NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm.
Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm.
Department of Biology, Molecular Genetics Unit, Western University, London, Ontario, Canada.
Benjamin J
BJ
Dickson
Department of Biology, Molecular Genetics Unit, Western University, London, Ontario, Canada.
Mohamed I
MI
Gatie
Department of Biology, Molecular Genetics Unit, Western University, London, Ontario, Canada.
Danielle M
DM
Spice
Department of Biology, Molecular Genetics Unit, Western University, London, Ontario, Canada.|Child Health Research Institute, London, Ontario, Canada.|Ontario Institute for Regenerative Medicine, Toronto, Ontario, Canada.
Gregory M
http://orcid.org/0000-0001-8612-364X
GM
Kelly
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D050993:GATA6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D060449:Wnt Signaling Pathway
D050993:GATA6 Transcription Factor; C494760:Gata6 protein, mouse; D017382:Reactive Oxygen Species; D014212:Tretinoin; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C422943:Nox4 protein, mouse