Edited by Vladimir SkulachevKeywords: Macrophage Hypoxia-inducible factor Peroxide signaling Peroxiredoxin Sestrin a b s t r a c t Reactive oxygen species not only serve as signaling molecules, they also contribute to oxidative stress and cell damage. The thioredoxin and glutaredoxin systems form along with peroxiredoxins a precisely regulated defense system to maintain the cellular redox homeostasis. There is evidence that nitric oxide (NO) protects cells from oxidative stress by preventing inactivation of peroxiredoxins by sulfinylation. Here we demonstrate that NO and hypoxia upregulate Sestrin2 by HIF-1-dependent and additional mechanisms and that Sestrin2 contributes to preventing peroxiredoxins from sulfinylation. We conclude that Sestrin2 plays a role in peroxide defense as a reductase for peroxiredoxins.

IntroductionMammalian hematopoiesis in the BM is regulated among others by the oxygen (O 2 ) availability. O 2 concentrations in the BM range from anoxia to 6% opposed to 4%-14% in well-oxygenated tissues, including the blood. 1,2 Recent data indicate that O 2 gradients within the BM participate in keeping hematopoietic stem cells (HSCs) in a low-replicating pluripotent state. HSCs are located in an extremely hypoxic niche as demonstrated by dye-perfusion and engraftment studies. 3,4 Hypoxia-inducible factors 1-3 (HIF-1-HIF-3) are stabilized by a low pO 2 to induce adaptive gene expression. They are heterodimers consisting of distinct O 2 -sensitive ␣-subunits and a stable common ␤-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT). 5 HIF-1 and HIF-2 were recently connected to HSC biology. In their hypoxic niche, HIF-1 maintains HSC quiescence, 6 whereas HIF-2 maintains their self-renewing capacity. 7 HIF-1 also affects embryonic hematopoiesis as demonstrated by defective myeloid and erythroid progenitor formation in HIF-1␣ Ϫ/Ϫ as well as ARNT Ϫ/Ϫ embryos. 8 In adult hematopoiesis, HIF-1 is essential for B-cell progenitor proliferation and mature B-cell subclass differentiation. 9 However, its involvement in mononuclear phagocyte development is unknown. Previous findings indicated defective development of human plasmacytoid dendritic cells (pDCs) under hypoxia in vitro. 10 Therefore, we asked whether HIF-1 regulates DC lineage differentiation in mice. Methods AnimalsHIF-1␣ fl/fl or HIF-2␣ fl/fl mice 11,12 were bred with LysM-Cre transgenic mice 13 in the C57BL/6 background. Age-matched C57BL/6 wild-type (WT) mice were controls. ID2 Ϫ/Ϫ mice and their respective WT control were in the NMRI background. 14 The guidelines of the Hessian animal care and use committee were followed. DC generation from BMFor DC generation in vitro, 2 ϫ 10 6 total BM cells/mL in RPMI 1640 with 10% FCS and 200 ng/mL recombinant murine fms-related tyrosine kinase 3-ligand (Flt3-L, PeproTech) were cultured in 6-well Ultra-Low attachment plates (Corning) for up to 9 days 15 at various O 2 levels as indicated, using a InVivo 2 400 hypoxia workstation (Ruskinn Technologies). Alternatively, cells were cultured with 100M dimethyloxallyl glycine (DMOG, from Biomol). Sorted monocyte/DC progenitors/common DC progenitors (MDPs/ CDPs; 10 4 cells/well) were cultured with 200 ng/mL Flt3-L in 24-well Ultra-Low attachment plates. Flow cytometry and cell sortingBM cells, spleen, or whole blood cells were stained with fluorochromeconjugated antibodies and analyzed on a LSRII/Fortessa flow cytometer (BD Biosciences). MDP/CDP were sorted from lineage Ϫ cell-enriched BM (lineage cell depletion kit and AutoMACS cell separator from Miltenyi Biotec) using a FACSAria III cell sorter (BD Biosciences). For details (antibodies, surface markers, intracellular transcription factor staining procedures), see supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). pDCs were i...

Background: Microarray analysis still is a powerful tool to identify new components of the transcriptosome. It helps to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis.

Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1αLysM−/− macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1αLysM−/− macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.

MΦ show a highly versatile phenotype depending on the receiving microenvironmental stimuli. MΦ phenotypes are grouped in three subcategories. One is classically activated MΦ (after stimulation with LPS or IFN-γ), and two are alternatively activated forms, known as wound-healing MΦ (induced by IL-4/IL-13) and regulatory MΦ (induced by IL-10/TGF-β). Besides cytokines, hypoxia defines MΦ functions, as shown for classically activated cells. Yet, little is known about the role of hypoxia and HIF-1 and -2 in wound-healing or regulatory MΦ. HIF target genes (such as ADM), analyzed in alternatively activated MΦ from WT and HIF-/- mice, were regulated predominantly by HIF-1 and consistently showed reduced hypoxic induction in MΦ stimulated with IL-4. To gain mechanistic insights, we analyzed HIF expression in polarized MΦ. Classically activated MΦ are characterized by the induction of HIF-1α but reduction of HIF-2α mRNA and protein, whereas wound-healing MΦ decreased HIF-1α protein expression without altering mRNA levels. Analysis of protein stability and expression after proteasomal inhibition pointed to translational regulation of HIF-1α in wound-healing MΦ. Following angiogenic-sprouting using embryonic stem cells exposed to supernatants of MΦ incubated with IL-4 under hypoxia, shorter sprouts were revealed compared with supernatants of hypoxic MΦ without IL-4. Conclusively, IL-4 reduces HIF-1α translation and thus, its activity in MΦ and concomitantly, attenuates their ability to promote angiogenesis under hypoxic conditions.