Binding of Pleomorphic Adenoma Gene-like 2 to the Tumor Necrosis Factor (TNF)-α-responsive Region of the NCF2 Promoter Regulates p67 Expression and NADPH Oxidase Activity

Abstract

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox , is up-regulated by TNF-␣, and we recently mapped a region in the NCF2 promoter that was required for this TNF-␣-dependent response. Because this TNF-␣-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-␣ treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67 phox protein expression, and subsequent superoxide production in response to TNF-␣. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-␣ treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.The NADPH oxidase is a multicomponent, enzyme complex consisting of both membrane-bound and cytosolic components and is found in all phagocytic leukocytes, such as monocytes and neutrophils, where it plays an essential role in host defense against pathogens (reviewed in Ref. phox are sufficient for a functional oxidase; however, in vivo the system is clearly more complicated (reviewed in Ref. 6). Although the specific role of p67 phox in enzyme function is still being determined, it has been proposed that p67phox is an NADPH-binding protein (7), and Dang et al. (8) subsequently showed that p67 phox was able to directly bind NADPH. In addition, several groups have shown that p67 phox binding to flavocytochrome b 558 induces conformational changes in flavocytochrome b 558 , resulting in initiation of electron flow (9 -11). The physiological importance of p67 phox in NADPH oxidase function is demonstrated by certain forms of chronic granulomatous disease (CGD), which are caused by autosomal mutations in NCF2 (12). Patients with CGD have an inactive NADPH oxidase, making them susceptible to recurrent bacterial and fungal infections (reviewed in Ref. 13).Regulation of p67 phox expression appears distinct from other phox proteins, as p67 phox is the last phox protein to be expressed during cellular differentiation, and its expression correlates the closest with the acquisition of oxidase activity (14). Thus, it has been proposed that p67 phox may be the rate-limiting cofactor in NADPH oxidase activation (10,15,16). A number of stud...

PLAGL2 translocation and SP-C promoter activity—A cellular response of lung cells to hypoxia

“…In some cells, it was observed to concentrate at the perinuclear region ( Figure 3B, block arrow). A recent report also showed that PLAGL2 was in both cytosolic and nuclear fractions of leucocytes [13]. Interestingly, upon exposure to CoCl 2 , the signal of PLAGL2 became more intense in the nuclei, indicating the translocation of PLAGL2 from the cytoplasm into the nucleus (Figure 3C and D).…”

“…More interestingly, PLAGL2 also modulates the promoter activation of NCF2 gene [13], which encodes p67 phox , a cytosolic protein involved in the NADPH oxidase electron transfer from NADPH to molecular oxygen. Taken together with our results that PLAGL2 responds to hypoxia and could prevent lung cells from hypoxia-induced functional and cytotoxic damages, it further raises an inevitable role of PLAGL2 in mediating the cellular response to oxidative stress.…”

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages

“…However, the lack of LPS-mediated induction of p67phox transcripts throughout the differentiation process suggests a lack of Phox-mediated oxidative response at the transcriptional level in cells undergoing repeated stimulation with LPS. In mammals, the regulation of p67phox is delayed in maturing myeloid cells and thus is considered a rate-limiting cofactor in Phox activation [55]. Therefore, the aforementioned gradual, long-lasting and iterative onset of respiratory burst typical of macrophages upon continuous stimulation must depend on the activation of other ROS-generating enzymatic complexes, such as nitric oxide synthases (NOS).…”

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

“…Numerous studies have demonstrated that various subunits or cofactors can be upregulated or downregulated by different stimuli such as cytokines, inflammatory factors, hormones, autocrines, paracrines, physical stress, and some drugs, which may be involved in transcriptional or posttranscriptional regulation of gene expression and translational or posttranslational regulation of proteins (12,408).…”