Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells.
The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found that TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production on TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3 dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not affected. Our experiments further indicate that Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta-treated MDA-MB-231and MCF10A cells. Collectively, these data indicate that Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for the progression of the EMT in breast epithelial cells, and thereby has therapeutic implications.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD, USA.
Howard E
HE
Boudreau
Benjamin W
BW
Casterline
Balazs
B
Rada
Agnieszka
A
Korzeniowska
Thomas L
TL
Leto
D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics*; D005260:Female; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; D005353:Fibronectins; D007546:Isoquinolines; D011725:Pyridines; D011758:Pyrroles; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human