Unknown,Transcriptomics,Genomics,Proteomics

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Reduced Treg suppressive function and HIF-1Î± expression in hypoxia

ABSTRACT: Recently, hypoxia via the transcription factor HIF-1a has been implicated to play an important role for the fate of the adaptive immune response by regulatory T cells (Treg) and T helper 17 cells (TH17) in the mouse model. However, the reports on the effect of HIF-1a are conflicting and so far no functional data in the human system are available. Therefore, we analyzed the effect of hypoxia and HIF-1a on Treg and TH17 in the human system. FACS, western blot and reporter assays clearly demonstrated that hypoxia does not up-regulate the level of HIF-1a in CD4+ T cells (THC) and microarray analysis revealed no change of the transcriptome comparing normoxia vs. hypoxia. Furthermore, we could show that HIF-1a is almost exclusively regulated via NF-kB and NFAT, whereas hydroxylation and subsequent degradation of HIF-1a had little to no effect. In addition, we showed that HIF-1a is essential for nTreg mediated suppression and for IL-17A secretion of TH17, but not for TH17 lineage commitment measured by RORÎ³t expression. In conclusion, our results demonstrated that THC have a distinct regulation of HIF-1a protein levels, which was absolutely essential for Treg and TH17 function. 3 patients, 2 cell type, 2 treatments = 12 arrays

ORGANISM(S): Homo sapiens

SUBMITTER: Claudia Pommerenke

PROVIDER: E-GEOD-41999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells

Project description:HIF-1a activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HET HIF-1a fracture calluses are larger, stronger and stiffer than WT HIF-1a calluses due to decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia following fracture activates hypoxia-inducible factor 1 alpha (HIF-1a) leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1a is a key regulator of bone regeneration Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1a deficiency (HET HIF-1a) and wild type littermates (WT HIF-1a). Fracture calluses and intact contralateral femurs from post fracture day (PFD) 21 and 28 (N=5-10) were subjected to MicroCT evaluation and 4-point bending in order to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10 and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. Results: Surprisingly, fracture calluses from HET HIF-1a mice exhibit greater mineralization and are larger, stronger and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, demonstrating higher expression of pro-apoptotic PP2A and lower expression of anti-apoptotic BCL2 in WT HIF-1a calluses. Subsequent TUNEL staining demonstrates that WT HIF-1a calluses contain larger numbers of TUNEL positive chondrocytes and osteoblasts than HET HIF-1a calluses. Conclusions: We conclude that partial HIF-1a deficiency results in decreased chondrocytic and osteoblastic apoptosis; thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration. Keywords: Bone, Fracture, Apoptosis, Hypoxia, Microarray

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Reduced Treg suppressive function and HIF-1Î± expression in hypoxia

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