Transdifferentiation Requires iNOS Activation: Role of RING1A S-Nitrosylation.
BACKGROUND
We have previously shown that innate immunity is necessary for transdifferentiation of fibroblasts to endothelial cells. A major signaling molecule involved in innate immunity is inducible nitric oxide synthase (iNOS). Accordingly, we hypothesized that iNOS-generated nitric oxide (NO) might enhance transdifferentiation.


OBJECTIVE
To elucidate the role of NO in epigenetic plasticity during transdifferentiation.


RESULTS
We exposed the BJ fibroblasts to transdifferentiation formulation that included endothelial growth factors and innate immune activator polyinosinic:polycytidylic acid to induce endothelial cells. Generation of transdifferentiated endothelial cells was associated with iNOS expression and NO elaboration. In the absence of polyinosinic:polycytidylic acid, or in the presence of antagonists of NFκB (nuclear factor kappa B) or iNOS activity, NO synthesis and induce endothelial cell generation was reduced. Furthermore, genetic knockout (in murine embryonic fibroblasts) or siRNA knockdown (in BJ fibroblasts) of iNOS nearly abolished transdifferentiation, an effect that could be reversed by iNOS overexpression. Notably, polyinosinic:polycytidylic acid induced nuclear localization of iNOS, and its binding to, and nitrosylation of, the epigenetic modifier ring finger protein 1A (RING1A) as assessed by immunostaining, Co-IP, and mass spectrometry. Nitrosylation of RING1A reduced its binding to chromatin and reduced global levels of repressive histone marker H3K27 trimethylation. Overexpression of a mutant form of RING1A (C398A) lacking the nitrosylation site almost abrogated transdifferentiation.


CONCLUSIONS
Overall, our data indicate that during transdifferentiation, innate immune activation increases iNOS generation of NO to S-nitrosylate RING1A, a key member of the polycomb repressive complex. Nitrosylation of RING1A reduces its binding to chromatin and decreases H3K27 trimethylation level. The release of epigenetic repression by nitrosylation of RING1A is critical for effective transdifferentiation.
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX.
Shu
S
Meng
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX.
Gang
G
Zhou
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX.
Qilin
Q
Gu
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX.
Palas K
PK
Chanda
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX.
Frank
F
Ospino
From the Department of Cardiovascular Sciences, Center for Cardiovascular Regeneration, Houston Methodist Research Institute, TX. jpcooke@tmhs.org.
John P
JP
Cooke
D000818:Animals; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000502:physiology*; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology; D008297:Male; D051379:Mice; D009569:Nitric Oxide / Q000276:immunology / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000276:immunology; D063150:Polycomb Repressive Complex 1 / Q000276:immunology / Q000378:metabolism*
D009569:Nitric Oxide; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D063150:Polycomb Repressive Complex 1; C489840:Ring1 protein, mouse
chromatin; endothelial cell; epigenetic repression; innate immunity; mass spectrometry