Bone Vol. 17, No. 2, Supplement August 1995:33S-38S ELSEVIER New Understanding of the Molecular Mechanism of Receptor-Mediated Genomic Actions of the Vitamin D Hormone M. R. H A U S S L E R , P. W. J U R U T K A , J.-C. H S I E H , P. D. T H O M P S O N , S. H. S E L Z N I C K , C. A. H A U S S L E R , and G. K. W H I T F I E L D Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, AZ, USA of the receptor as a DNA-bound active heterodimer of liganded hVDR and unoccupied RXR. (Bone 17:33S-38S; The nuclear vitamin D receptor (VDR) binds the 1,25dihydroxyvitamin D 3 [I,25(OH)2D3] hormone with high aff'mity and elicits its actions to regulate gene expression in target cells by binding to vitamin D-responsive elements (VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, [~3-integrin, and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the greatest affinity as a heterodimer with one of the family of retinoid X receptors (RXRs). VDR consists of an N-terminal zinc finger domain that determines DNA binding, a "hinge" segment and a C-terminal hormone binding domain which also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the 1,25(OH)2D 3 ligand in transcriptional activation by the VDR-RXR heterodimer is to alter the conformation of the hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away from being the silent partner of VDR to instead form RXR homodimers. Recent data reveal that after binding RXR, a subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor liB (TFIIB). VDR can be shown to bind directly to TFIIB, in vitro, and synergizes with it in transcriptional control by 1,25(OH)2D 3 in transfected cells, thus unveiling a molecular mechanism whereby 1,25(OH)2D 3 activates the transcription machine. Finally, natural mutations in hVDR that confer 1,25(OH)2D 3 resistance in a number of patients have been characterized. The mutations fall into three categories: (i) DNA binding/ nuclear localization; (ii) hormone binding; and (iii) RXR heterodimerization. These natural mutations are consistent with the structure/function analysis of hVDR via biochemical and molecular biological approaches and confirm the basic model 1995) Key Words: Vitamin D receptor; Vitamin D-responsive element; Retinoid X receptors; Transcriptional control; 1,25Dihydroxyvitamin D3; Hereditary hypocalcemic vitamin D-resistant rickets. Introduction As depicted in Figure 1, vitamin D obtained from diet or photoactivation in the skin is metabolized to its active form, 1,25dihydroxyvitamin D 3 [I,25(OH)2D3], by sequential hydroxylations in the liver and kidney. The 1,25(OH)2D 3 hormone is a member of the subfamily of steroid hormone lipophilic ligands that enters the nucleus directly and binds to a DNA-associated aporeceptor. 8 Similar to the vitamin D receptor (VDR), the thyroid hormone (TR) and retinoic acid receptors (RAR) are localized in the nucleus even in the unoccupied state. VDR is a phosphoprotein which undergoes additional phosphorylation upon 1,25(OH)2D 3 binding. 8 Human VDR (hVDR) is phosphorylated by protein kinase C at serine-51, in a basic nuclear localization domain between the two zinc fingers in the DNA-binding region, and by casein kinase II (CKII) at serine-208, a residue near the N-terminus of the hormone-binding domain which is potentially involved in positive modulation of transactivation. 7 The association of 1,25(OH)2D 3 with VDR elicits the recruitment of a retinoid X receptor (RXR), forming a VDR-RXR heterodimer with high affinity for a number of vitamin D-responsive elements (VDREs) which lie in the regulatory regions of 1,25(OH)2D3-controlled genes. 17 Altered expression of these genes (Figure 1) results in target cell modification and the ensemble of biologic effects of vitamin D including bone remodeling, intestinal Ca" ÷ and PO43- absorption, PTH suppression, and catabolism of the 1,25(OH)2D 3 hormone by 25(OH)vitamin D-24-hydroxylase (24-OHase). As detailed in what follows, Figure 1 also illustrates that the novel vitamin A metabolite which serves as a natural ligand for RXR, namely 9-cis retinoic acid (9-cis RA), negatively modulates 1,25(OH)2D3-dependent transcription by diverting the RXR silent partner away from its complex with VDR. The opposing effects of 1,25(OH)2D 3 and 9-cis RA could have profound effects on the cell differentiation and mineral homeostatic functions of vitamin D, 16 but this requires further elucidation in defined cell culture and with in vivo systems. Address for correspondence and reprints: Dr. Mark R. Haussler, Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, AZ 85724, USA. © 1995 by Elsevier Science Inc. 33S 8756-3282/95/$9.50 SSDI 8756-3282(95)00205-R 34S M.R. Haussler et al. Structure/function of the vitamin D receptor Liver Vitamin D Constitutive ) 25tOH~D~ ~ i .~ Bone Vol. 17, No. 2, Supplement August 1995:33S-38S Kidney ." 1,25(OH)2D3 .... ~"PTH/1 25 Hormone Hegulalea ~ Ca/P64 Target Cell uc,eu o,c < \ + ~r hnRNA ~ :riptional Control J Osteoblast function (Osteocaicin 1"; Osteopontin 1"; Collagen J,)~ Macrophage/Osteociast differentiation (c-myc J,; ~3 Integrin 1 " ) ) Intestinal Ca/PO4 absorption (CaBP28k 1"; CaBPgk 1`) Renal vitamin D catabolism (24-OHase 1') Parathyroid hormone synthesis (PTH ,I,) Immunomodulation (IL-2 J,)/Cell Differentiation L ( / J VDREs Identify Primary Vitamin D Target Genes Table 1 lists the VDREs in 1,25(OH)2D3-controlled genes that have thus far been definitively identified by combinations of promoter-reporter construct transfections, mutagenesis, and in vitro VDR binding. Positive VDREs consist of hexanucleotide direct repeats spaced by three nucleotides (DR3s) with the half element being similar but not identical to the A G G T C A estrogen-responsive half-element. 26 Recent data from studies of VDR-RXR heterodimers on a DR3 element ~2 reveal that the RXR partner lies on the 5' half-element. This orientation of heterodimers has been observed as well with TR and RAR, 29 which form RXR-containing heterodimers on positively controlled DR4 and DR5 elements, respectively. Thus, in all cases of positive control by the VDR/TR/RAR subfamily characterized to date, the RXR partner lies on the 5' half-element. Viewing the 3' VDR half-element in Table 1 (top portion), it is evident that the guanine in the second position is absolutely conserved and that there are two variants of the VDRE half-element. The first type, characteristic of rat and human osteocalcin, ~s'28 avian 133 integfin, 2 and the proximal 24-OHase 22 VDRE, possesses pu- Table 1. Selected natural vitamin D responsive elements Gene controlled Type of regulation 5'-half element "Spacer" 3'-half element + + + + GGGTGA GGGTGA GGTTCA GGTTCA ATG ACG CGA GCG AGGACA GGGGCA GGTTCA GGTGCG + + AGGTGA GAGGCA GTG GAA AGGGCG GGGAGA Rat osteocalcin Human osteocalcin Mouse osteopontin Rat 24-OHase-distal Rat 24-OHaseproximal Avian 133 integrin Positive VDRE consensus . . . . . . . . . . . . . . . . . . Human PTH Avian PTH Rat bone sialoprotein . . + . . . . . . . . . . . . . . . . GGGTCA . . . . . . . . . . . . . . . . . . . . . . GTG . . . . . . . . . . . . GGGGCA . . . . . . . . . . . . . . . - GGTTCA GGGTCA AAG GGA CAGACA GGGTGT - AGGGTT TAT AGGTCA a ~Overlaps an inverted TATA box " mRNA ,j,, Figure 1. Overview of vitamin D metabolism and action to regulate transcription in target cells. ProteinsBi°active ~1~ Altered Cell Functions / / J fines (primarily guanine) in the third and fourth positions. In contrast, the mouse osteopontin 21 and distal 24-OHase 3° VDREs contain, for the most part, thymine bases in these positions. In spite of these differences, a consensus-positive VDRE can be tentatively assigned and is listed in Table I. It is important to note that all natural VDREs identified to date that function as strong enhancers of transcription have the core DR3 motif, and that all bind the RXR-VDR heterodimer. While alternative VDRE motifs are not excluded, they will likely turn out to be weaker elements which may require additional adjacent ciselements that bind cooperating accessory transacting factors. Several negative VDREs have also been characterized (Table 1). For example, the rat bone sialoprotein VDRE overlaps an inverted TATA box in the basal promoter, illuminating occlusion of TATA-binding protein as one mechanism for negative regulation. 14 Avian 13 and human 5 PTH VDREs differ in that the former is quite similar to a positive VDRE, whereas the latter has a very weak 3' half-element that could affect VDR conformation or even bind a repressor partner, with VDR being relegated to the transcriptionally inactive 5' half-element. It is probable that negative regulation by VDR will have a number of mechanisms, some perhaps not even involving DNA binding, but instead resulting from a type of transcription factor squelching. What is the Molecular Role of 1,25(OH)2D3? From published VDR DNA-binding experiments carried out by gel mobility band retardation assays, it is clear that when physiologic quantities of VDR are utilized, the receptor only binds efficiently to DR3 VDREs when purified RXRs or suitable nuclear extract sources of RXRs are supplied. 2'13'17As"E2,2s However, there has been some confusion and controversy over whether the 1,25(OH)2D 3 hormone is required for such binding. 17.~s,22,24,2s One problem in interpretation of gel mobility shift assays is that often vast excesses of expressed VDR/RXR are included in the reaction and only the minuscule fraction that actually binds to the labeled VDRE probe is visualized. Thus, even in the absence of 1,25(OH)2D3, a small fraction of VDR will exist in the active conformation and produce a positive band shift. The role of 1,25(OH)2D 3 is probably to shift the conformational equilibrium to favor the active species, much like an allosteric activator stimulates enzyme activity. A second problem is that nonphysiologic concentrations of salt (~< 0.1 mol/L KCI) have been used in some studies, allowing the formation of Bone Vol. 17, No. 2, Supplement August 1995:33S-38S M.R. Haussler et al. Structure/function of the vitamin D receptor relatively low affinity protein-DNA complexes. In reactions that best mimic physiologic conditions, namely 0.15-0.2 mol/L KC117'22'28 and limiting concentrations of VDR/RXR, the 1,25(OH)2D 3 hormone can be shown to be required for heterodimeric VDR-RXR binding to DR3 VDREs. The model depicted in Figure 2 illustrates our concept of VDR-DNA binding, the essence of which is that there are two distinct dimerizing forms of RXR-VDR, with the 1,25(OH)2D 3 hormone eliciting a transformation of the weakly associated beterodimer into a more stable complex. This "sliding hinge" model includes several hypotheses not previously put forth. We propose (Figure 2, top) that in the absence of 1,25(OH)2D 3 (resting state), a significant fraction of VDRs exist as preheterodimerized species bound weakly to DNA. Weak dimerization is, by analogy with RXR-TR and RXR-RAR, 29 proposed to result from contact between the D-box in the second zinc finger of RXR and the first zinc finger or prefinger region of VDR. This weakly dimerized form is configured such that it can slide along DNA but is unable to penetrate deeply enough into the major groove of DNA to recognize and bind to DR3 VDREs (Figure 2, top). By traversing in the plane of DNA, the VDR-RXR heterodimer is positioned within reach of its target sites and is thus poised to accept ligands. This model therefore represents an interesting analogy to the plasma membrane dimeric receptors for several growth factors that lie in the plasma membrane awaiting circulating ligands. It should be noted that, as detailed below, VDR-RXR heterocomplexes can also form in solution (presumably in the nucleoplasm?) in a process accelerated by 1,25(OH)2D 3. Therefore, all activation events need not occur on DNA per se. Upon 1,25(OH)2D 3 binding (Figure 2, center), we propose Resting State 35S that a strong dimerization interface is exposed in the hormonebinding domain of VDR. Heterodimerization then conformationally alters or " h i n g e s " the RXR-VDR complex such that the zinc finger regions of each receptor now can not only penetrate into the major groove of DNA to contact fully the half elements, but also are configured such that they correctly discriminate the spacing of a DR3, as opposed to DR4s or DR5s that contain similar half-elements. We can therefore summarize the role of the 1,25(OH)2D 3 hormone as follows: the primary function of 1,25(OH)2D 3 is to activate allosterically the RXR-VDR heterocomplex and allow it to specifically recognize the VDRE. Although others assert that the ligand for RXR, 9-cis RA, either synergizes with 1,25(OH)2D 3 in activating transcription in insect cells 3 or has no significant effect in mammalian cells, 6 w e 17 and two additional groups 4"t2 have clearly shown that 9-cis RA exerts a negative influence on 1,25(OH)2D 3 actions. Accordingly, 9-cis RA prevents RXR-VDR from full association with DR3 VDREs, 4"17 whereas 9-cis RA and a more specific RXR ligand partially repress 1,25(OH)2D3-stimulated transcription in transfected cells, t 2. ~7 This effect of 9-cis RA binding to the VDR silent partner is depicted in the lower portion of Figure 2. If added or present prior to 1,25(OH)2D 3, 9-cis RA is a potent suppressor of RXR-VDR binding to the VDRE, acting by diverting RXR from forming heterodimeric RXR complexes. 4']7 This diversionary action by 9-cis RA does not absolutely require the presence of an RXRE (Thompson and Haussler, unpublished data), but as illustrated in Figure 2 (bottom), RXR homodimer binding to the DR 1 RXRE can then occur. Most importantly, and likely explaining the conflicting data in experiments involving coaddition of 1,25(OH)2D 3 and 9-cis RA to gel mobility shift and transfected cell transcription reactions, we (Thompson and Haussler, unpublished data) have observed that if 1,25(OH)2D 3 initially occupies VDR to facilitate RXR-VDR heterodimers, this species is relatively resistant to 9-cis RA binding and inhibition (Figure 2, lower right). Therefore, as in classic physiologic systems of opposing hormones, the state of activation will be dictated by the individual concentrations and order of appearance of the 1,25(OH)2D 3 and 9-cis RA nuclear ligands. Structure/Function of Human VDR and Naturally Occurring Mutations _ Activated State x~,25(OH)2D 3 ~ p x~,25(OH)2D3 P VDREI D, VDRE2 Partially Repressed State 9-cis RA • RXRE D' P VDREI P 9-cis RA P P VDRE2 Figure 2. Sliding hinge model for 1,25(OH)2D3-VDR binding to the VDRE and its diversion by 9-cis RA. Illustrated in Figure 3 is a schematic map of human VDR. In addition to their classic roles in DNA binding and hormone binding, respectively, the N-terminal and C-terminal domains of hVDR subserve other functions. For example, the zinc finger region contains two nuclear localization signals, one mapping to the T-box region just C-terminal to the second finger ]5 and a second residing in an area of five basic amino acids between the two fingers. 9 Heterodimerization with RXR is a property of the C-terminal 1,25(OH)2D3-binding domain and the relevant portion of VDR appears to consist of at least a bipartite region encompassing a conserved sequence known as E123 as well as several heptad repeats within a leucine zipperlike domain, particularly the fourth and ninth heptads. 2° Until X-ray crystallographic data are available for the actual tertiary structure of VDR and its contacts with RXR and the VDRE, models of VDR will by necessity be derived from biochemical and artificial mutagenesis studies. However, a number of natural mutations in hVDR have been characterized that produce the autosomal recessive disorder of hereditary hypocalcemic vitamin D-resistant rickets (HHVDRR) (reviewed in refs. 16 and 25). These mutations are pictured in Figure 3 and their characterization provides 36S M.R. Haussler et al. Structure/function of the vitamin D receptor Bone Vol. 17, No. 2, Supplement August 1995:33S-38S DNA Binding Domain Hormone Binding (E) Domain ~,_~.,,~.~ H3+Nq I (~) \ 1/21 \ [ tr Localization _ H35Q ~ ~ E1 R274L R73Q HeptadRepeats I314S Y295stop/ ]l c~ ~ L t I i-T--- Q152stop ~ I Transcriptional Activation Heterodimerization Domains , [ N u c l e ation" r G33D , Hing_......~e , I R73stop ' Hormone /| 1 Binding Mutations I j9 t 427 R391C P a t i e n t #2) (p NovelMutation M~ Novel affecting predon predominantlyRXR hetero heterodimerization K45E r F47I R50Q DNABindingandNuclearLocalizationMutations Figure 3. Schematic structure/function map of hVDR and summary of natural mutations that cause the hereditary hypocalcemic vitamin D-resistant rickets phenotype. An encircled P indicates a site of phosphorylation. Patient No. 2 (R391C) refers to the study of Selznick et al. 27 strong independent verification of the present model for VDR. The first and most abundant class of mutations resides in the zinc finger region and affects DNA binding and nuclear localization. Interestingly, these altered amino acids are for the most part in residues highly conserved in the steroid receptor superfamily and fall into three subcategories: (i) the tip of the first zinc finger which may constitute a weak dimerization interface with the D-box of the RXR partner on DNA29; (ii) the " k n u c k l e " region just C-terminal of finger 1 which comprises an a-helix in the estrogen receptor (ER) that recognizes the ERE half-element26; and (iii) the a-helical region(s) on the C-terminal side of finger 2 which make contacts with the DNA phosphate backbone in the ER-ERE structure. 26 A second class of naturally mutated VDRs are those possessing missense or nonsense (stop) codons that preclude 1,25(OH)2D 3 hormone binding via alteration or truncation, respectively, of the C-terminal domain (Figure 3). Finally, we have recently discovered a novel mutation (R391C) in an HHVDRR patient which compromises primarily RXR heterodimerization. 27 This latter substitution in hVDR appears to establish yet a third class of mutant VDRs and unequivocally demonstrates that RXR-VDR heterodimerization is obligatory to prevent the HHVDRR phenotype. The natural mutations identified in the lower portion of Figure 3 therefore support both the schematic structure/function of hVDR pictured in the top portion of this figure and the model for vitamin D action depicted in Figure 2. The paramount conclusion from the characterization of natural mutants of hVDR is that, to function biologically, VDR must localize to the nucleus and bind to three entities: 1,25(OH)2D3, an RXR isoform, and DNA. How Does VDR Regulate the Transcription Machine? In the basal state of DNA transcription, the TATA-box binding protein (TFIID) and its associated factors (TAFs) are bound to the TATA box at approximately position - 20 in the 5' region of controlled genes, but the frequency of transcriptional initiations is very low because the RNA polymerase II-basal transcription factor liB (TFIIB) enzyme complex is not stably associated with TFIID-TAFs. The recruitment of the TFIIB-RNA polymerase II complex appears to be the rate-limiting step in preinitiation complex formation, and is stimulated dramatically when a transacting factor or factors bind to upstream enhancers. In a process involving DNA looping, transactivators are thought to attract TFIIB and also interact with TAFs, forming a stable preinitiation complex that executes repeated rounds of productive transcription. Recent data indicate that the activation function in the hormone-binding domain of the estrogen receptor, AF-2, associates specifically with a TAF known as TAFn30 ~1 and that the ER binds to TFIIB in vitro, l° In collaboration with the groups of Ozato at the National Institutes of Health and Tsai and O'Malley at the Baylor College of Medicine, we have observed that hVDR specifically associates with hTFIIB, l In that work, Blanco et al. showed that VDR binds to a TFIIB-glutathione S-transferase fusion protein linked to glutathione-laden beads.l It was also observed that both TRI3 and R A R a , but not RXR, interact with hTFIIB. These results indicate that the RXR silent partner is not actively engaged in TFIIB contact, but that the ligand-binding partners in the VDR/TR/RAR subfamily provide a hard-wired connection to the assembly and enhancement of the transcription machine. Moreover, simultaneous independent data obtained by MacDonald et al. 19 using the powerful yeast two-hybrid system to detect protein-protein interactions also revealed that hVDR binds efficiently to TFIIB. MacDonald et al. 19 further exploited the yeast two-hybrid system to prove that, although hVDR and RXR interact, no homodimeric association occurs for hVDR alone, providing conclusive evidence against the existence of VDR homodimers in biologically relevant settings. Utilizing fusion protein technology, they also showed that VDR interacts directly with RXR to form a heterodimer in solution and not just on DNA; this process was enhanced eightfold by the presence Bone Vol. 17, No. 2, Supplement August 1995:33S-38S o f 1,25(OH)2D 3 h o r m o n e . 19 B e c a u s e h V D R - T F I I B association is not d e p e n d e n t o n the 1 , 2 5 ( O H ) 2 D 3 ligand, 1"~9 the role o f 1,25(OH)2D 3 c a n n o w be further r e s o l v e d to an early participation in c o n f o r m i n g V D R s u c h that it attracts R X R followed by the targeting o f the resulting R X R - V D R b e t e r o d i m e r to V D R E s (see Figure 2). A l t h o u g h we h a v e yet to e x a m i n e w h e t h e r the p r e s e n c e o f R X R further facilitates V D R - T F I I B association, it is probable that the s i m p l e p o s i t i o n i n g o f a certain d o m a i n or dom a i n s o f h V D R i m m e d i a t e l y u p s t r e a m o f target structural g e n e s constitutes the direct physical link to T F I I B a n d transcriptional e n h a n c e m e n t by 1,25(OH)2D 3. B l a n c o et al. ~ h a v e also reported functional studies w h i c h for the first time s h o w the interaction o f T F I I B with a n y m e m b e r o f the steroid receptor s u p e r f a m i l y in l i g a n d - d e p e n d e n t activation o f transcription in intact cells. In pluripotent P19 m o u s e e m b r y onal c a r c i n o m a cells, transfection o f h V D R or hTFIIB alone p r o d u c e d no better t h a n a t w o f o l d i n d u c t i o n o f V D R E - l u c i f e r a s e reporter e x p r e s s i o n b y 1 , 2 5 ( O H ) 2 D 3. H o w e v e r , w h e n transfected together, h V D R a n d h T F I I B m e d i a t e d a synergistic transcriptional response of approximately 30-fold when 1,25(OH)2D 3 w a s added, a n d this effect w a s absolutely dependent on the p r e s e n c e o f the V D R E in the luciferase construct. It s h o u l d be n o t e d that the V D R - T F I I B positive cooperation appears to be cell-specific b e c a u s e similar e x p e r i m e n t s in contacti n h i b i t e d N I H / 3 T 3 S w i s s m o u s e e m b r y o c e l l s r e s u l t e d in hTFIIB-elicited s q u e l c h i n g o f transcription. Therefore, in m o r e differentiated cells, p e r h a p s i n c l u d i n g osteoblasts or fibroblasts, a c c e s s o r y coactivators m a y be p r e s e n t to m o d u l a t e TFIIB or bridge b e t w e e n V D R a n d TFIIB. In c o n c l u s i o n , V D R and TFIIB are h y p o t h e s i z e d to exist in a m u l t i - s u b u n i t transcription c o m plex w h i c h also c o n t a i n s T A F s and/or coactivators that m a y be p r o m o t e r - or t i s s u e - s p e c i f i c . F u r t h e r c h a r a c t e r i z a t i o n o f this c o m p l e x will require the d i s c o v e r y o f cell type a n d promoterspecific c o m p o n e n t s via transfection a n d b i o c h e m i c a l interaction studies. U l t i m a t e l y an in vitro transcription s y s t e m m u s t be dev i s e d that utilizes d e f i n e d c o m p o n e n t s to faithfully replicate 1,25(OH)2D3-stimulated g e n e e x p r e s s i o n . Conclusion and Perspectives T h e p r e c e d i n g description o f the m o l e c u l a r m e c h a n i s m w h e r e b y the v i t a m i n D h o r m o n e controls g e n e e x p r e s s i o n is as yet i n c o m plete. To a d v a n c e our u n d e r s t a n d i n g , a physical characterization o f the structure o f V D R via X - r a y c r y s t a l l o g r a p h y is required. F u r t h e r m o r e , to c o m p r e h e n d the g e n o m i c action o f v i t a m i n D in c a l c i u m h o m e o s t a t i c a n d o t h e r target cells it will be n e c e s s a r y to elucidate the detailed i n v o l v e m e n t o f various R X R i s o f o r m s , specific T A F s , a n d n o v e l coactivators that m i g h t i n f l u e n c e the regulation o f different v i t a m i n D - c o n t r o l l e d promoters. Additional d e v e l o p m e n t a l a n d biologic insights into the v i t a m i n D endocrine s y s t e m will no d o u b t arise w h e n a V D R g e n e k n o c k o u t is created in m i c e . All o f this i n f o r m a t i o n m a y assist in determ i n i n g if the n o n g e n o m i c actions o f v i t a m i n D c o m p o u n d s that have b e e n o b s e r v e d in vitro are biologically relevant a n d perhaps will aid in e v a l u a t i n g the potential role for V D R in the pathop h y s i o l o g y o f osteoporosis. Acknowledgments: This work was supported by NIH Grant Nos. DK33351 and AR15781 to M.R.H. and DK40372 to G.K.W. References 1. Blanco, J. C. G., Wang, I.-M., Tsai, S. Y., Tsai, M.-J., O'Malley, B. W., Jurutka, P. W., Haussler, M. R., and Ozato, K. Transcription factor TFIIB M . R . Haussler et al. Structure/function of the vitamin D receptor 37S and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proc Natl Acad Sci USA 92:1535-1539; 1995. 2. Can, X., Ross, F. P., Zhang, L., MacDonald, P. N., Chappel, J., and Teitelbantu, S. L. 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