Original Contribution
Transcriptional regulation of the NADPH oxidase isoform, Nox1, in colon epithelial cells: Role of GATA-binding factor(s)

https://doi.org/10.1016/j.freeradbiomed.2005.08.022Get rights and content

Abstract

Nonphagocytic NADPH oxidases (Noxs) are major sources of reactive oxygen species (ROS) and exist as a family of isoenzymes with tissue-restricted expression and functions. Nox1, expressed in colon epithelium and vascular smooth muscle, is suggested to be involved in innate immune defense and cell growth or proliferation. The transcriptional regulation of Nox1 appears to be particularly important in the modulation of its activity but the underlying mechanisms are unknown. Here we have identified the functional Nox1 promoter in human colon epithelial Caco-2 cells, and show that a 520-bp genomic fragment encompassing the CAP site is sufficient to direct high levels of expression of a linked reporter gene in these cells. Deletion analyses together with electrophoretic mobility-shift assays (EMSAs) suggest that maximal promoter activity is dependent on a GATA-binding site, conserved between human and mouse, within the proximal promoter region. The ability of mouse GATA factors to transactivate the Nox1 promoter was demonstrated in Cos-7 cells and site-directed mutagenesis of the conserved GATA-binding site further demonstrates that the regulation of Nox1 transcription is mediated by the direct binding of a GATA factor to the Nox1 proximal promoter. We also identified more distal, upstream regions which act to repress significantly expression from the Nox1 promoter.

Introduction

The regulated production of reactive oxygen species (ROS, such as superoxide and hydrogen peroxide) by specialized enzymes plays a role in diverse processes including innate immune defense, oxygen sensing, and the modulation of redox-sensitive intracellular signal transduction pathways involved in cell growth, proliferation, and matrix remodeling [1], [2], [3]. The best example of an enzyme whose primary purpose is the generation of ROS is the phagocytic NADPH oxidase which comprises a membrane-bound cytochrome b558 catalytic core, composed of one gp91phox and one p22phox subunit, and at least four cytosolic regulatory subunits (p47phox, p67phox, p40phox, and Rac) that translocate to the cytochrome to activate the enzyme [3], [4]. More recently, it has been shown that the expression of NADPH oxidase is not restricted to phagocytes, but is more widespread [5], and several homologues of gp91phox, termed Nox1-5 (with gp91phox = Nox2), have been identified, which are each encoded by separate genes and show distinctive tissue-restricted patterns of gene expression [6]. Thus it has now become clear that a family of superoxide-generating NADPH oxidases is a major source of ROS in many diverse cell types. Nox1 is expressed in colon epithelium, prostate, uterus, and vascular smooth muscle [7], [8], while low levels of gp91phox(Nox 2) have been detected in many cardiovascular cells [5] in addition to the high levels expressed in phagocytes. Nox3 is found in the inner ear and in fetal kidney [9], [10] and Nox4, while first identified in the kidney [10], now appears to be widely expressed [11]. Finally Nox5 is found in spleen, sperm, and mammary glands [12].

The basic domain structure of Nox1 is similar to Nox2, being approximately 56% identical at the amino acid level [7]. However, its activation mechanisms differ significantly from those that are well established for Nox2. In particular, it has recently been determined that Nox1 normally binds to two novel regulatory proteins named NOXA1 and NOXO1, rather than their analogues p67phox and p47phox, respectively [13], [14], [15]. Whereas the phosphorylation of p47phox and its subsequent binding to gp91phox-p22phox are key events in the activation of the phagocytic Nox2 oxidase, NOXO1 appears to be constitutively associated with Nox1 and does not require cell activation for this binding [16]. Rather, there are several lines of evidence to suggest that regulation at the level of gene transcription may be particularly important in the control of Nox1 activity. First, the overexpression of Nox1 alone in NIH 3T3 fibroblasts resulted in an increased constitutive production of both superoxide and hydrogen peroxide, while transfection of rat aortic vascular smooth muscle cells with antisense Nox1 resulted in a decrease in superoxide generation [7]. In addition, a large body of data implicates an increase in NADPH oxidase-derived ROS, and corresponding significant increases in the levels of Nox1 mRNA, in vascular pathologies such as atherosclerosis and restenosis after angioplasty [4], [17], [18], [19].

The precise function(s) of Nox1, and Nox1-generated ROS, remain unclear. It has been suggested that within the colon epithelium Nox1 plays a role in host defense, analogous to that of Nox2 in phagocytes [8]. This idea is supported by both of the observations that an increase in the level of Nox1 mRNA is induced by the inflammatory mediator IFN-γ and that Nox1 is activated by Type I Helicobacter pylori lipopolysaccharide through Toll-like receptor 4 [20], [21].

Many studies, in a variety of cell types, have also implicated a role for Nox1 in cell growth and differentiation. Thus in vascular smooth muscle cells, Nox1 mRNA is strongly induced by growth factors which effect cellular hypertrophy, such as angiotensin II, platelet-derived growth factor, and prostaglandin F in association with an increase in oxidase activity [17], [22]. By contrast, a forced decrease in Nox1 expression results in a reduction in the rate of serum-dependent growth [7]. Immortalized human keratinocytes display higher levels of Nox1 at proliferating, compared to quiescent confluent stages [23], and similarly, Nox1 oxidase activity was found to be higher in proliferating compared to confluent colon epithelial Caco-2 cells [24]. A correlation between Nox1-derived ROS and cellular transformation to a tumorigenic phenotype in certain fibroblast cell lines also initially suggested that increased Nox1 activity may be mitogenic [7], [25]. More recent studies, however, have failed to confirm these results either in fibroblasts [3] or in a variety of tumor samples including colon carcinoma [20]. Nonetheless, Nox1 overexpression has been reported to increase growth and tumorigenicity of prostate epithelial cells [25], while a recent study has demonstrated that targeted inhibition of Nox1 expression suppresses Ras-induced transformation of fibroblast cells [26]. Thus while Nox 1 may not by itself be mitogenic, it can clearly mediate growth signals, and the close association between altered Nox1 expression and cell growth suggests a causal and functional relationship.

An understanding of the molecular mechanisms that underlie the transcriptional regulation of Nox1 is essential in view of the importance of alterations of Nox1 mRNA expression in its activation, and would help to inform strategies to target increased Nox1 activity in, for instance, vascular pathologies. In the present study, we have investigated the regulation of the Nox1 promoter in Caco-2 cells. We have identified the transcriptional initiation site of the human Nox1 gene in Caco-2 cells, and undertaken initial characterization of the regulatory elements within the promoter that mediate Nox1 expression in these cells.

Section snippets

Chemicals and reagents

Standard chemicals, custom-designed primers, culture medium, antibiotics, Glutamax, and trypsin were purchased from Sigma; fetal calf serum (FCS) from Invitrogen; and dihydroethidium from Molecular Probes.

RNA isolation, cDNA preparation, and real-time reverse transcriptase (RT)-PCR

Total RNA from cell cultures was isolated using the SV total RNA isolation kit (Promega), according to the manufacturer's protocol. RNA was reverse-transcribed with AMV RT (Promega) according to the manufacturer's instructions, using random decamers. Negative control reactions were also

Transcriptional regulation of Nox1

The Nox isoform, Nox1, is expressed both in the colon epithelial cell line, Caco-2, and in the vascular smooth muscle cell line, A7r5. However, the relative levels of expression within the two cell lines are very different, with Nox1 mRNA being approximately 80-fold more abundant in Caco-2 cells than A7r5 cells. This suggests that distinct regulatory mechanisms may effect expression in the two different cell types. Transcriptional regulation of the Nox1 gene is increasingly being understood to

Acknowledgments

We are grateful to Dr. Lewis Mahadevan, for providing the pEF pLINK II 4.9 expression vector, and to Dr. Edward Morrisey for providing mouse pcDNAG4 and pcDNAG5 expression clones. This work was supported by the British Heart Foundation.

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