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The active form of vitamin D, 1,25-dihydroxyvitamin D3, [1,25(OH)2D3] has potent actions on innate and adaptive immunity. Although endocrine synthesis of 1,25(OH)2D3 takes place in the kidney, the enzyme that catalyzes this, 25-hydroxyvitamin D-1α-hydroxylase (CYP27b1 in humans, Cyp27b1 in mice), is expressed at many extra-renal sites including the colon. We have shown previously that colonic expression of CYP27b1 may act to protect against the onset of colitis. To investigate this further, we firstly characterized changes in Cyp27b1 expression in a mouse model of colitis. Mice treated with dextran sodium sulfate (DSS) showed weight loss, histological evidence of colitis, and increased expression of inflammatory cytokines. This was associated with decreased renal expression of Cyp27b1 (5-fold, P = 0.013) and lower serum 1,25(OH)2D3 (51.8 ± 5.9 pg/nl vs. 65.1 ± 1.6 in controls, P < 0.001). However, expression of CYP27b1 was increased in the proximal colon of DSS mice (4-fold compared with controls, P < 0.001). Further studies were carried out using Cyp27b1 null (−/−) mice. Compared with +/− controls the Cyp27b1 −/− mice showed increased weight loss (4.9% vs. 22.8%, P < 0.001) and colitis. This was associated with raised IL-1 in the distal colon and IL-17 in the proximal and distal colon. Conversely, DSS-treated Cyp27b1−/− mice exhibited lower IL-10 in the proximal colon and toll-like receptors 2 and 4 in the distal colon. These data indicate that both local and endocrine synthesis of 1,25(OH)2D3 affect colitis in DSS-treated mice. Lack of Cyp27b1 exacerbates disease in this model, suggesting that similar effects may occur with vitamin D deficiency.

Tissue availability of the active vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] is dependent on expression of the activating enzyme 1alpha-hydroxylase (CYP27b1) and its catabolic counterpart 24-hydroxylase (CYP24). The activity of these two enzymes is in turn controlled by factors including affinity of the serum vitamin D-binding protein (DBP) for 25-hydroxyvitamin D [25(OH)D]; the availability of enzyme cofactors; and the relative amount of hydroxylase gene product expressed. In recent years, it has become clear that directed trafficking of substrate and enzyme is also a pivotal component of the regulated process of hormone synthesis by both renal and extrarenal tissues expressing the CYP27b1 and CYP24 genes. Extracellular regulatory trafficking events are defined by the quantity of substrate 25(OH)D entering the circulatory pool. Entry into some target cells in vivo, such as the macrophage and proximal renal tubular epithelial cells, requires 25(OH)D binding to serum DBP, followed by recognition, internalization, and intracellular release. The "released" intracellular substrate is moved to specific intracellular destinations (i.e., the mitochondrial CYP enzymes and the vitamin D receptor [VDR]) by the hsc70 family of chaperone proteins. Synthesis of 1,25(OH)(2)D is also regulated by CYP24 and its metabolically inactive splice variant CYP24-SV. Finally, initiation of transcription of 1,25(OH)(2)D-regulated genes, such as the CYP24, requires movement of the CYP27b1 product, 1,25(OH)(2)D, to the VDR in the same cell for intracrine action or export to another cell for paracrine action. In either case, the 1,25(OH)(2)D ligand is required for the VDR to heterodimerize with the retinoid x receptor and compete away the dominant-negative acting, heterogeneous nuclear ribonucleoprotein (hnRNP)-related, vitamin D response element-binding proteins that inhibit hormone-directed transactivation of genes. In this review, we use vitamin D-directed events in the human innate immune response to Mycobacterium tuberculosis as a physiologically relevant model system in which to highlight the importance of these intracellular traffic patterns.

Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease.

Using vitamin D-resistant New World primates as model of natural diversity for sterol/steroid action and metabolism, two families of novel intracellular vitamin D regulatory proteins have been discovered and their human homologs elucidated. The first family of proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), initially considered to function only as pre-mRNA-interacting proteins, have been demonstrated to be potent cis-acting, trans-dominant regulators of vitamin D hormone-driven gene transactivation. The second group of proteins bind 25-hydroxylated vitamin D metabolites. Their overexpression increases vitamin D receptor (VDR)-directed target gene expression. We found that these intracellular vitamin D binding proteins (IDBPs) are homologous to proteins in the heat shock protein-70 family. Our ongoing studies indicate directly or indirectly through a series of protein interactions that the IDBPs interact with hydroxylated vitamin D metabolites and facilitate their intracellular targeting.

We undertook an investigation of an outbreak of rachitic bone disease in the Emperor Tamarin New World primate colony at the Los Angeles Zoo in the mid-1980s. The disease phenotype resembled that observed in humans with an inactivating mutation of the vitamin D receptor (VDR), hypocalcemia, high 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels, and rickets in rapidly growing adolescent primates. In contrast to the human disease, the New World primate VDR was functionally normal in all respects. The proximate cause of vitamin D hormone resistance in New World primates was determined to be the constitutive overexpression of a heterogeneous nuclear ribonucleoprotein in the A family which we coined the vitamin D response element binding protein (VDRE-BP). VDRE-BP competed in trans with the VDR-retinoid X receptor (RXR) for binding to the vitamin D response element. VDRE-BP-legislated resistance to 1,25-(OH)2D was antagonized (i.e., compensated) by another set of constitutively overexpressed proteins, the hsp-70-related intracellular vitamin D binding proteins (IDBPs). IDBPs, present but expressed at much lower levels in Old World primates including man, exhibited a high capacity for 25-hydroxylated vitamin D metabolites and functioned to traffic vitamin Ds to specific intracellular destinations to promote their action and metabolism. J. Cell. Biochem. 88: 308–314, 2003. © 2002 Wiley-Liss, Inc.

N-Terminal analogs of PTH-related protein (PTHrP) and PTH bind to a common receptor and exhibit similar biological properties. However, recent studies suggest that certain midregion and C-terminal PTHrP peptides have activities distinct from those of PTH in the placenta and in osteoclasts, respectively. In this study we determined the biological activities of full-length recombinant PTHrP-(1-141) and several synthetic N-terminal, midregion, and C-terminal PTHrP fragments in two PTHrP-producing cell types. Peptides were tested for their ability to stimulate cAMP production and raise intracellular free calcium ([Ca2+]i) in primary rat aortic smooth muscle cells (VSMC) and UMR-106 rat osteoblast-like (UMR) cells. In UMR cells PTHrP-(1-34)NH2, PTHrP-(1-141), and bovine PTH-(1-34) all increased cAMP (approximately 50 fold) and [Ca2+]i (180 nM). By contrast, in VSMC, these N-terminal peptides increased cAMP (3-fold) but had no detectable effect on [Ca2+]i. PTHrP-(1-34) and PTHrP-(1-141) significantly blunted the angiotensin II-induced rise in cAMP (but not the calcium signal) consistent with the concept that PTHrP opposes angiotensin II activity in VSMC. PTHrP-(67-86)NH2, PTHrP-(107-138)NH2, and PTHrP-(107-111)NH2 had no effect on either cAMP or [Ca2+]i in either cell type. VSMC and UMR-106 cells both expressed a 2.5-kilobase PTH/PTHrP receptor messenger RNA (mRNA) transcript. However, high affinity specific binding of 125I-labeled [Tyr36] PTHrP-(1-36)NH2 was detected in UMR cells but not in VSMC. We conclude that the PTH-like, N terminus of the PTHrP molecule is critical in induction of cAMP and [Ca2+]i pathways in UMR cells, and for cAMP stimulation in VSMC. In addition, PTHrP, like other established vasodilators, signals in VSMC mainly (if not exclusively) by increasing the production of cAMP.

A general chronology of growth hormone-releasing peptide (GHRP) and ghrelin and their shared receptor is shown in Table 1 [1] [3]. The discovery of the natural hormone ghrelin appears to be the exciting beginning of a new unusual hormone system. Initially, in 1980, GH-releasing activity of the unnatural GHRPs was thought to represent the activit y of the as-yet unidentified natural hypophysiotrophic hormone GH-releasing hormone (GHRH). However, in 1982, GHRH was isolated from a pancreatic tumour and the hypothalamus and chemically identified as 44 and 40 amino acid linear peptides. Although the GH-releasing activity of GHRPs and GHRH are similar, it is also apparent that they are definitely chemically and functionally different because of the uniqueness of the action of GHRP on GH secretion in vitro as well as in vivo in a variety of animal species. In 1984, it was postulated that GHRP reflected the activity of another new hypothalamic hormone involved in the increased secretion of GH and thus was in need of isolation.

The cytochrome P450 25-hydroxyvitamin D3-1α-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)2D3 synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)2D3 by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P < 0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P < 0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology.

Seven phospholipids, modified with ester groups in their hydrophobic chains, were synthesized and examined for their ability to promote sodium ion flux across vesicular membranes. It was found by 23Na NMR that only the phospholipids having short chain segments beyond their terminal ester groups catalyze sodium ion transfer by up to 2 orders of magnitude relative to a conventional phospholipid, POPC. The rates increase with the concentration of the ester−phospholipid admixed with POPC in the bilayer. More surprisingly, the rates increase with the time allowed for the vesicles to age. This was attributed to ester−phospholipid migrating in the bilayers to form domains that solubilize the sodium ion within the hydrocarbon interior of the membrane. Such membrane domains explain why shift reagent-modified NMR spectra display three 23Na signals representing sodium outside the vesicles, sodium within the vesicular water pools, and sodium within the membranes themselves.

In the Eocene period, 50–100 million yr ago, the great southern hemispheric landmass, Pangea, broke apart. These tectonic events resulted in the American land mass and Madagascar moving away from Africa. This continental separation occurred early in the process of primate evolution, trapping primordial primates in South America, Africa, and Madagascar, respectively. As a consequence, the three major primate infraorders, platyrrhines or New World primates, catarrhines or Old World primates and lemurs, evolved independently of one another (1) (Fig. 1A). Unlike Old World primates, including our own species, which have populated virtually every land mass on our planet, New World primates have remained confined to Central and South America for the last 50 million yr. Compared to Old World primates, especially some of the terrestrial species such as gorillas, New World primates are smaller in stature. This is a characteristic well suited to their lifestyle as plant-eating, arboreal sunbathers, residing in the canopy of the periequatorial rain forests of the Americas.

Previously recognized intracellular proteins with an affinity for vitamin D metabolites include the vitamin D receptor and the cytochrome P-450based vitamin D metabolizing mixed-function oxidases. We recently characterized a third set of high-capacity, intracellular vitamin D binding proteins (IDBPs) in the inducible heat shock protein-70 (hsp-70) family. Here we report the cloning and expression of cDNAs coding for two IDBPs. The full-length cDNAs for IDBP-1 and IDBP-2 demonstrated 95% and 94% nucleotide homology, respectively, with the cDNAs for human constitutively expressed heat shock protein 70 (hsc-70) and hsp-70. Transient expression of the IDBP cDNAs in a vitamin D-responsive primate cell line increased extractable 25-hydroxylated vitamin D metaboliteIDBP-binding 25-fold. Transfection experiments also demonstrated that the majority of the constitutively expressed 25-hydroxylated vitamin D metabolite binding activity was attributable to expression of the hsc-70-related IDBP-1 and that metabolite binding activity sublocalized to the highly conserved ATP-binding/ATPase domain of hsp70s. Stable overexpression of IDBP-1 in wild-type cells enhanced vitamin D-directed responsiveness of endogenous vitamin D-24-hydroxylase, osteopontin, and osteocalcin genes by several-fold over that observed in cells transfected with an empty vector. These results suggest that IDBP-1 facilitates the intracellular localization of active vitamin D metabolites and vitamin D receptor-mediated transactivation. (Molecular Endocrinology 14

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is believed to act as a local vasorelaxant by activating either the classical PTH or a unique PTHrP receptor. We used a newly cloned complementary DNA encoding the rat PTH/PTHrP receptor to study the expression of its messenger RNA (mRNA) in primary aortic vascular smooth muscle cells (VSMC) and in UMR-106 osteoblast-like cells under basal conditions and in response to treatment with agonists. Both cell types expressed a 2.4-kilobase PTH/PTHrP receptor mRNA transcript and exhibited hormone-induced desensitization of PTHrP-(1-34)NH2-stimulated cAMP. In VSMC, angiotensin-II, which induces PTHrP expression, also rapidly (30 min) desensitized the cAMP response and down-regulated (75-90%) receptor mRNA within 1 h. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) mimicked these effects, whereas neither PTHrP-(1-34)NH2, forskolin, nor (Bu)2cAMP altered receptor mRNA expression. By contrast, in UMR-106 cells, PTHrP-(1-34)NH2 induced time- and dose-dependent decreases in receptor mRNA that were preceded by pronounced desensitization (cAMP and ligand binding) of cell surface receptors. These effects were mimicked by (Bu)2cAMP and forskolin, but not by phorbol 12-myristate 13-acetate, suggesting that both receptor mRNA down-regulation and receptor desensitization in UMR cells were mediated through a protein kinase-A pathway. We suggest that VSMC and UMR cells express a common receptor, which is subject to cell-specific regulation. Such diversity in the PTH/PTHrP receptor regulatory mechanisms provides a means for restricting the length and duration of the cellular response to hormone in a cell/tissue-specific manner.