No abstract

cTumor 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/EBP␤ dependent. The involvement of STAT3 and C/EBP␤ was shown by chromatin immunoprecipitation (ChIP) and ChIP-Western analysis, as well as decoy oligonucleotides scavenging both STAT3 and C/EBP␤ 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/EBP␤ 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.

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bMacrophages 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 hypoxiainducible 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% O 2 ). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1␣ or -2␣ in primary human macrophages pointed to an HIF-2␣ 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.C hronic diseases such as diabetes, atherosclerosis, and cancer are characterized by hypoxic areas resulting, for example, from compromised perfusion of narrowed or leaky vessels. Cells of the immune system are involved in the outcome of these diseases. As part of the innate immune system, macrophages actively regulate inflammation but also the resolution of inflammation as well as tissue regeneration and remodeling. Macrophages invade hypoxic areas attracted by a number of cytokines produced by hypoxic cells. To survive and operate in a hypoxic environment, cells need a variety of adaptive mechanisms (1, 2).Hypoxia-inducible factors (HIFs) are important to coordinate hypoxic responses and consist of a constitutively expressed ␤-subunit and an oxygen-regulated ␣-subunit. Both are members of the helix-loop-helix/Per, ARNT, and SIM (PAS) transcription factor family (1, 3). Among the ␣-subunits, HIF-1␣ and HIF-2␣ are best characterized. Both contain an oxygen-dependent degradation domain (ODD) with two conserved prolyl residues that are hydroxylated by prolyl hydroxylases (PHDs) 1 to 3 when sufficient oxygen is available, allowing their proteasomal degradation (4, 5). PHDs are impaired under hypoxia, which in turn causes accumulation and translocation of HIF-␣ into the nucleus. The ␣-subunit forms a heterodimer with the ␤-subunit and binds to hypoxiaresponsive elements (HRE) in regulatory regions of target genes (6). By recruiting cofactors like p300 or CBP, the HIF proteins enhance transcription of about 400 target genes (7,8). Although HIF abundance is mostly regulated by protein stability, regulation of HIF-1␣ mRNA via binding of tristetraprolin (TTP) to AU-rich elements (AREs) in the 3...

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).

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.

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.