Halloran, Mark ( LH ) Rar PORTABLE

The next step in the bioactivation of D2 and D3, hydroxylation to 25OHD, takes place primarily in the liver although a number of other tissues express this enzymatic activity. 25OHD is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. DeLuca and colleagues were the first to identify 25OHD and demonstrate its production in the liver over 30 years ago, but ambiguity remains as to the actual enzyme(s) responsible for this activity. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities appear to differ indicating different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1. However, in mouse knockout studies and in humans with mutations in these enzymes, only CYP2R1 loss is associated with decreased 25OHD levels (18,19). However, deletion or mutation of CYP2R1 does not totally eliminate 25OHD production These are mixed function oxidases, but differ in apparent Kms and substrate specificities.

Halloran, Mark ( LH ) rar

The vitamin D metabolites bound to DBP are in general not available to most cells. Thus, the free or unbound concentration is that which is critical for cellular uptake as postulated by the free hormone hypothesis. Support for the concept that the role of DBP is to provide a reservoir for the vitamin D metabolites but that it is the free concentration that enters cells and exerts biologic function comes from studies in mice in which DBP has been deleted and in humans in which the gene is mutated. In DBP knockout mice the vitamin D metabolites are presumably all free and/or bioavailable. These mice do not show evidence of vitamin D deficiency unless placed on a vitamin D deficient diet despite having very low levels of serum 25OHD and 1,25(OH)2D (107). Tissue levels of 1,25(OH)2D were found to be normal in the DBP knockout mice as were markers of vitamin D action such as expression of intestinal TRPV6, calbindin 9k, PMCA1b, and renal TRPV5 (108). Recently a family in which a large deletion of the coding portion of the DBP gene (and adjacent NPFFR2 gene) has been reported (109). The proband had normal calcium, phosphate and PTH levels with vitamin D supplementation despite very low levels of 25OHD, 24,25(OH)2D, and 1,25(OH)2D that were not responsive to massive doses of vitamin D (oral or parenteral). The free 25OHD was nearly normal. The carrier sibling had vitamin D metabolite levels between those of the proband and the normal sibling. Thus, both the studies in DBP null mice and humans support the free hormone hypothesis while also supporting the role of DBP as a circulating reservoir for the vitamin D metabolites. Therefore, there is currently a debate as to whether the free concentration of 25OHD, for example, is a better indicator of vitamin D nutritional status than total 25OHD, given that DBP levels, and hence total 25OHD levels, can be influenced by liver disease, nephrotic syndrome, pregnancy, and inflammatory states (110,111). However, certain tissues such as the kidney, placenta, and parathyroid gland express the megalin/cubilin complex which is able to transport vitamin D metabolites bound to DBP into the cell. This is critical for preventing renal losses of the vitamin metabolites (112) and may be important for vitamin D metabolite transport into the fetus and regulation of PTH secretion. Indeed, mice lacking the megalin/cubilin complex have poor survival with evidence of osteomalacia indicating its role in vitamin D transport into critical cells involved with vitamin D signaling

The VDR was discovered in 1969 (121) (although only as a binding protein for an as yet unknown vitamin D metabolite subsequently identified as 1,25(OH)2D), and was eventually cloned and sequenced in 1987 (122,123). Inactivating mutations in the VDR result in hereditary vitamin D resistant rickets (HVDRR) (124). Animal models in which the VDR has been knocked out (125) (126) have the full phenotype of severe vitamin D deficiency indicating that the VDR is the major mediator of vitamin D action. The one major difference is the alopecia seen in HVDRR and VDR knockout animals, a feature not associated with vitamin D deficiency, suggesting that the VDR may have functions independent of 1,25(OH)2D at least in hair follicle cycling. The VDR is a member of a large family of proteins (over 150 members) that includes the receptors for the steroid hormones, thyroid hormone, vitamin A family of metabolites (retinoids), and a variety of cholesterol metabolites, bile acids, isoprenoids, fatty acids and eicosanoids. A large number of family members have no known ligands, and are called orphan receptors. VDR is widely, although not universally, distributed throughout the different tissues of the body (127). Many of these tissues were not originally considered target tissues for 1,25(OH)2D. The discovery of VDR in these tissues along with the demonstration that 1,25(OH)2D altered function of these tissues has markedly increased our appreciation of the protean effects of 1,25(OH)2D.

Nuclear hormone receptors including the VDR are further regulated by protein complexes that can be activators or repressors (135). The role of corepressors in VDR function has been demonstrated (136) but is less well studied than the role of coactivators. One such corepressor, hairless, is found in the skin and may regulate 1,25(OH)2D mediated epidermal proliferation and differentiation as well as ligand independent VDR regulation of hair follicle cycling (137-139). The coactivators, which are essential for VDR function, form two distinct complexes, the interaction of which remains unclear (129). The SRC family has three members, SRC 1-3, all of which can bind to the VDR in the presence of ligand (1,25(OH)2D) (140). These coactivators recruit additional coactivators such as CBP/p300 and p/CAF that have histone acetyl transferase activity (HAT), an enzyme that by acetylation of lysines within specific histones appears to help unravel the chromatin allowing the transcriptional machinery to do its job. The domain in these molecules critical for binding to the VDR and other nuclear hormone receptors is called the NR box, and has as its central motif LxxLL where L stands for leucine and x for any amino acid. Each SRC family member contains three well conserved NR boxes in the region critical for nuclear hormone receptor binding. The DRIP (Mediator) complex is comprised of 15 or so proteins several of which contain LxxLL motifs (141). However, DRIP205 (Mediator 1) is the protein critical for binding the complex to VDR. It contains 2 NR boxes. Different NR boxes in these coactivators show specificity for different nuclear hormone receptors (142). Unlike the SRC complex, the DRIP complex does not have HAT activity (129). Rather the DRIP complex spans the gene from the VDRE to the transcription start site linking directly with RNA polymerase II and its associated transcription factors. DRIP and SRC appear to compete for binding to the VDR. In keratinocytes DRIP binds preferentially to the VDR in undifferentiated cells, whereas SRC 2 and 3 bind in the more differentiated cells in which DRIP levels have declined (143). Thus in these cells DRIP appears to regulate the early stages of 1,25(OH)2D induced differentiation, whereas SRC may be more important in the later stages, although overlap in gene specificity is also observed (144,145) (146). These coregulators are not specific for VDR, but interact with a large number of other transcription factors. The DRIP (Mediator) complex can mark regions in the genome containing large numbers of sites for transcription factors including VDREs. These sites are known as super enhancers often regulating genes involved with cell fate determination (147). Recently, SMAD 3, a transcription factor in the TGF-β pathway, has been found to complex with the SRC family members and the VDR, enhancing the coactivation process (148). Phosphorylation of the VDR may also control VDR function (149). Furthermore, VDR has been shown to suppress β-catenin transcriptional activity (150), whereas β-catenin enhances that of VDR (151). Thus, control of VDR activity may involve crosstalk between signaling pathways originating in receptors at the plasma membrane as well as within the nucleus.

1,25(OH)2D regulates transcellular calcium transport using a combination of genomic and nongenomic actions. The first step, calcium entry across the BBM, is accompanied by changes in the lipid composition of the membrane including an increase in linoleic and arachidonic acid (196,197) and an increase in the phosphatidylcholine:phosphatidylethanolamine ratio (198). These changes are associated with increased membrane fluidity (197), which we have shown results in increased calcium flux (199). The changes in lipid composition occur within hours after 1,25(OH)2D administration and are not blocked by pretreatment with cycloheximide (198). In addition, an epithelial specific calcium channel, TRPV6, is expressed in the intestinal epithelium (200). This channel has a high degree of homology to TRPV5, a channel originally identified in the kidney (201,202). The tissue distributions of these channels are overlapping and can be found in other tissues, but TRPV6 appears to be the main form in the intestine (203,204). TRPV6 mRNA levels in the intestine of vitamin D deficient mice are markedly increased by 1,25(OH)2D, although similar changes are not found in the kidney (205). Mice null for TRPV6 have decreased intestinal calcium transport (206).

Bone develops intramembranously (e.g., skull) or from cartilage (endochondral bone formation, e.g., long bones with growth plates). Intramembranous bone formation occurs when osteoprogenitor cells proliferate and produce osteoid, a type I collagen rich matrix. The osteoprogenitor cells differentiate into osteoblasts which then deposit calcium phosphate crystals into the matrix to produce woven bone. This bone is remodeled into mature lamellar bone. Endochondral bone formation is initiated by the differentiation of mesenchymal stem cells into chondroblasts that produce the proteoglycan rich type II collagen matrix. These cells continue to differentiate into hypertrophic chondrocytes that shift from making type II collagen to producing type X collagen. These cells also initiate the degradation and calcification of the matrix by secreting matrix vesicles filled with degradative enzymes such as metalloproteinases and phospholipases, alkaline phosphatase (thought to be critical for the mineralization process), and calcium phosphate crystals. Vascular invasion and osteoclastic resorption are stimulated by the production of VEGF and other chemotactic factors from the degraded matrix. The hypertrophic chondrocytes also begin to produce markers of osteoblasts such as osteocalcin, osteopontin, and type I collagen resulting in the initial deposition of osteoid. Moreover, at least some of these chondrocytes further differentiate (or trans differentiate) into osteoblasts (251). Terminal differentiation of the hypertrophic chondrocytes and the subsequent calcification of the matrix are markedly impaired in vitamin D deficiency leading to the flaring of the ends of the long bones and the rachitic rosary along the costochondral junctions of the ribs, classic features of rickets. Although supply of adequate amounts of calcium and phosphate may correct most of these defects in terminal differentiation and calcification, the vitamin D metabolites, 1,25(OH)2D and 24,25(OH)2D, have been shown to exert distinct roles in the process of endochondral bone formation. 041b061a72