TY - JOUR
T1 - Transdifferentiation Requires iNOS Activation
T2 - Role of RING1A S-Nitrosylation
AU - Meng, Shu
AU - Zhou, Gang
AU - Gu, Qilin
AU - Chanda, Palas K.
AU - Ospino, Frank
AU - Cooke, John P.
N1 - Funding Information:
Sources of Funding This work supported, in part, by grants to Dr Cooke from the National Institutes of Health (U01 HL100397) and the Cancer Prevention and Research Institute (RP150611). S. Meng is supported by a postdoctoral fellowship from the American Heart Association.
Publisher Copyright:
© 2016 American Heart Association, Inc.
PY - 2016/10/14
Y1 - 2016/10/14
N2 - Rationale: 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. Methods and 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.
AB - Rationale: 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. Methods and 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.
KW - chromatin
KW - endothelial cell
KW - epigenetic repression
KW - innate immunity
KW - mass spectrometry
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U2 - 10.1161/CIRCRESAHA.116.308263
DO - 10.1161/CIRCRESAHA.116.308263
M3 - Article
C2 - 27623813
AN - SCOPUS:84991444219
SN - 0009-7330
VL - 119
SP - e129-e138
JO - Circulation Research
JF - Circulation Research
IS - 9
ER -