Characterization of the Promoter and the Transcriptional Regulation of theLipoxin A4 Receptor(FPR2/ALX) Gene in Human Monocytes and Macrophages

Abstract

The lipoxin A4 receptor FPR2/ALX plays an important part in host defense and inflammation. The receptor binds structurally diverse agonistic ligands, which mainly regulate chemotaxis and activation of leukocytes. However, little is known about the promoter region of the FPR2/ALX gene and its transcriptional regulation in leukocytes. We identified two TATA-less promoter regions, separated by 224 bp, that drive the expression of FPR2/ALX in macrophages. Both promoter regions increased transcription in a reporter assay, and the basal transcription factors OCT1 and SP1 were shown to bind the first and the second promoter, respectively, and to transactivate transcription. Although monocytes expressed high levels of FPR2/ALX mRNA from the second promoter region, differentiation into macrophages abrogated FPR2/ALX expression. Stimulation of macrophages with a set of cytokines revealed that only IFN-γ and LPS increased FPR2/ALX expression from the first promoter to levels similar to those detected in monocytes. The upregulation by IFN-γ is in part mediated by the interaction of IFN regulatory factor 1 with an IFN-responsive sequence element transcription factor binding site located in the first promoter region of the FPR2/ALX gene. However, this upregulation on the mRNA level did not translate into FPR2/ALX protein expression in macrophages owing to reduced translation of the longer mRNA from the first promoter. In contrast, FPR2/ALX mRNA transcribed from the second promoter was translated into surface expression of FPR2/ALX in monocytes. These data support a model in which FPR2/ALX plays a role in chemotaxis and activation of monocytes; however, they also suggest that its function in resident tissue macrophages is limited.

Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32

“…We have previously shown that the FPR2/ALX receptor is absent in primary human macrophages (22), suggesting that LXA4 proresolution actions may be mediated by other receptors in these cells (40,41), and we show in this article that RvD1 signaling in human macrophages is mediated by GPR32, involving calcium release from intracellular stores. In contrast, RvD1 signaling in human neutrophils differs from that of macrophages.…”

“…Bars indicate the mean and SD of three independent experiments. *p , 0.05 compared with basal migration of macrophages without stimuli, levels as we have previously done for other G protein-coupled receptors triggered by lipid mediators (22,25,27). GPR32 is transcribed and expressed on the surface of resting macrophages (Fig.…”

“…Of interest, GPR32 has no mouse ortholog, suggesting that the effects seen in mice are dependent on FPR2/ALX. In contrast, we have previously shown that the FPR2/ALX receptor is expressed on primary human monocytes, but not on macrophages, because of alternative promoter usage in macrophages and reduced translation of this mRNA (22), suggesting that GPR32 may be the key player in mediating RvD1 effects in human macrophages.…”

“…These in vitro proresolution effects of RvD1 on primary human macrophages match the time-point at which this potent lipid mediator is found in inflammatory exudates, namely, the later stages of the resolution of inflammation, at which further recruitment of immune cells may not be necessary (11). In contrast, LXA4, which is produced earlier during the resolution timeline, recruits monocytes via the FPR2/ALX receptor (22) and primes these monocytes toward a proresolution macrophage phenotype (22,44). After these initial proresolution effects mediated by LXA4, it seems reasonable that the sequential rise in RvD1 during the later stages of the resolution of inflammation would further promote the polarization of macrophages toward a proresolution phenotype with increased phagocytic activity, but without, however, recruiting more leukocytes.…”

Differential regulation of monocytic expression of leukotriene and lipoxin receptors

“…Previous studies revealed that IFN-γ up-regulates FPR2/ALXmRNA in microglia from mice [19], and in human enterocytes [20] and macrophages [21]. In addition, FPR2/ALX has been shown to be up-regulated by IL-13 [20], a cytokine also produced mainly In contrast to the up-regulation of FPR2/ALX mRNA levels in THP-1 cells after pro-inflammatory stimuli, but in line with the results of activated CD4 + cells /macrophage co-cultures (Fig 1B), the expression of the BLT 1 receptor once more displayed a different profile.…”