RESUMO
Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoyl-coenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD.
Assuntos
Receptores ErbB/antagonistas & inibidores , Fast Foods , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers. CONCLUSION: MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711-1724).
Assuntos
Receptores ErbB/fisiologia , Falência Hepática/etiologia , Regeneração Hepática/fisiologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Animais , Masculino , Camundongos , Transdução de SinaisRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) is the most commonly diagnosed form of liver cancer with high morbidity and mortality. Copy number variation (CNV) analysis of human HCC revealed that leukocyte-specific protein 1 (LSP1) had the highest number of cases with CNV. LSP1, a F-actin-binding protein, is expressed in hematopoietic cells and interacts with kinase suppressor of Ras (KSR), a scaffold for the extracellular signal-related kinase/mitogen-activated protein kinase pathway. Expression of LSP1 in liver, and its role in normal hepatocellular function and carcinogenesis, remains unknown. Therefore, LSP1 messenger RNA and protein levels were analyzed in normal hepatocytes in culture, rat liver following partial hepatectomy (PHx), and hepatoma cell lines. In culture and after PHx, LSP1 increased after the termination of hepatocyte proliferation. To investigate LSP1 function in HCC, short hairpin RNA was utilized to stably knock down LSP1 expression in the JM1 rat hepatoma cell line. Loss of LSP1 in JM1 cells resulted in dramatic up-regulation of cyclin D1 and phosphorylated ERK2, increased cell proliferation, and migration. Coimmunoprecipitation and immunofluorescence analysis displayed an interaction and colocalization between LSP1, KSR, and F-actin in JM1 cells and liver during regeneration. Conversely, expression of LSP1 in the JM2 rat hepatoma cell line led to decreased proliferation. Enhanced expression of LSP1 in mouse hepatocytes during liver regeneration after injection of an LSP1 expression plasmid also led to decreased hepatocyte proliferation. CONCLUSION: LSP1 is expressed in normal hepatocytes and liver after PHx after termination of proliferation. In rat hepatoma cell lines and mouse liver in vivo, LSP1 functions as a negative regulator of proliferation and migration. Given the high frequency of LSP1 CNV in human HCC, LSP1 may be a novel target for diagnosis and treatment of HCC.