Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction.

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity.

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A-C and JMJD2A-D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO2 below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.

Inactivation of tristetraprolin in chronic hypoxia provokes the expression of cathepsin B.

Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.

IL-4 reduces the proangiogenic capacity of macrophages by down-regulating HIF-1α translation.

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-ß). 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.

Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus.

Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

Interleukin-10-induced neutrophil gelatinase-associated lipocalin production in macrophages with consequences for tumor growth.

Tumor cell-derived factors, such as interleukin 10 (IL-10), polarize macrophages toward a regulatory M2 phenotype, characterized by the expression of anti-inflammatory cytokines and protumorigenic mediators. Here we explored molecular mechanisms allowing IL-10 to upregulate the protumorigenic protein NGAL in primary human macrophages. Reporter assays of full-length or deletion constructs of the NGAL promoter provided evidence that NGAL production is STAT3 dependent, activated downstream of the IL-10-Janus kinase (Jak) axis, as well as being C/EBPß dependent. The involvement of STAT3 and C/EBPß was shown by chromatin immunoprecipitation (ChIP) and ChIP-Western analysis, as well as decoy oligonucleotides scavenging both STAT3 and C/EBPß in human macrophages. Furthermore, the production of NGAL in macrophages in response to IL-10 induces cellular growth and proliferation of MCF-7 breast cancer cells. We conclude that both STAT3 and C/EBPß are needed to elicit IL-10-mediated NGAL expression in primary human macrophages. Macrophage-secreted NGAL shapes the protumorigenic macrophage phenotype to promote growth of MCF-7 breast cancer cells. Our data point to a macrophage-dependent IL-10-STAT3-NGAL axis that might contribute to tumor progression.

Proline metabolism in the moderately halophilic bacterium Halobacillus halophilus: differential regulation of isogenes in proline utilization.

The amino acid proline is not only synthesized as a compatible solute but also used as a carbon and energy source by the moderately halophilic bacterium Halobacillus halophilus. Growth on proline was not affected by the salinity of the medium. Proline was degraded by proline dehydrogenase (ProDH) and Δ(1) -pyrroline-5-carboxylate dehydrogenase (P5CDH) to glutamate via Δ(1) -pyrroline-5-carboxylate. The basic biochemical parameters for ProDH and P5CDH activities were obtained for both in cell free extracts. The encoding genes were identified. H. halophilus has two isogenes each for prodh and p5cdh. prodh2 and p5cdh2 form an operon (put operon) whose mRNA is highly abundant in proline-grown cells. Expression of the put operon was also upregulated by salinity and late growth phase in glucose-grown cells. Similarly, ProDH and P5CDH activities increased in late exponential growth phase. This observation is in line with the previous notion that the compatible solute proline is degraded in stationary phase (in glucose grown cultures).