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Hypoxia induces macrophage inflammatory protein-2 (MIP-2) gene expression in murine macrophages via NF-B: the prominent role of p42/ p44 and PI3 kinase pathways

A. ZAMPETAKI^*, S. A. MITSIALIS^*, J. PFEILSCHIFTER and STELLA KOUREMBANAS^*,1

^* Department of Medicine, Division of Newborn Medicine, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston Massachusetts, USA; and
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitaet, Frankfurt am Main, Germany

¹Correspondence: Division of Newborn Medicine, Children’s Hospital, 300 Longwood Ave, Enders 9, Boston, MA 02115, USA. E-mail: stella.kourembanas{at}childrens.Harvard.edu

SPECIFIC AIMS

Expanding on our recent reports that hypoxia induces a pronounced inflammatory response in the mouse lung, we explored cellular types and molecular mechanisms that are mobilized by hypoxic signaling to elevate lung levels of cytokines. Using MIP-2 (CXCL2) as a model chemokine system, we investigate the effects of hypoxia on the expression of this inflammatory molecule in macrophages and outline the pathways that implement and transmit hypoxic signals to activate MIP-2 gene expression.

PRINCIPAL FINDINGS

1. Hypoxia induces MIP-2 gene expression in macrophages
We have reported that mice exposed to hypoxia exhibit elevated levels of MIP-2 protein in the lung. To determine whether monocytes represent a significant source of lung MIP-2 production under hypoxia, a mouse macrophage cell line, RAW264.7, was exposed to hypoxia and MIP-2 protein levels in the supernatant media were measured by ELISA. Elevated levels of MIP-2 protein were detected as early as 2 h and persisted in the media even after 24 h of hypoxic exposure (Fig. 1 ). Induction of MIP-2 protein by hypoxia was also investigated in mouse pulmonary macrophages (isolated from BAL) and in peritoneal macrophages. As with RAW264.7 cells, hypoxia caused a 3-fold up-regulation in MIP-2 protein levels in primary peritoneal macrophages (Fig. 1 , Inset); similar results were obtained with primary pulmonary macrophage cultures.

View larger version (18K): [in this window] [in a new window]   Figure 1. Hypoxia induces MIP-2 protein expression in macrophages. RAW 264.7 were exposed to normoxia (21% O2) or hypoxia (0% O2) and MIP-2 protein was measured in the media supernatant by ELISA. Inset: MIP-2 protein in the media of primary mouse peritoneal macrophage cultures exposed to hypoxia for 18 h. Values are means ± SD. Samples were run in triplicate for each condition and normalized to total cellular protein. *P <0.05 vs. normoxia.

To examine whether hypoxic induction of MIP-2 was associated with increased accumulation of MIP-2 mRNA, total RNA was extracted from hypoxic RAW264.7 cells and analyzed by RNase Protection Assay. MIP-2 mRNA was detectable as early as 1 h after hypoxia and quantification of the expression levels showed a 2-fold increase that was sustained even after 24 h of hypoxic stimulation.

2. Hypoxia induces MIP-2 gene transcription via NF-B
To determine transcriptional control elements in the MIP-2 promoter that contribute to the hypoxic response, RAW 264.7 cells were transiently transfected with expression plasmids harboring a luciferase gene under the control of wild-type or mutant variants of MIP-2 gene promoter. After cells were exposed to hypoxia (0% O₂) for 24 h, a strong induction (9- to 17-fold) was observed in all promoter variants with the exception of a construct harboring a mutation targeted to an NF-B binding site (pGL3-MIP2253b), implicating NF-B as a key regulator of MIP-2 gene induction by hypoxia.

An oligonucleotide corresponding to the putative binding site of NF-B on the MIP-2 promoter region was used as a probe in EMSA analyses. A constitutive complex was detected in nuclear extracts from normoxic and hypoxic RAW 264.7 cells. Hypoxia led to the appearance of a slower migrating complex. This hypoxia-inducible complex is specific for NF-B, since binding was efficiently competed by an unlabeled RelA oligonucleotide. Supershift analyses of the EMSA complexes indicated that the hypoxia inducible complex is a heterodimer of p65-p50 whereas the constitutive complex is apparently a p50 heterodimer.

3. The hypoxic induction of MIP-2 requires p42/p44 and PI3 kinase but not p38 kinase signaling
To evaluate the contribution of kinase signaling pathways in hypoxic induction of the MIP-2 gene, RAW264.7 cells were pretreated with kinase inhibitors before exposure to hypoxia. The optimal inhibitor concentration was determined empirically in pilot experiments and estimated to result in 70% inhibition of kinase activity as assayed in vitro using extracts from treated cells. In all subsequent experiments, the concentration of inhibitors used was 1 µM for SB203580, 10 µM for PD98059, and 25 µM for LY294002. Levels of MIP-2 protein were only mildly affected by the presence of a p38 inhibitor (SB203580), but hypoxic induction was abolished by inhibitors of the p42/p44 (PD98059) or PI3 kinase (LY294002) pathways, indicating that these kinases play a crucial role in the hypoxic induction of MIP-2 protein.

We characterized the effect of the kinase pathways on MIP-2 promoter activity in hypoxia through transient transfections of RAW264.7 cells. The p38 inhibitor decreased MIP-2 promoter activity by <20%. p42/p44 and PI3 kinase pathway inhibitors had a more profound effect and blocked the activation of the MIP-2 promoter by hypoxia. These results agreed with studies of endogenous MIP-2 gene expression in RAW264.7 cells. As expected, PI3K and p42/44 pathway inhibitors abrogated the hypoxic induction of MIP-2 mRNA but no effect was observed in the presence of the p38 inhibitor. To insure that p38 kinase was inhibited, we assayed RAW 264.7 cell extracts from treated and untreated normoxic cultures. Hypoxia significantly increased p38 kinase activity in RAW 264.7 cells, and the concentration of inhibitor used (1 µM SB203580) was sufficient to reduce the activity to levels observed under normoxia.

4. Hypoxic signaling through p42/44 and PI3 kinase pathways affects p65 transactivation potential but not nuclear localization of NF-B
To test whether hypoxic MAPK and PI3 kinase signaling affects nuclear localization of NF-B, nuclear extracts from cells treated with kinase inhibitors were prepared and EMSA was performed using a labeled MIP-2 NF-B oligonucleotide as probe. Kinase inhibitors did not decrease NF-B binding of the hypoxia-inducible complex, indicating that p42/p44 and PI3K signaling occurs downstream of (or parallel to) NF-B dissociation from IB and subsequent nuclear translocation. Supershift studies showed that the composition of the two NF-B complexes in the hypoxic nucleus did not change upon kinase inhibitor treatment.

Cytokine activation of kinase signaling has been described to modulate NF-B activity by affecting the transactivation potential of p65. To determine whether this mechanism is involved in NF-B activation by hypoxia, we transfected RAW264.7 cells with a construct encoding a Gal4-p65 fusion protein in which the yeast Gal-4 DNA binding domain has been fused to the activation domain (amino acids 298–551) of p65. A Gal-4-responsive luciferase reporter was cotransfected and luciferase activity was assayed after exposing cells to hypoxia for 24 h. Transcription under the control of the fusion protein was enhanced significantly by hypoxia, indicating that the transactivation domain of p65 is a direct target of hypoxic signaling. In contrast to nuclear localization, hypoxia-induced p65 transactivation potential was abrogated in the presence of p42/p44 and PI3K inhibitors, in agreement with the MIP-2 gene expression results, p38 kinase inhibition had no significant effect on hypoxic p65 activation.

CONCLUSIONS AND SIGNIFICANCE

A variety of stimuli causing pulmonary inflammation can lead to acute lung injury and functional impairment of gas transfer in the lung. The hallmark of pulmonary inflammation is the presence of infiltrating leukocytes. Their recruitment has been shown to require intercellular communication and activation events that include secretion of early response cytokines, expression of cell surface adhesion molecules, and the production of chemokines.

Infiltration of neutrophils in the alveolar space is a key phase in acute lung injury. In rodents, neutrophil accumulation was shown to be mainly mediated by the C-X-C chemokine MIP-2. Intraperitoneal or intrapulmonary instillation of anti-MIP-2 antibody significantly decreased the influx of neutrophils in the lung and attenuated lung injury in rats injected with LPS. We previously demonstrated that chronic hypoxia induces elevated levels of MIP-2 mRNA as part of a pronounced inflammatory response in the mouse lung. To identify the cellular sources of MIP-2 protein and the mechanisms of its regulation, we exposed cultured macrophages to hypoxia. Our results indicate that, in the hypoxic lung, macrophages can represent a significant source of MIP-2.

Hypoxia regulates the expression of diverse gene targets. The key transcription factor mediating most hypoxic responses is hypoxia-inducible factor-1 (HIF-1), but there is no obvious HIF-1 response element in the promoter region of the MIP-2 gene. Our results indicate that hypoxic MIP-2 induction occurs exclusively via the NF-B pathway. NF-B plays a pivotal role in the induction of a variety of genes involved in immune and inflammatory responses. Increased NF-B binding was observed under hypoxia; supershift EMSA analyses identified this inducible species as a heterodimer of the p65 and p50 subunits. Activation of NF-B, translocation to the nucleus, and increased DNA binding, however, are not sufficient to induce MIP-2 gene expression in hypoxia; our studies using MARK and PI3K pharmacological inhibitors demonstrated that p42/44 and PI3 kinase signaling pathways play a critical role. Blocking these pathways prevented the hypoxic induction of MIP-2 mRNA and protein without significantly affecting the hypoxic induction of NF-B nuclear translocation.

Previous studies of hypoxic NF-B activation have not addressed the cross-talk between kinase signaling and NF-B activity in hypoxia. Our studies establish that in hypoxia, p42/44 and PI3 kinase pathway signaling control NF-B-dependent transcription by mechanisms downstream of nuclear translocation. Our data demonstrate that the transactivation potential of p65 is increased by hypoxia. We show that, in contrast to the hypoxia-induced nuclear localization of NF-B, this enhanced ability of p65 to activate transcription in hypoxia depends on p42/p44 and PI3 kinase signaling. The increase in p65 transactivation potential appears to be the crucial step in hypoxic mobilization of NF-B, as hypoxia-induced nuclear translocation of this factor is not sufficient to induce MIP-2 gene expression. Figure 2 depicts a schematic representation of hypoxic signaling relevant to NF-B mobilization and a comparison with HIF induction mechanisms.

View larger version (72K): [in this window] [in a new window]   Figure 2. Schematic of hypoxia signaling pathways relevant to this study. Hypoxia directly inhibits proline hydroxylases (PHD), resulting in stabilization of HIF1 protein, and activates kinase cascades through an indirect relay system (R) that is probably mitochondrial-produced ROS, but may also use unidentified messenger molecules. Confirmed direct actions are depicted with solid arrows, postulated or indirect actions by dashed arrows. Orange circles represent any post-translational modification, including, but not limited to, phosphorylation events. Sites of action of the inhibitors used (SB, PD, & LY) are indicated. The PI3K pathway has been reported to enhance levels of stable HIF1 protein, and the p38 and p42/p44 MAPK pathways have been reported to enhance HIF1 transactivation potential. We postulate that hypoxia mobilizes NF-B in two distinct steps. The first, IB release and nuclear migration of p65/p50, does not depend on the above-mentioned kinase pathways. The second step is a crucial enhancement of p65 transactivation potential and depends on PI3K and p42/p44 activity.

Little is known about the molecular mechanisms by which hypoxia may increase tissue inflammation vs. other physiologic processes such as erythropoiesis, angiogenesis, and glycolysis, under the control of HIF-1. The inflammatory pathways elicited by hypoxia are mediated at least in part by NF-B. Studies are needed to elucidate the specific role and significance of the pathways that transmit and implement the hypoxic signaling in monocytes; a better understanding of such molecular mechanisms will be invaluable in developing therapeutic strategies to attenuate inflammatory responses and lung injury.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0991fje; doi: 10.1096/fj.03-0991fje

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This Article Full Text (PDF) All Versions of this Article: 18/10/1090 03-0991fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZAMPETAKI, A. Articles by KOUREMBANAS, S. Search for Related Content PubMed PubMed Citation Articles by ZAMPETAKI, A. Articles by KOUREMBANAS, S.