title: Nox2 and Nox4 influence neonatal c-kit(+) cardiac precursor cell status and differentiation.
abstract: Redox status has emerged as critical in modulating stemness and lineage commitment in several precursor cell types. However, a role for redox genes, specifically NADPH oxidases (Nox), in cardiac precursor cells (CPCs) has not been established. We tested whether CPCs marked by type III receptor tyrosine kinase c-kit (c-kit(+)) exhibit a unique NADPH oxidase signature that confers precursor status and whether alterations in this profile are functionally linked to changes in lineage specification. Dihydroethidium (DHE) microfluorography indicated reduced basal reactive oxygen species (ROS) formation within early postnatal c-kit(+) CPCs. Real-time quantitative PCR revealed downregulation of ROS generator Nox2 and its subunit p67(phox) in c-kit(+) CPCs under basal conditions but upregulation of Nox2 and Nox4 over the course of differentiation. Adenoviral silencing of Nox2 and Nox4 increased expression of CPC markers c-kit and Flk-1 and blunted smooth and cardiac muscle differentiation, respectively, while overexpression of Nox2 and Nox4 significantly reduced c-kit expression. These changes were accompanied by altered expression of transcription factors regulating cardiac lineage commitment, Gata6 and Gata4, and cytokine transforming growth factor (TGF)-β1. Similar to other precursor cell types, RT(2)Profiler PCR Arrays revealed that c-kit(+) CPCs also exhibit enhanced antioxidant capacity at the mRNA level. In conclusion, we report that c-kit(+) CPCs demonstrate reduced Nox2 expression and ROS levels and that increases in Nox2 and Nox4 influence their differentiation into mature cells. We speculate that ROS generators Nox2 and Nox4, along with the antioxidant genes identified by PCR Arrays, may be novel targets in CPCs that could prove useful in cell-based therapy of the heart.
authors.affiliation: Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York; and.
authors.forename: Alyson S
authors.initials: AS
authors.lastname: Nadworny
authors.forename: Mallik R
authors.initials: MR
authors.lastname: Guruju
authors.forename: Daniel
authors.initials: D
authors.lastname: Poor
authors.forename: Robert M
authors.initials: RM
authors.lastname: Doran
authors.forename: Ram V
authors.initials: RV
authors.lastname: Sharma
authors.forename: Michael I
authors.initials: MI
authors.lastname: Kotlikoff
authors.forename: Robin L
authors.initials: RL
authors.lastname: Davisson
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D002478:Cells, Cultured; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D019009:Proto-Oncogene Proteins c-kit
keywords: cardiac differentiation; reactive oxygen species; redox; stem cells