2D3 action involves the direct binding of the 1,25(OH)2D3 activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Numerous VDR co-regulatory proteins have been identified, and genome-wide studies have shown that the actions of 1,25(OH)2D3 involve regulation of gene activity at a range of locations many kilobases from the transcription start site. The structure of the liganded VDR/RXR complex was recently characterized using cryoelectron microscopy, X-ray scattering, and hydrogen deuterium exchange. These recent technological advances will result in a more complete understanding of VDR coactivator interactions, thus facilitating cell and gene specific clinical applications. Although the identification of mechanisms mediating VDR-regulated transcription has been one focus of recent research in the field, other topics of fundamental importance include the identification and functional significance of proteins involved in the metabolism of vitamin D. CYP2R1 has been identified as the most important 25-hydroxylase, and a critical role for CYP24A1 in humans was noted in studies showing that inactivating mutations in CYP24A1 are a probable cause of idiopathic infantile hypercalcemia. In addition, studies using knockout and transgenic mice have provided new insight on the physiological role of vitamin D in classical target tissues as well as evidence of extraskeletal effects of 1,25(OH)2D3 including inhibition of cancer progression, effects on the cardiovascular system, and immunomodulatory effects in certain autoimmune diseases. Some of the mechanistic findings in mouse models have also been observed in humans. The identification of similar pathways in humans could lead to the development of new therapies to prevent and treat disease.

Vitamin D: metabolism.

The biologically active metabolite of vitamin D, 1,25(OH)(2)D(3), affects mineral homeostasis and has numerous other diverse physiologic functions including effects on growth of cancer cells and protection against certain immune disorders. This article reviews the role of vitamin D hydroxylases in providing a tightly regulated supply of 1,25(OH)(2)D(3). The role of extrarenal 1alpha(OH)ase in placenta and macrophages is also discussed, as well as regulation of 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.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k.

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 target proteins: Function and regulation

Recent findings have indicated that calbindin‐D28k, the first known target of vitamin D action, is present in osteoblasts and protects against TNF and glucocorticoid induced apoptosis of osteoblastic cells. Cytokine mediated destruction of pancreatic β cells, a cause of insulin dependent diabetes, is also inhibited by calbindin‐D28k. In calbindin‐D28k transfected pancreatic β cells free radical formation by cytokines is inhibited by calbindin. Thus, besides its role as a facilitator of calcium diffusion, calbindin has a major role in protecting against cellular degeneration in different cell types. Besides calbindin, the other known pronounced effect of 1,25(OH)2D3 in intestine and kidney is increased synthesis of 25(OH)D3 24‐hydroxylase (24(OH)ase) which is involved in the catabolism of 1,25(OH)2D3. We have noted that CCAAT enhancer binding protein β (C/EBPβ) is induced by 1,25(OH)2D3 in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)2D3. These studies establish C/EBPβ as a novel 1,25(OH)2D3 target gene and indicate a role for C/EBPβ in 24(OH)ase transcription. These studies extend our previous studies related to factors that affect vitamin D receptor (VDR) mediated 24(OH)ase transcription (YY1, TFIIB, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment. J. Cell. Biochem. 88: 238–244, 2003.

Vitamin D: molecular mechanism of action.

Abstract:  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 β 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‐D9k null mutant mice as well as mice lacking the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin‐D9k and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)2D3 in intestine and kidney is 25(OH)D3 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)2D3. In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP β, 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 β as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)2D3 and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)2D3 mediates its multiple biological effects.

Functional cooperation between CCAAT/enhancer-binding proteins and the vitamin D receptor in regulation of 25-hydroxyvitamin D3 24-hydroxylase

ABSTRACT 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces the synthesis of 25-hydroxyvitamin D3 24-hydroxylase [24(OH)ase], an enzyme involved in its catabolism, thereby regulating its own metabolism. Here we demonstrate that CCAAT enhancer binding protein β (C/EBPβ) is induced by 1,25(OH)2D3 in kidney and in osteoblastic cells and is a potent enhancer of vitamin D receptor (VDR)-mediated 24(OH)ase transcription. Transfection studies indicate that 1,25(OH)2D3 induction of 24(OH)ase transcription is enhanced a maximum of 10-fold by C/EBPβ. Suppression of 1,25(OH)2D3-induced 24(OH)ase transcription was observed with dominant negative C/EBP or osteoblastic cells from C/EBPβ−/− mice. A C/EBP site was identified at positions −395 to −388 (−395/−388) in the rat 24(OH)ase promoter. Mutation of this site inhibited C/EBPβ binding and markedly attenuated the transcriptional response to C/EBPβ. We also report the cooperation of CBP/p300 with C/EBPβ in regulating VDR-mediated 24(OH)ase transcription. We found that not only 1,25(OH)2D3 but also parathyroid hormone (PTH) can induce C/EBPβ expression in osteoblastic cells. PTH potentiated the induction of C/EBPβ and 24(OH)ase expression in response to 1,25(OH)2D3 in osteoblastic cells. Data with the human VDR promoter (which contains two putative C/EBP sites) indicate a role for C/EBPβ in the protein kinase A-mediated induction of VDR transcription. From this study a fundamental role has been established for the first time for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in 1,25(OH)2D3-induced transcription. These findings also indicate a novel role for C/EBPβ in the cross talk between PTH and 1,25(OH)2D3 that involves the regulation of VDR transcription.

Vitamin D and intestinal calcium absorption

The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.

Evidence for a Role of Prolactin in Calcium Homeostasis: Regulation of Intestinal Transient Receptor Potential Vanilloid Type 6, Intestinal Calcium Absorption, and the 25-Hydroxyvitamin D3 1α Hydroxylase Gene by Prolactin

Increased calcium transport has been observed in vitamin D-deficient pregnant and lactating rats, indicating that another factor besides 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is involved in intestinal calcium transport. To investigate prolactin as a hormone involved in calcium homeostasis, vitamin D-deficient male mice were injected with 1,25(OH)(2)D(3), prolactin, or prolactin + 1,25(OH)(2)D(3). Prolactin alone (1 microg/g body weight 48, 24, and 4 h before termination) significantly induced duodenal transient receptor potential vanilloid type 6 (TRPV6) mRNA (4-fold) but caused no change in calbindin-D(9k). Combined treatment with 1,25(OH)(2)D(3) and prolactin resulted in an enhancement of the 1,25(OH)(2)D(3) induction of duodenal TRPV6 mRNA, calbindin-D(9k) mRNA, and an induction of duodenal calcium transport [P < 0.05 compared with 1,25(OH)(2)D(3) alone]. Because lactation is associated with an increase in circulating 1,25(OH)(2)D(3), experiments were done to determine whether prolactin also has a direct effect on induction of 25-hydroxyvitamin D(3) 1alpha hydroxylase [1alpha(OH)ase]. Using AOK B-50 cells cotransfected with the prolactin receptor and the mouse 1alpha(OH)ase promoter -1651/+22 cooperative effects between prolactin and signal transducer and activator of transcription 5 were observed in the regulation of 1alpha(OH)ase. In addition, in prolactin receptor transfected AOK B-50 cells, prolactin treatment (400 ng/ml) and signal transducer and activator of transcription 5 significantly induced 1alpha(OH)ase protein as determined by Western blot analysis. Thus, prolactin, by multiple mechanisms, including regulation of vitamin D metabolism, induction of TRPV6 mRNA, and cooperation with 1,25(OH)(2)D(3) in induction of intestinal calcium transport genes and intestinal calcium transport, can act as an important modulator of vitamin D-regulated calcium homeostasis.

Expression and activities of ethylene biosynthesis enzymes during ripening of banana fruits and effect of 1-MCP treatment

The respiratory climacteric, ethylene evolution and activities and expression of ethylene biosynthesis enzymes show a characteristic pattern in banana during ethylene induced ripening. A unique biphasic respiratory climacteric with a 10 and 6 fold increment in respiration rates on days 2 and 6 respectively after ethylene treatment is preceded by ethylene evolution on days 1 and 4 with 8.3 and 6.93 fold increments respectively. This represents a unique feature of ripening in banana. While ACC synthase transcript accumulation matched the respiratory climacteric, the ACC synthase activity and ACC oxidase transcript accumulation showed a different pattern. The most significant observation was the effect of 1-MCP on ACC content and in vitro ACC oxidase activity. Though 1-MCP treated fruit did not show any respiratory climacteric or burst in ethylene production, it did not inhibit completely ACC accumulation and in vitro ACC oxidase activity. No transcript accumulation of ACC synthase was observed at any time in 1-MCP treated fruits, whereas a basal level of ACC oxidase transcript was detected throughout. It is concluded that ethylene induced ripening of banana is characteristically different from that of other climacteric fruits and that ethylene biosynthesis may have more than one mechanisms operating during ripening which are tightly controlled at various levels.