ZEB1 protects skeletal muscle from damage and is required for its regeneration

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration

The adaptive antioxidant response during fasting-induced muscle atrophy is oppositely regulated by ZEB1 and ZEB2

Reactive oxygen species (ROS) serve important homeostatic functions but must be constantly neutralized by an adaptive antioxidant response to prevent supraphysiological levels of ROS from causing oxidative damage to cellular components. Here, we report that the cellular plasticity transcription factors ZEB1 and ZEB2 modulate in opposing directions the adaptive antioxidant response to fasting in skeletal muscle. Using transgenic mice in which Zeb1 or Zeb2 were specifically deleted in skeletal myofibers, we show that in fasted mice, the deletion of Zeb1, but not Zeb2, increased ROS production and that the adaptive antioxidant response to fasting essentially requires ZEB1 and is inhibited by ZEB2. ZEB1 expression increased in fasted muscles and protected them from atrophy; conversely, ZEB2 expression in muscles decreased during fasting and exacerbated muscle atrophy. In fasted muscles, ZEB1 reduces mitochondrial damage and increases mitochondrial respiratory activity; meanwhile, ZEB2 did the opposite. Treatment of fasting mice with Zeb1-deficient myofibers with the antioxidant triterpenoid 1[2-cyano- 3,12- dioxool- eana- 1,9( 11)-dien- 28- oyl] trifluoro-ethylamide (CDDO-TFEA) completely reversed their altered phenotype to that observed in fasted control mice. These results set ZEB factors as potential therapeutic targets to modulate the adaptive antioxidant response in physiopathological conditions and diseases caused by redox imbalance.The different parts of this study were independently funded by grants to A.P. from Duchenne Parent Project Spain (DPPE/01_2018), the Catalan Agency for Management of University and Research Grants (AGAUR) (2021- SGR 01328), and the Spanish State Research Agency (AEI) of the Spanish Ministry of Science and Innovation (PID2020- 116338RB- I00) as part of the 2021- 2023 Plan for Scientific and Technical Research and Innovation (PEICTI), which is co- financed by the European Regional Development Fund of the European Union Commission. L.H. is supported by a PhD scholarship from the China Scholarship Council (202208110041

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded by grants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) as part of MICINN’s National Scientific and Technical Research and Innovation 2021-2023 Plan, which is cofinanced by the European Regional Development Fund (ERDF) of the European Union Commission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514

Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1WT/ApoeKO), male mice with Zeb1 ablation in their myeloid cells (Zeb1∆M/ApoeKO) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1∆M/ApoeKO mice display more pronounced systemic metabolic alterations than Zeb1WT/ApoeKO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1∆M/ApoeKO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.S

Regulation of skeletal muscle atrophy by the ZEB1 transcription factor

Muscle atrophy, which is characterized by excessive protein catabolism, is one of the major adaptive processes that occur in several physiopathological and clinical conditions, to counteract stressing stimuli. Skeletal muscle atrophy is triggered by the induction of a group of proteins (atrogenes) that includes components of the ubiquitin–proteasome and autophagy-lysosomal systems. Atrogenes are induced by FOXO transcription factors, but their regulation had not been fully dissected. In this dissertation, it has been studied the role of the transcription factor ZEB1, best known for promoting tumor progression, in muscle atrophy induced by disuse and fasting. It was found that, in both conditions, ZEB1 inhibited muscle atrophy, but through different mechanisms. In disuse-induced atrophy, ZEB1 antagonized FOXO3- mediated induction of atrogenes, while during fasting ZEB1 promoted the expression of NRF1 and NRF2, two important mitochondrial and oxidative stress regulatory genes. During hindlimb immobilization, global Zeb1 heterozygous deletion results in enhanced muscle atrophy and higher expression of a number of atrogenes, including Atrogin-1/Fbxo32 and MuRF1/Trim63. Mechanistically, ZEB1 directly represses in vitro and in vivo Fbxo32 and Trim63 promoter transcription in a stage-dependent manner and in a reverse pattern with MYOD1. ZEB1 binds to the Fbxo32 promoter in undifferentiated myoblasts and atrophic myotubes, but not in non-atrophic myotubes, where it is displaced by MYOD1. ZEB1 represses both promoters through CtBP- mediated inhibition of FOXO3 transcriptional activity. Using a conditional muscle-specific Zeb1 knockout mouse model, it was found that ZEB1 promoted the formation of oxidative slow-type I fibers, through the induction of MEF2C and PGC1ß. During fasting-induced muscle atrophy, the specific knock out of Zeb1 in myofibers induced higher muscle atrophy (Zeb1 KO muscles have an increased number of fibers with lower CSA), lower mitochondrial respiration, due to mitochondrial complex III dysfunction, and higher ROS production. ZEB1 directly binds to Nrf1 and Nrf2 promoters, two key regulatory genes of mitochondrial biogenesis and oxidative stress. Altogether, these results set ZEB1 as a key driver of muscle atrophy, highlighting its importance as a possible new target in therapeutic approaches to clinical conditions causing muscle mass loss.La atrofia muscular, patología caracterizada por una progresiva pérdida de masa muscular, representa uno de los mayores procesos adaptativos a numerosas afecciones fisiopatológicas y clínicas, como el desuso, el cáncer y el ayuno. Estos procesos desencadenan la atrofia del músculo esquelético a través de la inducción de un grupo de proteínas, denominadas atrogenes, que incluyen componentes de los sistemas de degradación autofágico y del complejo proteasoma. Los atrogenes están inducidos por factores de transcripción FOXO, pero su regulación aún no se conoce completamente. En esta tesis se ha estudiado el papel del factor de transcripción ZEB1, mejor conocido por promover la progresión tumoral, en la atrofia muscular inducida por el desuso y el ayuno. En ambas condiciones, ZEB1 inhibe la atrofia muscular pero lo hace a través de diferentes mecanismos. En la atrofia inducida por el desuso, ZEB1 antagoniza la inducción de los atrogenes mediada por FOXO3, mientras que durante el ayuno ZEB1 promueve la expresión de NRF1 y NRF2, dos genes reguladores de la actividad mitocondrial y de la respuesta a estrés oxidativo. La inmovilización de una de las extremidades posteriores en ratones heterocigóticos para ZEB1, desencadena una mayor atrofia muscular y un aumento de la expresión de varios atrogenes, incluidos Atrogin1/Fbxo32 y MuRF1/Trim63. A nivel mecanístico, ZEB1 se une y reprime la transcripción de los genes Fbxo32 y Trim63 in vitro e in vivo de manera dependiente del estado del músculo y con un patrón inverso a MYOD1 (un factor importante en la transcripción muscular). De ésta manera, ZEB1 se une al promotor Fbxo32 en mioblastos indiferenciados y miotubos atróficos, pero no en miotubos no atróficos, donde MYOD1 lo desplaza. Además, ZEB1 reprime los promotores de Fbxo32 y Trim63 a través de la inhibición, mediada por el co-factor CtBP, de la actividad transcripcional de FOXO3. Por otro lado, en un modelo de ratón knock out (KO) para ZEB1 específico de músculo, ZEB1 promueve la formación de más fibras oxidativas de tipo I, a través de la inducción de MEF2C y PGC1ß, que son factores necesarios para la formación de fibras de tipo I y IIx, respectivamente. Durante la atrofia muscular inducida por el ayuno, la ausencia específica de Zeb1 en las miofibras induce una mayor atrofia muscular (los músculos Zeb1 KO tienen un mayor número de fibras con menor diámetro), una respiración mitocondrial más baja, debido a la disfunción del complejo mitocondrial III, y una mayor producción de especies reactivas del oxígeno (del inglés, ROS). ZEB1 se une e induce por unión directa al ADN la transcripción de NRF1 y NRF2, dos genes reguladores clave de la biogénesis mitocondrial y el estrés oxidativo, destacando la importancia de ésta vía durante la atrofia muscular inducida por el ayuno. En conjunto, estos resultados establecen ZEB1 como un impulsor clave de la atrofia muscular, destacando su importancia como un posible nuevo objetivo en los enfoques terapéuticos para las condiciones clínicas que causan la pérdida de masa muscular

Regulation of skeletal muscle atrophy by the ZEB1 transcription factor

[eng] Muscle atrophy, which is characterized by excessive protein catabolism, is one of the major adaptive processes that occur in several physiopathological and clinical conditions, to counteract stressing stimuli. Skeletal muscle atrophy is triggered by the induction of a group of proteins (atrogenes) that includes components of the ubiquitin–proteasome and autophagy-lysosomal systems. Atrogenes are induced by FOXO transcription factors, but their regulation had not been fully dissected. In this dissertation, it has been studied the role of the transcription factor ZEB1, best known for promoting tumor progression, in muscle atrophy induced by disuse and fasting. It was found that, in both conditions, ZEB1 inhibited muscle atrophy, but through different mechanisms. In disuse-induced atrophy, ZEB1 antagonized FOXO3- mediated induction of atrogenes, while during fasting ZEB1 promoted the expression of NRF1 and NRF2, two important mitochondrial and oxidative stress regulatory genes. During hindlimb immobilization, global Zeb1 heterozygous deletion results in enhanced muscle atrophy and higher expression of a number of atrogenes, including Atrogin-1/Fbxo32 and MuRF1/Trim63. Mechanistically, ZEB1 directly represses in vitro and in vivo Fbxo32 and Trim63 promoter transcription in a stage-dependent manner and in a reverse pattern with MYOD1. ZEB1 binds to the Fbxo32 promoter in undifferentiated myoblasts and atrophic myotubes, but not in non-atrophic myotubes, where it is displaced by MYOD1. ZEB1 represses both promoters through CtBP- mediated inhibition of FOXO3 transcriptional activity. Using a conditional muscle-specific Zeb1 knockout mouse model, it was found that ZEB1 promoted the formation of oxidative slow-type I fibers, through the induction of MEF2C and PGC1ß. During fasting-induced muscle atrophy, the specific knock out of Zeb1 in myofibers induced higher muscle atrophy (Zeb1 KO muscles have an increased number of fibers with lower CSA), lower mitochondrial respiration, due to mitochondrial complex III dysfunction, and higher ROS production. ZEB1 directly binds to Nrf1 and Nrf2 promoters, two key regulatory genes of mitochondrial biogenesis and oxidative stress. Altogether, these results set ZEB1 as a key driver of muscle atrophy, highlighting its importance as a possible new target in therapeutic approaches to clinical conditions causing muscle mass loss.[spa] La atrofia muscular, patología caracterizada por una progresiva pérdida de masa muscular, representa uno de los mayores procesos adaptativos a numerosas afecciones fisiopatológicas y clínicas, como el desuso, el cáncer y el ayuno. Estos procesos desencadenan la atrofia del músculo esquelético a través de la inducción de un grupo de proteínas, denominadas atrogenes, que incluyen componentes de los sistemas de degradación autofágico y del complejo proteasoma. Los atrogenes están inducidos por factores de transcripción FOXO, pero su regulación aún no se conoce completamente. En esta tesis se ha estudiado el papel del factor de transcripción ZEB1, mejor conocido por promover la progresión tumoral, en la atrofia muscular inducida por el desuso y el ayuno. En ambas condiciones, ZEB1 inhibe la atrofia muscular pero lo hace a través de diferentes mecanismos. En la atrofia inducida por el desuso, ZEB1 antagoniza la inducción de los atrogenes mediada por FOXO3, mientras que durante el ayuno ZEB1 promueve la expresión de NRF1 y NRF2, dos genes reguladores de la actividad mitocondrial y de la respuesta a estrés oxidativo. La inmovilización de una de las extremidades posteriores en ratones heterocigóticos para ZEB1, desencadena una mayor atrofia muscular y un aumento de la expresión de varios atrogenes, incluidos Atrogin1/Fbxo32 y MuRF1/Trim63. A nivel mecanístico, ZEB1 se une y reprime la transcripción de los genes Fbxo32 y Trim63 in vitro e in vivo de manera dependiente del estado del músculo y con un patrón inverso a MYOD1 (un factor importante en la transcripción muscular). De ésta manera, ZEB1 se une al promotor Fbxo32 en mioblastos indiferenciados y miotubos atróficos, pero no en miotubos no atróficos, donde MYOD1 lo desplaza. Además, ZEB1 reprime los promotores de Fbxo32 y Trim63 a través de la inhibición, mediada por el co-factor CtBP, de la actividad transcripcional de FOXO3. Por otro lado, en un modelo de ratón knock out (KO) para ZEB1 específico de músculo, ZEB1 promueve la formación de más fibras oxidativas de tipo I, a través de la inducción de MEF2C y PGC1ß, que son factores necesarios para la formación de fibras de tipo I y IIx, respectivamente. Durante la atrofia muscular inducida por el ayuno, la ausencia específica de Zeb1 en las miofibras induce una mayor atrofia muscular (los músculos Zeb1 KO tienen un mayor número de fibras con menor diámetro), una respiración mitocondrial más baja, debido a la disfunción del complejo mitocondrial III, y una mayor producción de especies reactivas del oxígeno (del inglés, ROS). ZEB1 se une e induce por unión directa al ADN la transcripción de NRF1 y NRF2, dos genes reguladores clave de la biogénesis mitocondrial y el estrés oxidativo, destacando la importancia de ésta vía durante la atrofia muscular inducida por el ayuno. En conjunto, estos resultados establecen ZEB1 como un impulsor clave de la atrofia muscular, destacando su importancia como un posible nuevo objetivo en los enfoques terapéuticos para las condiciones clínicas que causan la pérdida de masa muscular

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation

Acknowledgements: We are grateful to Dr. F. Sanchez-Madrid (Hospital Princesa and CNIC, Madrid, Spain), Dr. D. Cebrian (CNIC, Madrid, Spain), and Dr. A. Valledor (University of Barcelona, Spain) for helpful insights on early versions of the manuscript. We thank Dr. C. Stephan-Otto Attolini (BIST-IRB, Barcelona, Spain) and Dr. J. Rios (IDIBAPS, Hospital Clinic, and Autonomous University of Barcelona, Barcelona, Spain) for their expert guidance on the statistical analyses of the data in the study. We also thank Dr. L. Ribas de Pouplana (BIST-IRB, Barcelona, Spain) for advice on mitochondrial translation experiments. We acknowledge technical assistance by staff in the Flow Cytometry Unit at IDIBAPS, the Molecular Interactions Services Unit at the Biomedical Research Institute of Bellvitge (IDIBELL), and the Transmission Electron Microscopy Unit at the University of Barcelona School of Medicine. We also thank A Téllez (Hospital Clinic, Barcelona, Spain) for his help in collecting samples from septic patients, and Dr. MJ Fernández-Aceñero (Hospital Clinico San Carlos, Madrid, Spain) for help in collecting skin samples from healthy controls, psoriatic patients, and melanoma patients. We are also thankful to Dr. DC Dean (University of Louisville, KY, USA) for his generous gift of an anti-ZEB1 polyclonal antibody. We thank Dr. A. Garcia for the artistic drawing of schematics in the article. IDIBAPS is partly funded by the CERCA Programme of Generalitat de Catalunya. The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded by grants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) as part of MICINN’s National Scientific and Technical Research and Innovation 2021-2023 Plan, which is co-financed by the European Regional Development Fund (ERDF) of the European Union Commission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514).Funder: Government of Catalonia | Agència de Gestió d&apos;Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)AbstractAcute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.</jats:p

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation

Acknowledgements: We are grateful to Dr. F. Sanchez-Madrid (Hospital Princesa and CNIC, Madrid, Spain), Dr. D. Cebrian (CNIC, Madrid, Spain), and Dr. A. Valledor (University of Barcelona, Spain) for helpful insights on early versions of the manuscript. We thank Dr. C. Stephan-Otto Attolini (BIST-IRB, Barcelona, Spain) and Dr. J. Rios (IDIBAPS, Hospital Clinic, and Autonomous University of Barcelona, Barcelona, Spain) for their expert guidance on the statistical analyses of the data in the study. We also thank Dr. L. Ribas de Pouplana (BIST-IRB, Barcelona, Spain) for advice on mitochondrial translation experiments. We acknowledge technical assistance by staff in the Flow Cytometry Unit at IDIBAPS, the Molecular Interactions Services Unit at the Biomedical Research Institute of Bellvitge (IDIBELL), and the Transmission Electron Microscopy Unit at the University of Barcelona School of Medicine. We also thank A Téllez (Hospital Clinic, Barcelona, Spain) for his help in collecting samples from septic patients, and Dr. MJ Fernández-Aceñero (Hospital Clinico San Carlos, Madrid, Spain) for help in collecting skin samples from healthy controls, psoriatic patients, and melanoma patients. We are also thankful to Dr. DC Dean (University of Louisville, KY, USA) for his generous gift of an anti-ZEB1 polyclonal antibody. We thank Dr. A. Garcia for the artistic drawing of schematics in the article. IDIBAPS is partly funded by the CERCA Programme of Generalitat de Catalunya. The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded by grants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) as part of MICINN’s National Scientific and Technical Research and Innovation 2021-2023 Plan, which is co-financed by the European Regional Development Fund (ERDF) of the European Union Commission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514).Funder: Government of Catalonia | Agència de Gestió d&apos;Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression