Cutting Edge: Aire Is a Coactivator of the Vitamin D Receptor.

Vitamin D deficiency is associated with the development of autoimmunity, which arises from defects in T cell tolerance to self-antigens. Interactions of developing T cells with medullary thymic epithelial cells, which express tissue-restricted Ags, are essential for the establishment of central tolerance. However, vitamin D signaling in the thymus is poorly characterized. We find that stromal and hematopoietic cells in the mouse thymus express the vitamin D receptor (Vdr) and Cyp27b1, the enzyme that produces hormonal 1,25-dihydroxyvitamin D (1,25D). Treatment of cultured thymic slices with 1,25D enhances expression of the critical medullary thymic epithelial cell transcription factor autoimmune regulator (Aire), its colocalization with the Vdr, and enhances tissue-restricted Ag gene expression. Moreover, the Vdr interacts with Aire in a 1,25D-dependent manner and recruits Aire to DNA at vitamin D response elements, where it acts as a Vdr coactivator. These data link vitamin D signaling directly to critical transcriptional events necessary for central tolerance.

Identification of a forkhead box protein transcriptional network induced in human neutrophils in response to inflammatory stimuli.

Introduction: Neutrophils represent the largest proportion of circulating leukocytes and, in response to inflammatory stimuli, are rapidly recruited to sites of infection where they neutralize pathogens. Methods and results: We have identified a novel neutrophil transcription network induced in response to inflammatory stimuli. We performed the first RNAseq analysis of human neutrophils exposed to lipopolysaccharide (LPS), followed by a meta-analysis of our dataset and previously published studies of LPS-challenged neutrophils. This revealed a robustly enhanced transcriptional network driven by forkhead box (FOX) transcription factors. The network is enriched in genes encoding proinflammatory cytokines and transcription factors, including MAFF and ATF3, which are implicated in responses to stress, survival and inflammation. Expression of transcription factors FOXP1 and FOXP4 is induced in neutrophils exposed to inflammatory stimuli, and potential FOXP1/FOXP4 binding sites were identified in several genes in the network, all located in chromatin regions consistent with neutrophil enhancer function. Chromatin immunoprecipitation (ChIP) assays in neutrophils confirmed enhanced binding of FOXP4, but not FOXP1, to multiple sites in response to LPS. Binding to numerous motifs and transactivation of network genes were also observed when FOXP proteins were transiently expressed in HEK293 cells. In addition to LPS, the transcriptional network is induced by other inflammatory stimuli, indicating it represents a general neutrophil response to inflammation. Discussion: Collectively, these findings reveal a role for the FOXP4 transcription network as a regulator of responses to inflammatory stimuli in neutrophils.

Vitamin D-regulated Gene Expression Profiles: Species-specificity and Cell-specific Effects on Metabolism and Immunity.

Vitamin D has pleiotropic physiological actions including immune system regulation, in addition to its classical role in calcium homeostasis. Hormonal 1,25-dihydroxyvitamin D (1,25D) signals through the nuclear vitamin D receptor, and large-scale expression profiling has provided numerous insights into its diverse physiological roles. To obtain a comprehensive picture of vitamin D signaling, we analyzed raw data from 94 (80 human, 14 mouse) expression profiles of genes regulated by 1,25D or its analogs. This identified several thousand distinct genes directly or indirectly up- or downregulated in a highly cell-specific manner in human cells using a 1.5-fold cut-off. There was significant overlap of biological processes regulated in human and mouse but minimal intersection between genes regulated in each species. Disease ontology clustering confirmed roles for 1,25D in immune homeostasis in several human cell types, and analysis of canonical pathways revealed novel and cell-specific roles of vitamin D in innate immunity. This included cell-specific regulation of several components of Nucleotide-binding Oligomerization Domain-like (NOD-like) pattern recognition receptor signaling, and metabolic events controlling innate immune responses. Notably, 1,25D selectively enhanced catabolism of branched-chain amino acids (BCAAs) in monocytic cells. BCAA levels regulate the major metabolic kinase mammalian Target of Rapamycin (mTOR), and pretreatment with 1,25D suppressed BCAA-dependent activation of mTOR signaling. Furthermore, ablation of BCAT1 expression in monocytic cells blocked 1,25D-induced increases in autophagy marker LAMP1. In conclusion, the data generated represents a powerful tool to further understand the diverse physiological roles of vitamin D signaling and provides multiple insights into mechanisms of innate immune regulation by 1,25D.

Endocrine aryl hydrocarbon receptor signaling is induced by moderate cutaneous exposure to ultraviolet light.

Links between solar UV exposure and immunity date back to the ancient Greeks with the development of heliotherapy. Skin contains several UV-sensitive chromophores and exposure to sunlight can produce molecules, such as vitamin D3, that act in an endocrine manner. We investigated the role of the aryl hydrocarbon receptor (AHR), an environmental sensor and ligand-regulated transcription factor activated by numerous planar compounds of endogenous, dietary or environmental origin. 15- to 30-minute exposure of cells to a minimal erythemal dose of UVB irradiation in vitro induced translocation of the AHR to the nucleus, rapidly inducing site-specific DNA binding and target gene regulation. Importantly, ex vivo studies with Ahr wild-type or null fibroblasts showed that serum from mice whose skin was exposed to a 15 min UVB dose, but not control serum, contained agonist activity within 30 min of UV irradiation, inducing AHR-dependent gene expression. Moreover, a 15-min cutaneous UVB exposure induced AHR site-specific DNA binding and target gene regulation in vivo within 3-6 hr post-irradiation in blood and in peripheral tissues, including intestine. These results show that cutaneous exposure of mice to a single minimal erythemic dose of UVB induces rapid AHR signaling in multiple peripheral organs, providing compelling evidence that moderate sun exposure can exert endocrine control of immunity through the AHR.

The Tumor Suppressor FBW7 and the Vitamin D Receptor Are Mutual Cofactors in Protein Turnover and Transcriptional Regulation.

The E3 ligase and tumor suppressor FBW7 targets drivers of cell-cycle progression such as the oncogenic transcription factor c-MYC, for proteasomal degradation. Vitamin D signaling regulates c-MYC expression and turnover in vitro and in vivo, which is highly significant as epidemiologic data link vitamin D deficiency to increased cancer incidence. We hypothesized that FBW7 and the vitamin D receptor (VDR) controlled each other's function as regulators of protein turnover and gene transcription, respectively. We found that hormonal 1,25-dihydroxyvitamin D3 (1,25D) rapidly enhanced the interaction of FBW7 with VDR and with c-MYC, whereas it blocked FBW7 binding to c-MYC antagonist MXD1. 1,25D stimulated the recruitment of FBW7, SCF complex subunits, and ubiquitin to DNA-bound c-MYC, consistent with 1,25D-regulated c-MYC degradation on DNA. 1,25D also accelerated the turnover of other FBW7 target proteins such as Cyclin E, c-JUN, MCL1, and AIB1, and, importantly, FBW7 depletion attenuated the 1,25D-induced cell-cycle arrest. Although the VDR contains a consensus FBW7 recognition motif in a VDR-specific insertion domain, its mutation did not affect FBW7-VDR interactions, and FBW7 ablation did not stabilize the VDR. Remarkably, however, FBW7 is essential for optimal VDR gene expression. In addition, the FBW7 and SCF complex subunits are recruited to 1,25D-induced genes and FBW7 depletion inhibited the 1,25D-dependent transactivation. Collectively, these data show that the VDR and FBW7 are mutual cofactors, and provide a mechanistic basis for the cancer-preventive actions of vitamin D. IMPLICATIONS: The key findings show that the VDR and the E3 ligase FBW7 regulate each other's functions in transcriptional regulation and control of protein turnover, respectively, and provide a molecular basis for cancer-preventive actions of vitamin D.Visual Overview: http://mcr.aacrjournals.org/content/17/3/709/F1.large.jpg.

Ligand-Dependent Corepressor (LCoR) Is a Rexinoid-Inhibited Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor α Coactivator.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is an essential regulator of placental development. To gain deeper insights into placental PPARγ signaling, we dissected its regulation of the Muc1 promoter. We find that, unlike prototypic target activation by heterodimeric receptors, which is either stimulated by or refractory to retinoid X receptor (RXR) ligands (rexinoids), the induction of Muc1 by liganded PPARγ requires RXRα but is inhibited by rexinoids. We demonstrate that this inhibition is mediated by the activation function 2 (AF2) domain of RXRα and that Muc1 activation entails altered AF2 structures of both PPARγ and RXRα. This unique regulation of Muc1 reflects specific coactivation of PPARγ-RXRα heterodimers by the transcription cofactor ligand-dependent corepressor (LCoR), corroborated by significant downregulation of Muc1 in Lcor-null placentas. LCoR interacts with PPARγ and RXRα in a synergistic fashion via adjacent noncanonical protein motifs, and the AF2 domain of ligand-bound RXRα inhibits this interaction. We further identify the transcription factor Krüppel-like factor 6 (KLF6) as a critical regulator of placental development and a component of Muc1 regulation in cooperation with PPARγ, RXRα, and LCoR. Combined, these studies reveal new principles and players in nuclear receptor function in general and placental PPARγ signaling in particular.

Hormonal vitamin D up-regulates tissue-specific PD-L1 and PD-L2 surface glycoprotein expression in humans but not mice.

PD-L1 (programmed death ligand 1) and PD-L2 are cell-surface glycoproteins that interact with programmed death 1 (PD-1) on T cells to attenuate inflammation. PD-1 signaling has attracted intense interest for its role in a pathophysiological context: suppression of anti-tumor immunity. Similarly, vitamin D signaling has been increasingly investigated for its non-classical actions in stimulation of innate immunity and suppression of inflammatory responses. Here, we show that hormonal 1,25-dihydroxyvitamin D (1,25D) is a direct transcriptional inducer of the human genes encoding PD-L1 and PD-L2 through the vitamin D receptor, a ligand-regulated transcription factor. 1,25D stimulated transcription of the gene encoding PD-L1 in epithelial and myeloid cells, whereas the gene encoding the more tissue-restricted PD-L2 was regulated only in myeloid cells. We identified and characterized vitamin D response elements (VDREs) located in both genes and showed that 1,25D treatment induces cell-surface expression of PD-L1 in epithelial and myeloid cells. In co-culture experiments with primary human T cells, epithelial cells pretreated with 1,25D suppressed activation of CD4+ and CD8+ cells and inhibited inflammatory cytokine production in a manner that was abrogated by anti-PD-L1 blocking antibody. Consistent with previous observations of species-specific regulation of immunity by vitamin D, the VDREs are present in primate genes, but neither the VDREs nor the regulation by 1,25D is present in mice. These findings reinforce the physiological role of 1,25D in controlling inflammatory immune responses but may represent a double-edged sword, as they suggest that elevated vitamin D signaling in humans could suppress anti-tumor immunity.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection.

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

Engagement of the Aryl Hydrocarbon Receptor in Mycobacterium tuberculosis-Infected Macrophages Has Pleiotropic Effects on Innate Immune Signaling.

Understanding the mechanisms of host macrophage responses to Mycobacterium tuberculosis is essential for uncovering potential avenues of intervention to boost host resistance to infection. Macrophage transcriptome profiling revealed that M. tuberculosis infection strongly induced the expression of several enzymes controlling tryptophan catabolism. These included IDO1 and tryptophan 2,3-dioxygenase, which catalyze the rate-limiting step in the kynurenine pathway, producing ligands for the aryl hydrocarbon receptor (AHR). The AHR and heterodimeric partners AHR nuclear translocator and RELB are robustly expressed, and AHR and RELB levels increased further during infection. Infection enhanced AHR/AHR nuclear translocator and AHR/RELB DNA binding and stimulated the expression of AHR target genes, including that encoding the inflammatory cytokine IL-1ß. AHR target gene expression was further enhanced by exogenous kynurenine, and exogenous tryptophan, kynurenine, or synthetic agonist indirubin reduced mycobacterial viability. Comparative expression profiling revealed that AHR ablation diminished the expression of numerous genes implicated in innate immune responses, including several cytokines. Notably, AHR depletion reduced the expression of IL23A and IL12B transcripts, which encode subunits of IL-23, a macrophage cytokine that stimulates production of IL-22 by innate lymphoid cells. AHR directly induced IL23A transcription in human and mouse macrophages through near-upstream enhancer regions. Taken together, these findings show that AHR signaling is strongly engaged in M. tuberculosis-infected macrophages and has widespread effects on innate immune responses. Moreover, they reveal a cascade of AHR-driven innate immune signaling, because IL-1ß and IL-23 stimulate T cell subsets producing IL-22, another direct target of AHR transactivation.

The enhancement of biodesulfurization activity in a novel indigenous engineered Pseudomonas putida.

BACKGROUND: The combustion of sulfur-rich fossil fuels leads to release of sulfur oxide pollution in the environment. In biodesulfurization process, an organism is able to remove sulfur from fossil fuels without decreasing the caloric value of those substrates. The main aim of this research was to design a recombinant microorganism to remove the highest amount of sulfur compounds in fossil fuels. METHODS: Three genes (dszA,B,C) from dsz operon are responsible for the 4S pathway (biodesulfurization pathway) in Rhodococcus erythropolis IGTS8 were inserted into the chromosome of a novel indigenous Pseudomonas putida. The reaction catalyzed by products of dszA,B,C genes require FMNH2 supplied by dszD enzyme. Thus, pVLT31 vector harboring dszD gene was transferred into this recombinant strain. RESULTS: The results demonstrated a higher biodesulfurization activity when the flavin reductase gene was transferred into recombinant P. putida harboring dszA,B,C. These results were approved by the Gibbs test and HPLC analysis. CONCLUSION: These analyses showed that this novel indigenous engineered P. putida could be a promising candidate for an industrial and environmental application for Biodesulfurization process.