Jui Cheng Hsieh

Molecular Mechanisms of Vitamin D Action

The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP9k, and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.

Vitamin D receptor : molecular signaling and actions of nutritional ligands in disease prevention

The human vitamin D receptor (VDR) is a key nuclear receptor that binds nutritionally derived ligands and exerts bioeffects that contribute to bone mineral homeostasis, detoxification of exogenous and endogenous compounds, cancer prevention, and mammalian hair cycling. Liganded VDR modulates gene expression via heterodimerization with the retinoid X receptor and recruitment of coactivators or corepressors. VDR interacts with the corepressor hairless (Hr) to control hair cycling, an action independent of the endocrine VDR ligand, 1,25-dihydroxyvitamin D(3). We report novel dietary ligands for VDR including curcumin, gamma-tocotrienol, and essential fatty acid derivatives that likely play a role in the bioactions of VDR.

Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling

Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORα. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORα binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo.

1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism.

1,25-Dihydroxyvitamin D(3) (1,25D) is known primarily as a regulator of calcium, but 1,25D also promotes phosphate absorption from intestine, reabsorption from kidney, and bone mineral resorption. FGF23 is a newly discovered phosphaturic hormone that, like PTH, lowers serum phosphate by inhibiting renal reabsorption via Npt2a. We show that 1,25D strongly upregulates FGF23 in bone. FGF23 then represses 1alpha-OHase activity in kidney, thus preventing spiraling induction of FGF23 by 1,25D. We also report that LRP5, Runx2, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic, are transcriptionally regulated by 1,25D. This coordinated regulation together with that of FGF23 and PTH allows 1,25D to play a central role in maintaining calcium and phosphate homeostasis and bone metabolism. In the cases of LRP5, Runx2, TRPV6, and Npt2c we show that transcriptional regulation results at least in part from direct binding of VDR near the relevant gene promoter. Finally, because 1,25D induces FGF23, and FGF23 in turn represses 1,25D synthesis, a reciprocal relationship is established with FGF23 indirectly curtailing 1,25D-mediated intestinal absorption and counterbalancing renal reabsorption of phosphate. This newly revealed FGF23/1,25D/Pi axis is comparable in significance to phosphate and bone metabolism as the PTH/1,25D/Ca axis is to calcium homeostasis.

Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands.

The vitamin D hormone, 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], binds with high affinity to the nuclear vitamin D receptor (VDR), which recruits its retinoid X receptor (RXR) heterodimeric partner to recognize vitamin D responsive elements (VDREs) in target genes. 1,25(OH)2D3 is known primarily as a regulator of calcium, but it also controls phosphate (re)absorption at the intestine and kidney. Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced in osteoblasts that, like PTH, lowers serum phosphate by inhibiting renal reabsorption through Npt2a/Npt2c. Real‐time PCR and reporter gene transfection assays were used to probe VDR‐mediated transcriptional control by 1,25(OH)2D3. Reporter gene and mammalian two‐hybrid transfections, plus competitive receptor binding assays, were used to discover novel VDR ligands. 1,25(OH)2D3 induces FGF23 78‐fold in osteoblasts, and because FGF23 in turn represses 1,25(OH)2D3 synthesis, a reciprocal relationship is established, with FGF23 indirectly curtailing 1,25(OH)2D3‐mediated intestinal absorption and counterbalancing renal reabsorption of phosphate, thereby reversing hyperphosphatemia and preventing ectopic calcification. Therefore, a 1,25(OH)2D3–FGF23 axis regulating phosphate is comparable in importance to the 1,25(OH)2D3–PTH axis that regulates calcium. 1,25(OH)2D3 also elicits regulation of LRP5, Runx2, PHEX, TRPV6, and Npt2c, all anabolic toward bone, and RANKL, which is catabolic. Regulation of mouse RANKL by 1,25(OH)2D3 supports a cloverleaf model, whereby VDR‐RXR heterodimers bound to multiple VDREs are juxtapositioned through chromatin looping to form a supercomplex, potentially allowing simultaneous interactions with multiple co‐modulators and chromatin remodeling enzymes. VDR also selectively binds certain ω3/ω6 polyunsaturated fatty acids (PUFAs) with low affinity, leading to transcriptionally active VDR‐RXR complexes. Moreover, the turmeric‐derived polyphenol, curcumin, activates transcription of a VDRE reporter construct in human colon cancer cells. Activation of VDR by PUFAs and curcumin may elicit unique, 1,25(OH)2D3‐independent signaling pathways to orchestrate the bioeffects of these lipids in intestine, bone, skin/hair follicle, and other VDR‐containing tissues.

The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the "Fountain of Youth" to mediate healthful aging.

The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D3 (1,25D), its high affinity renal endocrine ligand, to signal intestinal calcium and phosphate absorption plus bone remodeling, generating a mineralized skeleton free of rickets/osteomalacia with a reduced risk of osteoporotic fractures. 1,25D/VDR signaling regulates the expression of TRPV6, BGP, SPP1, LRP5, RANKL and OPG, while achieving feedback control of mineral ions to prevent age-related ectopic calcification by governing CYP24A1, PTH, FGF23, PHEX, and klotho transcription. Vitamin D also elicits numerous intracrine actions when circulating 25-hydroxyvitamin D3, the metabolite reflecting vitamin D status, is converted to 1,25D locally by extrarenal CYP27B1, and binds VDR to promote immunoregulation, antimicrobial defense, xenobiotic detoxification, anti-inflammatory/anticancer actions and cardiovascular benefits. VDR also affects Wnt signaling through direct interaction with beta-catenin, ligand-dependently blunting beta-catenin mediated transcription in colon cancer cells to attenuate growth, while potentiating beta-catenin signaling via VDR ligand-independent mechanisms in osteoblasts and keratinocytes to function osteogenically and as a pro-hair cycling receptor, respectively. Finally, VDR also drives the mammalian hair cycle in conjunction with the hairless corepressor by repressing SOSTDC1, S100A8/S100A9, and PTHrP. Hair provides a shield against UV-induced skin damage and cancer in terrestrial mammals, illuminating another function of VDR that facilitates healthful aging.

Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells

Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.

Novel nuclear localization signal between the two DNA-binding zinc fingers in the human vitamin D receptor†

The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA‐binding zinc fingers that is similar to well‐characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D‐responsive element in DNA, in vitro, and hVDR‐mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR‐specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS‐7 cells. Although wild‐type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg‐49 and Lys‐55 contains a novel constitutive nuclear localization signal, RRSMKRK. J. Cell. Biochem. 70:94‐109, 1998.

The role of vitamin D in the FGF23, klotho, and phosphate bone-kidney endocrine axis

Abstract1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.

Molecular and functional comparison of 1,25‐dihydroxyvitamin D3 and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT‐29 colon cancer cells, both LCA and 1,25(OH)2D3 induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA‐VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two‐hybrid assays, we observe that: (i) 1,25(OH)2D3 enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)2D3‐liganded VDR‐RXR heterodimer recruits full‐length SRC‐1 coactivator, whereas this interaction is minimal with LCA unless LXXLL‐containing fragments of SRC‐1 are employed, and (iii) both 1,25(OH)2D3 and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)2D3‐VDR, LCA‐VDR does not interact detectably with NCoA‐62 or TRIP1/SUG1, suggesting a different pattern of LCA‐VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)2D3‐ and/or LCA‐stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)2D3 and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)2D3‐liganded conformation facilitating high affinity endocrine actions, and the LCA‐liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon. J. Cell. Biochem. 94: 917–943, 2005.