Palmitic acid dysregulates the Hippo–YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS–STING–IRF3 signaling mechanism
Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo–Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo–YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS–STING–IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS–STING–IRF3 signaling are critically involved in PA-induced Hippo–YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes. Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo–Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo–YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS–STING–IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS–STING–IRF3 signaling are critically involved in PA-induced Hippo–YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes. Delayed wound healing and diabetic skin ulcer are major complications of diabetes that cause significant disability and mortality in these patients (1.Boulton A.J. Vileikyte L. Ragnarson-Tennvall G. Apelqvist J. The global burden of diabetic foot disease.Lancet. 2005; 366: 1719-1724Abstract Full Text Full Text PDF PubMed Scopus (1751) Google Scholar, 2.Brem H. Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes.J. Clin. Invest. 2007; 117: 1219-1222Crossref PubMed Scopus (1208) Google Scholar3.Margolis D.J. Hoffstad O. Nafash J. Leonard C.E. Freeman C.P. Hennessy S. Wiebe D.J. 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The Hippo pathway in intestinal regeneration and disease.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 324-337Crossref PubMed Scopus (178) Google Scholar) and its effector Yes-associated protein (YAP) 3The abbreviations used are: YAP, Yes-associated protein; PA, palmitic acid; MST1, mammalian Ste20-like kinases 1; mtDNA mitochondrial DNA; cGAS, cyclic GMP-AMP synthase; STING, stimulator of interferon genes; IRF3, interferon regulatory factor 3; HAEC, human aortic endothelial cell; CCCP, carbonyl cyanide m-chlorophenyl hydrazone. (20.Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development. 2014; 141: 1614-1626Crossref PubMed Scopus (469) Google Scholar) are important in regulating organ growth, tissue repair, and regeneration. The Hippo pathway contains mammalian Ste20-like kinases 1/2 (MST1/2), Salvador (SAV), and large tumor suppressor (LATS1/2). YAP and the transcriptional coactivator with PDZ-binding motif (TAZ) are the primary downstream effectors of the Hippo pathway (15.Pan D. The hippo signaling pathway in development and cancer.Dev. Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar16.Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration, and stem cell self-renewal.Nat. Cell. Biol. 2011; 13: 877-883Crossref PubMed Scopus (911) Google Scholar, 17.Zhou Q. Li L. Zhao B. Guan K.L. The hippo pathway in heart development, regeneration, and diseases.Circ. Res. 2015; 116: 1431-1447Crossref PubMed Scopus (153) Google Scholar, 18.Yu F.X. Zhao B. Guan K.L. Hippo pathway in organ size control, tissue homeostasis, and cancer.Cell. 2015; 163: 811-828Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar, 19.Hong A.W. Meng Z. Guan K.L. The Hippo pathway in intestinal regeneration and disease.Nat. Rev. Gastroenterol. Hepatol. 2016; 13: 324-337Crossref PubMed Scopus (178) Google Scholar20.Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development. 2014; 141: 1614-1626Crossref PubMed Scopus (469) Google Scholar). MST1/2 phosphorylates and activates LATS1/2, (15.Pan D. The hippo signaling pathway in development and cancer.Dev. Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar, 20.Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development. 2014; 141: 1614-1626Crossref PubMed Scopus (469) Google Scholar), which in turn phosphorylates YAP at Ser-127 or Ser-381, leading to its cytoplasmic retention and degradation, and subsequent transcription suppression of its target genes (15.Pan D. The hippo signaling pathway in development and cancer.Dev. Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar16.Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration, and stem cell self-renewal.Nat. Cell. Biol. 2011; 13: 877-883Crossref PubMed Scopus (911) Google Scholar, 17.Zhou Q. Li L. Zhao B. Guan K.L. The hippo pathway in heart development, regeneration, and diseases.Circ. Res. 2015; 116: 1431-1447Crossref PubMed Scopus (153) Google Scholar18.Yu F.X. Zhao B. Guan K.L. Hippo pathway in organ size control, tissue homeostasis, and cancer.Cell. 2015; 163: 811-828Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar). YAP promotes cell proliferation, survival, and angiogenesis (20.Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development. 2014; 141: 1614-1626Crossref PubMed Scopus (469) Google Scholar, 21.Choi H.J. Zhang H. Park H. Choi K.S. Lee H.W. Agrawal V. Kim Y.M. Kwon Y.G. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2.Nat. 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The Hippo pathway and YAP are key regulators in angiogenesis (21.Choi H.J. Zhang H. Park H. Choi K.S. Lee H.W. Agrawal V. Kim Y.M. Kwon Y.G. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2.Nat. Commun. 2015; 6: 6943Crossref PubMed Scopus (173) Google Scholar, 22.Marti P. Stein C. Blumer T. Abraham Y. Dill M.T. Pikiolek M. Orsini V. Jurisic G. Megel P. Makowska Z. Agarinis C. Tornillo L. Bouwmeester T. Ruffner H. Bauer A. et al.YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.Hepatology. 2015; 62: 1497-1510Crossref PubMed Scopus (167) Google Scholar) and wound healing (23.Elbediwy A. Vincent-Mistiaen Z.I. Thompson B.J. YAP and TAZ in epithelial stem cells: a sensor for cell polarity, mechanical forces and tissue damage.Bioessays. 2016; 38: 644-653Crossref PubMed Scopus (72) Google Scholar). the of the Hippo–YAP pathway in angiogenesis, we that dysregulation of this pathway is involved in inhibition of endothelial angiogenesis. In the study, we found that palmitic acid induced MST1 expression, and YAP and endothelial angiogenesis. PA induced MST1 expression through the of mitochondrial damage and mitochondrial DNA and activation of the cytosolic DNA sensor cyclic GMP-AMP genes protein regulatory factor the of stress endothelial angiogenesis, we human aortic endothelial cells with PA, the of free fatty acid in B. fatty acid in and the PubMed Scopus Google Scholar). found that of PA endothelial and and impaired endothelial were to However, PA treatment tube formation, angiogenesis that PA inhibits endothelial proliferation, migration, and angiogenesis. 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In this study, we show that PA the Hippo–YAP pathway and inhibits angiogenesis. PA induces mitochondrial damage and cytosolic release of which activates the cytosolic DNA sensor cGAS–STING–IRF3 Activated IRF3 to the MST1 promoter and induces MST1 expression, leading to YAP and angiogenesis suppression impaired angiogenesis and wound healing in diabetes is the understood. Hippo–YAP B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration, and stem cell self-renewal.Nat. Cell. Biol. 2011; 13: 877-883Crossref PubMed Scopus (911) Google Scholar, 22.Marti P. Stein C. Blumer T. Abraham Y. Dill M.T. Pikiolek M. Orsini V. Jurisic G. Megel P. Makowska Z. Agarinis C. Tornillo L. Bouwmeester T. Ruffner H. Bauer A. et al.YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.Hepatology. 2015; 62: 1497-1510Crossref PubMed Scopus (167) Google Scholar) signaling is a key pathway in regulating tissue repair and angiogenesis. YAP, by inducing the expression of P. Stein C. Blumer T. Abraham Y. Dill M.T. Pikiolek M. Orsini V. Jurisic G. Megel P. Makowska Z. Agarinis C. Tornillo L. Bouwmeester T. Ruffner H. Bauer A. et al.YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.Hepatology. 2015; 62: 1497-1510Crossref PubMed Scopus (167) Google Scholar) and A. S. Li J. Li L. Hippo signaling and are in the for 2016; Full Text Full Text PDF PubMed Scopus Google Scholar) promotes angiogenesis (21.Choi H.J. Zhang H. Park H. Choi K.S. Lee H.W. Agrawal V. Kim Y.M. Kwon Y.G. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2.Nat. Commun. 2015; 6: 6943Crossref PubMed Scopus (173) Google Scholar, 22.Marti P. Stein C. Blumer T. Abraham Y. Dill M.T. Pikiolek M. Orsini V. Jurisic G. Megel P. Makowska Z. Agarinis C. Tornillo L. Bouwmeester T. Ruffner H. Bauer A. et al.YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.Hepatology. 2015; 62: 1497-1510Crossref PubMed Scopus (167) Google Scholar). In the Hippo pathway, by inactivating YAP, inhibits angiogenesis and tissue repair G. The Hippo pathway in cellular and regeneration of Biol. 2016; PubMed Scopus Google Scholar). we show that dysregulation of Hippo–YAP signaling is for PA-induced inhibition of angiogenesis. 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Yarovinsky T.O. a of the 13: PubMed Scopus Google Scholar). that STING is involved in Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. Tenbrock K. Wittkowski H. Jones O.Y. Kuehn H.S. Lee C.R. DiMattia M.A. Cowen E.W. Gonzalez B. Palmer I. et al.Activated STING in a vascular and pulmonary syndrome.N. Engl. J. Med. 2014; 371: 507-518Crossref PubMed Scopus (915) Google Scholar, J. D. S. STING PubMed Scopus Google Scholar). in STING have been in an by and Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. Tenbrock K. Wittkowski H. Jones O.Y. Kuehn H.S. Lee C.R. DiMattia M.A. Cowen E.W. Gonzalez B. Palmer I. et al.Activated STING in a vascular and pulmonary syndrome.N. Engl. J. Med. 2014; 371: 507-518Crossref PubMed Scopus (915) Google Scholar) and in N. B. M. I. S. N. P. S. C. G.I. N. A. B. F. underlies a with Clin. Invest. 2014; PubMed Scopus Google Scholar). in that activation of the STING pathway is involved in angiogenesis inhibition. However, in are to this pathway a role in angiogenesis defects in or wound healing in the diabetic by or inhibition of has that PA the Hippo–YAP pathway and inhibits endothelial angiogenesis. PA induces mtDNA cytosolic release and activates the cytosolic DNA sensor cGAS–STING–IRF3 pathway, which promotes MST1 expression, leading to inhibition of YAP and endothelial angiogenesis. This mechanism may have in impaired wound healing in diabetes.