Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes

Abstract

Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun Nterminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…A similar dual interaction with the membrane is well described in the case of p47 phox , although both interactions are prevented in the absence of cell stimulation [34,36,42]. Nuclear localization of Nox1 and of p22 phox has been reported in transformed human gingival keratinocytes [43] and in human umbilical vein endothelial cells (HUVEC) [44], respectively. Although we could not detect p22 phox in the nucleus in HEK293 cells (Fig.…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…Proliferating keratinocytes showed higher ROS generation and Nox1 levels than quiescent cells [123]. Overexpression of Nox1 in several cell types is associated with increased cell division [46,138,139].…”

“…In studies over the past 2 decades, cancer and rapidly proliferating cells were frequently noted to overproduce reactive oxygen [122,123], and antioxidants and inhibitors of NADPHoxidases were associated with decreased cell proliferation [124][125][126]. In many of cases, the source of this ROS is Nox enzymes: this includes melanoma (Nox4, [127]), prostate cancer (Nox5 [128] and Nox1 [129]), glioblastoma (Nox4 and sometimes Nox5 [130]), H. pylorusinduced gastric inflammation leading to gastric cancer (Nox1 [131]) and Barrett's esophageal adenocarcinoma (Nox5 [132]).…”

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: Roles of NADPH oxidase, mitochondria, and lipid rafts

“…Nox1, a homologue of the gp91phox (Nox2) subunit in phagocyte NADPH oxidase, has been described in normal human keratinocytes [24,25]. Using two different sequences of Nox1 siRNA in SLO-HK, the Nox1 protein was significantly decreased 48 h after transfection (Fig.…”

“…Specific small interfering RNA (Steatlh™-siRNA duplex oligoribonucleotides from Invitrogen™ Life Technologies, Carlsbad, CA) were designed by selecting two different duplex sequences of the human Nox1 gene, which is the catalytic subunit of a non-phagocytic NADPH oxidase expressed by keratinocytes [24,25]. The first duplex primer used for Nox1 was designated as Nox1-A (sense 5'-ACAAUAGCCUUGAUUCUCAUGGUAA-3', anti-sense 5'-UUACCAUGAGAAUCAAGGCUAUUGU-3') starting at 750 bp, and the second duplex primer was designated as Nox1-B (sense 5'-GCAAUAUUGUUGGUCAUGCAGCAUU-3', anti-sense 5'-AAUGCUGCAUGACCAACAAUAUUGC-3') located at 1642 bp.…”