Myofibroblast YAP/TAZ is dispensable for liver fibrosis in mice

Severe fibrosis often leads to significant mortality as a result of liver failure and cirrhosis. Recent evidence arising in the Journal of Hepatology etc. shed light on the role of the mechanosensing Hippo/YAP/TAZ pathway during hepatic fibrosis.1Liu Y. Lu T. Zhang C. Xu J. Xue Z. Busuttil R.W. et al.Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injury.J Hepatol. 2019; 71: 719-730Google Scholar, 2Machado M.V. Michelotti G.A. Pereira T.A. Xie G. Premont R. Cortez-Pinto H. et al.Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease.J Hepatol. 2015; 63: 962-970Google Scholar, 3Martin K. Pritchett J. Llewellyn J. Mullan A.F. Athwal V.S. Dobie R. et al.PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis.Nat Commun. 2016; 7: 12502Google Scholar, 4Mooring M. Fowl B.H. Lum S.Z.C. Liu Y. Yao K. Softic S. et al.Hepatocyte stress increases expression of yes-associated protein and transcriptional coactivator with PDZ-binding motif in hepatocytes to promote parenchymal inflammation and fibrosis.Hepatology. 2020; 71: 1813-1830Google Scholar, 5Mannaerts I. Leite S.B. Verhulst S. Claerhout S. Eysackers N. Thoen L.F. et al.The Hippo pathway effector YAP controls mouse hepatic stellate cell activation.J Hepatol. 2015; 63: 679-688Google Scholar Those studies suggested that the YAP/TAZ pathway was activated in various mouse models of liver fibrosis and in human fibrotic liver. However, conflicting data exist for both positive and negative regulation of the YAP/TAZ pathway in liver fibrogenesis. The study by Liu et al. elegantly demonstrated that YAP inhibition with verteporfin exacerbated liver fibrogenesis.[1]Liu Y. Lu T. Zhang C. Xu J. Xue Z. Busuttil R.W. et al.Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injury.J Hepatol. 2019; 71: 719-730Google Scholar In contrast, several recent studies suggested that inhibition of YAP/TAZ signaling attenuated liver fibrosis, which highlighted YAP/TAZ as a critical driver of hepatic fibrosis.2Machado M.V. Michelotti G.A. Pereira T.A. Xie G. Premont R. Cortez-Pinto H. et al.Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease.J Hepatol. 2015; 63: 962-970Google Scholar, 3Martin K. Pritchett J. Llewellyn J. Mullan A.F. Athwal V.S. Dobie R. et al.PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis.Nat Commun. 2016; 7: 12502Google Scholar, 4Mooring M. Fowl B.H. Lum S.Z.C. Liu Y. Yao K. Softic S. et al.Hepatocyte stress increases expression of yes-associated protein and transcriptional coactivator with PDZ-binding motif in hepatocytes to promote parenchymal inflammation and fibrosis.Hepatology. 2020; 71: 1813-1830Google Scholar, 5Mannaerts I. Leite S.B. Verhulst S. Claerhout S. Eysackers N. Thoen L.F. et al.The Hippo pathway effector YAP controls mouse hepatic stellate cell activation.J Hepatol. 2015; 63: 679-688Google Scholar Such discrepancy has sparked debate about the function of YAP/TAZ in liver fibrogenesis. In particular, the role of YAP/TAZ in myofibroblasts, a key cell type driving extracellular matrix production during fibrosis, is unclear. Herein, we independently addressed this issue using a newly generated Postn-CreERT2 mouse line[6]Kaur H. Takefuji M. Ngai C.Y. Carvalho J. Bayer J. Wietelmann A. et al.Targeted ablation of periostin-expressing activated fibroblasts prevents adverse cardiac remodeling in mice.Circ Res. 2016; 118: 1906-1917Google Scholar to lineage trace activated myofibroblasts in vivo and genetically target Yap/Taz specifically in myofibroblasts within the fibrotic liver. Periostin is a secreted matricellular protein which has been reported as marker of the myofibroblasts expressed exclusively in area of tissue injury.[6]Kaur H. Takefuji M. Ngai C.Y. Carvalho J. Bayer J. Wietelmann A. et al.Targeted ablation of periostin-expressing activated fibroblasts prevents adverse cardiac remodeling in mice.Circ Res. 2016; 118: 1906-1917Google Scholar,[7]Kanisicak O. Khalil H. Ivey M.J. Karch J. Maliken B.D. Correll R.N. et al.Genetic lineage tracing defines myofibroblast origin and function in the injured heart.Nat Commun. 2016; 7: 12260Google Scholar To characterize whether Postn-CreERT2 targets endogenous myofibroblasts in the liver, we crossed the Postn-CreERT2 mice with the mT/mG reporter line, which switches from membrane-targeted Tomato expression to membrane-targeted GFP expression upon Cre-mediated recombination (Fig. 1A). We subjected Postn-CreERT2; mT/mG mice to a well-established model of carbon tetrachloride (CCl4)-induced liver fibrosis followed by tamoxifen induction (Fig. 1B). Postn-CreERT2; mT/mG mice showed abundant expression of periostin protein in the injured liver as well as Postn-CreERT2-dependent expression of GFP (Fig. 1C). At the histological level, periostin lineage-traced GFP+ cells were predominantly expressed in the portal area with the characteristic septal pattern of liver fibrosis (Fig. 1D). Approximately 95% of the GFP-positive cells were αSMA positive, while nearly 92% were collagen-positive and ~85% were desmin-positive but only few were hepatocyte marker HNF4α or endothelial marker CD31 reactive (Fig. 1D-E). Similarly, in bile duct-ligation (BDL, Fig. 1F) and ischemia-reperfusion injury (IRI, Fig. 1G) induced fibrosis models, Postn-CreERT2-induced GFP expression strongly overlapped with αSMA. Thus, Postn-CreERT2 transgenic mice exclusively mark myofibroblasts in the fibrotic liver and Postn-expressing myofibroblasts should be considered as a primary target for anti-fibrotic therapies. To test the contribution of YAP/TAZ in the regulation of liver fibrosis, we generated mice lacking YAP/TAZ in Postn-expressing myofibroblasts by crossing mice carrying floxed alleles of both Yap and Taz (Yapfl/fl; Tazfl/fl) with Postn-CreERT2 animals (Fig. 1H-I). Consistent with previous reports, we found that in CCl4-treated mice, YAP/TAZ was predominantly localized in the nucleus of αSMA-positive myofibroblasts but rarely present in the hepatocyte nucleus (Fig.1J). Myofibroblast-specific, tamoxifen-inducible YAP/TAZ deficient mice (Pn-Yap/Taz-KO) treated with CCl4 showed strong recombination of both floxed alleles, resulting in loss of YAP and TAZ proteins specifically in the liver myofibroblasts (90.8% in floxed littermates vs. 2.5% in KO co-localization with αSMA+ cell nucleus, Fig.1J). Western blot and RT-qPCR analyses confirmed the loss of YAP and TAZ in myofibroblasts isolated from Pn-Yap/Taz-KO (Fig.1K, N). We next examined the consequences of myofibroblast Yap/Taz knockout in liver fibrogenesis by histopathological H&E, Sirius red and Masson’s trichrome staining. After 6 weeks of CCl4 injections, we did not detect a difference in fibrosis formation between the Pn-Yap/Taz-KO and floxed littermates (Fig. 1L-M). Accordingly, the gross morphology, fibrous portal expansion, necrosis, inflammatory cell infiltration as well as extracellular matrix and collagen deposition were all unaltered in CCl4-treated Pn-Yap/Taz-KO mice (Fig.1L-M). To exclude that our data may be specific to the CCl4 model, we independently confirmed the dispensable role of myofibroblast YAP/TAZ in 2 well-established models of acute liver fibrosis, induced by BDL or IRI (Fig. 1I, L-M). In line with those in vivo observations, there was a comparable expression of the major profibrogenic genes such as αSMA, Col1a1, Col1a2, Fibronectin, Desmin in liver tissue and isolated hepatic stellate cells between Pn-Yap/Taz-KO mice and control littermates (Fig. 1N-O). In conclusion, periostin lineage-tracing identified myofibroblasts in the fibrotic liver and genetic deletion of Yap/Taz in periostin+ myofibroblasts did not affect liver fibrogenesis in vivo. Considering the growing clinical interest in pharmacologically targeting YAP/TAZ to ameliorate fibrosis in multiple organs including the liver, kidney and lung,1Liu Y. Lu T. Zhang C. Xu J. Xue Z. Busuttil R.W. et al.Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injury.J Hepatol. 2019; 71: 719-730Google Scholar, 2Machado M.V. Michelotti G.A. Pereira T.A. Xie G. Premont R. Cortez-Pinto H. et al.Accumulation of duct cells with activated YAP parallels fibrosis progression in non-alcoholic fatty liver disease.J Hepatol. 2015; 63: 962-970Google Scholar, 3Martin K. Pritchett J. Llewellyn J. Mullan A.F. Athwal V.S. Dobie R. et al.PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis.Nat Commun. 2016; 7: 12502Google Scholar, 4Mooring M. Fowl B.H. Lum S.Z.C. Liu Y. Yao K. Softic S. et al.Hepatocyte stress increases expression of yes-associated protein and transcriptional coactivator with PDZ-binding motif in hepatocytes to promote parenchymal inflammation and fibrosis.Hepatology. 2020; 71: 1813-1830Google Scholar, 5Mannaerts I. Leite S.B. Verhulst S. Claerhout S. Eysackers N. Thoen L.F. et al.The Hippo pathway effector YAP controls mouse hepatic stellate cell activation.J Hepatol. 2015; 63: 679-688Google Scholar,8Haak A.J. Kostallari E. Sicard D. Ligresti G. Choi K.M. Caporarello N. et al.Selective YAP/TAZ inhibition in fibroblasts via dopamine receptor D1 agonism reverses fibrosis.Sci Transl Med. 2019; 11Google Scholar, 9Liang M. Yu M. Xia R. Song K. Wang J. Luo J. et al.Yap/Taz deletion in Gli(+) cell-derived myofibroblasts attenuates fibrosis.J Am Soc Nephrol. 2017; 28: 3278-3290Google Scholar, 10Alsamman S. Christenson S.A. Yu A. Ayad N.M.E. Mooring M.S. Segal J.M. et al.Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.Sci Transl Med. 2020; 12Google Scholar our study has direct implications on the therapeutic application of such approaches and raises caution about the extrapolation of previously identified profibrotic or anti-fibrotic potential of YAP/TAZ. It remains to be assessed which cell types are targeted by the broadly used YAP inhibitor verteporfin during liver injury and fibrogenesis. As YAP/TAZ are known to be expressed at a barely detectable level in healthy livers, but are enriched in multiple cell types including myofibroblasts within fibrotic livers from mice and humans, further studies are required to determine the functional consequences and the mechanism of this enrichment. This work was supported by the National Natural Science Foundation of China (grant # 81870220 , # 81822004 , # 92049203 , # 81941005 ), Shaanxi Natural Science Fund for Distinguished Young Scholars of China ( S2020-JC-JQ-0239 ) and the Project of Innovation-driven Plan in Central South University, China (No. 2020CX017 ). L.X. performed experiments, analyzed data and wrote part of the manuscript; N.W., Y.B. and Z.Y. generated Postn-CreERT2 and Yap/Taz flox/flox mice; Y.B., Z.Y. and S.W. designed and supervised the study, analyzed data and wrote the manuscript. All authors commented on the manuscript. We thank Dr. Ying Hao at Instrument Analysis Center for Xi'an Jiaotong University for confocal imaging. All animal protocols were approved by the ethical committee of Xi’an Jiaotong University (XJTU2018-249 and XJTU2019-12). Animals were kept under controlled conditions with a stable room temperature, humidity (45–65%) and a 12 h day/night cycle with unrestricted access to food and water. The data that support the findings of this study are available from the corresponding authors upon request. The authors declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details. The following is the supplementary data to this article: Download .pdf (.18 MB) Help with pdf files Multimedia component 1 Activation of YAP attenuates hepatic damage and fibrosis in liver ischemia-reperfusion injuryJournal of HepatologyVol. 71Issue 4PreviewOrthotopic liver transplantation (OLT) is considered the standard treatment for end-stage liver disease. Following organ retrieval, cold preservation and warm ischemia-reperfusion injury (IRI) can lead to impaired graft function, including primary graft non-function, which may predispose patients to acute and chronic rejection. Indeed, by contributing to a shortage of available donor organs, IRI represents one of the most challenging problems in transplantation.1–4 Thus, novel therapeutic concepts to combat IRI are needed to improve OLT outcomes and expand the donor organ pool. Full-Text PDF Reply to: “Myofibroblast YAP/TAZ is dispensable for liver fibrosis in mice”Journal of HepatologyVol. 75Issue 1PreviewWe thank Dr. Xu et al. for their insightful letter1 and take advantage of this reply to clarify important aspects of our article,2 in which we employed a well-established mouse model of liver ischemia-reperfusion injury (IRI) to show that activation of YAP attenuated oxidative stress and innate immune responses in liver IRI. As activation of YAP prevented hepatocellular damage in liver IRI, it further mitigated IR-mediated liver fibrosis. Full-Text PDF