Elf-1 and PU.1 induce expression of gp91(phox) via a promoter element mutated in a subset of chronic granulomatous disease patients

The cytochrome b heavy chain (gp91(phox)) is the redox center of the NADPH-oxidase and is highly expressed in mature myeloid cells. Point mutations at -57, -55, -53, and -52 bp of the gp91(phox) promoter have been detected in patients with chronic granulomatous disease (CGD; Newburger et al, J Clin Invest 94:1205, 1994; and Suzuki et al, Proc Natl Acad Sci USA 95:6085, 1998). We report that Elf-1 and PU. 1, ets family members highly expressed in myeloid cells, bind to this promoter element. Either factor trans-activates the -102 to +12 bp gp91(phox) promoter when overexpressed in nonhematopoietic HeLa cells or the PLB985 myeloid cell line. However, no synergy of gp91(phox) promoter activation occurs when both Elf-1 and PU.1 are overexpressed. Introduction of the -57 bp or -55 bp CGD mutations into the gp91(phox) promoter significantly reduces the binding affinity of Elf-1 and PU.1 and also reduces the ability of these factors to trans-activate the promoter. These results indicate that Elf-1 and PU.1 contribute to directing the lineage-restricted expression of the gp91(phox) gene in phagocytes and that failure of these factors to effectively interact with this promoter results in CGD.     

Functional effects of NAD(P)H oxidase p22(phox) C242T mutation in human leukocytes and association with thrombotic cerebral infarction

BACKGROUND: Previous study showed that polymorphism of the NAD(P)H oxidase p22(phox) gene is associated with atherosclerosis, although others could not confirm such association. We investigated the association between p22(phox) C242T polymorphism and thrombotic cerebral infarction and the role of this polymorphism on superoxide-production activity in human neutrophils and promyelocytic HL-60 cells as a model system. METHODS: PCR-RFLP analysis revealed that genotype and allele frequencies of C242T polymorphism in 120 patients with thrombotic cerebral infarction and 177 control subjects. The superoxide-production activity in neutrophils was determined by cytochrome c reduction assay. To clarify the role of p22(phox) C242T polymorphism on NAD(P)H oxidase activity, we used transgenic HL-60 cells transfected with expression plasmids carrying p22(phox) cDNAs with or without C242T polymorphism. RESULTS: Genotype and allele frequencies of C242T polymorphism in patients and control subjects were not significantly different. The superoxide-production activity in neutrophils with T allele was higher than in neutrophils without T allele. Moreover, expression analysis showed that superoxide-production activity in p22(phox) C242T-expressing HL-60 cells were significantly higher than in p22(phox)-expressing HL-60 cells. CONCLUSIONS: We conclude that C242T of p22(phox) gene is not involved in thrombotic cerebral infarction but more likely in increased NAD(P)H oxidase activity in phagocytes.     

C/EBPalpha binds and activates the PU.1 distal enhancer to induce monocyte lineage commitment

The PU.1 gene contains a 237-base pair distal enhancer located 14 kilobases upstream of its promoter. We have identified 2 sites within the PU.1 enhancer that strongly bind C/EBPalpha in a gel shift assay, and interaction with endogenous C/EBPalpha was confirmed by chromatin immunoprecipitation. Mutation of these DNA elements reduced activity of a distal enhancer-promoter construct 2- or 5-fold in a myeloid cell line, while mutation of a weaker C/EBPalpha-binding site located in the promoter minimally reduced activity in this context. These findings strengthen the link between C/EBPalpha and PU.1 expression. Reduction of C/EBPalpha activity in cases of acute myeloid leukemia may therefore contribute to transformation by reducing PU.1 levels. In addition, induction of PU.1 by C/EBPalpha during normal hematopoiesis may contribute to stem cell commitment to the myeloid lineages and further commitment to monopoiesis. Consistent with a requirement for C/EBPalpha induction of PU.1 during myeloid development, we demonstrate that C/EBPalpha induces monocytic development when expressed in PU.1(+/+), PU.1(+/-), or PU.1(+/kd) marrow myeloid progenitors but induces granulocyte lineage commitment in PU.1(kd/kd) cells lacking the PU.1 distal enhancer and does not induce either lineage in PU.1(-/-) cells.