Songyang Ren

Vitamin D-Directed Rheostatic Regulation of Monocyte Antibacterial Responses

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D3 (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D2 twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D “insufficient” conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

Altered Endocrine and Autocrine Metabolism of Vitamin D in a Mouse Model of Gastrointestinal Inflammation

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-1alpha-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.

Novel regulators of vitamin D action and metabolism: Lessons learned at the Los Angeles zoo.

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.

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism.

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.

Substrate and Enzyme Trafficking as a Means of Regulating 1,25-Dihydroxyvitamin D Synthesis and Action: The Human Innate Immune Response†

Tissue availability of the active vitamin D metabolite, 1,25‐dihydroxyvitamin D [1,25(OH)2D] is dependent on expression of the activating enzyme 1α‐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)2D is also regulated by CYP24 and its metabolically inactive splice variant CYP24‐SV. Finally, initiation of transcription of 1,25(OH)2D‐regulated genes, such as the CYP24, requires movement of the CYP27b1 product, 1,25(OH)2D, 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)2D 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.