Effects Of NRG1/ErbB4 Signaling Pathway On Iron Metabolism Of Muscle Cells In Mice

Iron is an essential trace element in many biological processes,including oxygen delivery,protein synthesis,DNA replication,and oxidative phosphorylation.Maintaining cellular iron homeostasis is one of the key factors for cells to keep normal function.Iron deficiency leads to stagnant growth and even death,while excessive iron produces free radicals that destroy DNA,lipid membranes and proteins.In addition,dysfuction of iron homeostasis is a common cause of various neurodegenerative diseases,hemochromatosis,cancer and other diseases.Therefore,the body must strictly control the cellular iron homeostasis.Neuregulin 1(NRG1)is a trophic factor produced by nerves and muscles.NRG1 acts by binding to a tyrosine kinase(ErbB proteins 28)on the membrane.The ErbB protein contains four subtypes(ErbB1-4).Studies have reported that ErbB2 expression in breast cancer cells exceeds 30%of normal cells,and the iron content of cells also exceeds that of normal cells,and there is a correlation between the two.Previous laboratory work has shown that PV-Cre+/-;ErbB4-/-mice have abnormalities in iron metabolism.To further investigate the mechanism of iron-induced metabolic abnormalities in PV-Cre+/-;ErbB4-/-mice,and the role of the NRG1/ErbB4 signaling pathway in the process of iron metabolism disorders,we performed the following experiments.First,we verified whether the NRGl/ErbB4 signaling pathway affects cellular iron metabolism in PV-Cre+/-;ErbB4-/-mice.Because the number of PV intermediate neurons in the brain accounts for a low proportion of the total number of cells in the brain,we chose peripheral tissues as the research object,we used peripheral tissue of ErbB4-CreERT;tdTomato mice PV performed PV immunofluorescence staining and found a large number of ErbB4 and PV co-expressed in the rectus femoris.Through determination of iron content in the rectus muscle of PV-Cre+/-;ErbB4-/-mice and detection of mRNA levels of iron metabolism-related proteins.It was found that PV-Cre+/-;ErbB4-/-mice had more iron content and TFR mRNA than control mice.The level of expression decreased significantly.We then used embryonic fibroblast cell line(NIH-3T3)cells as an in vitro cell model to further validate whether the NRG1/ErbB4 signaling pathway can affect cellular iron metabolism.In the first step,we treated NIH3T3 cells with NRG1 to detect changes in iron content and mRNA levels of iron-related proteins.The results showed that after 24 hours of treatment with NRG1,the extracellular fluid was significantly lower than that of the control group and a significant increase in cellular iron compare to control group.The mRNA levels of transferrin receptor(TFR),ferritin heavy chain(FTH)and light chain(FTL)were significantly higher than those of the control group,and the level of iron transfer protein(FPN)mRNA was significantly lower than that of the control group.This result indicates that NRG1 treatment promotes the uptake and storage of cellular iron and inhibits the release of cellular iron.This result indicates that NRG1 treated cells can affect cellular iron metabolism.In the second step,we inhibited the expression of Erbb4 in NIH-3T3 cells by transfecting the Erbb4 plasmid(ErbB4-shRNA/con-shRNA)in NIH-3T3 cells,and then detecting the mRNA levels of iron-related protein after 24 hours of treatment with NRG1.The results showed that Erbb4-shRNA can inhibit the effect of NRG 1 on cellular iron metabolism.This result indicates that NRG1 activates its downstream pathway through Erbb4 and thus affects cellular iron metabolism.In this study,we investigated the effects of NRG1/ErbB4 signaling pathway on cellular iron metabolism in PV-Cre+/-;ErbB4-/-mouse muscles and on NIH-3T3 cells,and found that NRG1/ErbB4 signaling pathway affects cellular iron metabolism.To a certain extent,we explained the phenomenon that PV-Cre+/-;ErbB4-/-mice performance abnormal iron metabolism in our previous experiments,and also found a new function of the NRG l/ErbB4 signaling pathway.