Expression And Anti-apoptosis Effects Of Heregulin/ErbB Signal Conduction System In Mesenchymal Stem Cells

Despite the advances made in the management of acute myocardial infarction, the effects of drug treatment for congestive heart failure secondary to ventricular remodeling following infarction are limited.Nowadays,Bone marrow Mesenchymal stem cells(MSCs)transplantation is a promising strategy to promote structural and functional repair in the heart via stem cell plasticity,Instead of or besides the replacement of lost cardiomyocytes by transdifferentiated MSCs,the secretion of paracrine factors by MSCs,which exert beneficial effects on surrounding cells,may be important for functional improvement.However,this approach is hampered by their poor viability after transplantation,the majority of MSCs are dead within four days after grafting into injured hearts.Ischemia is the main reason for stem cell death in ischemic heart.Therefore,cytoprotection is critical for successful cellular therapy.Neuregulins(NRG)are a family of growth and differentiation factors related to epidermal growth factor,whose receptors are the ErbB family of tyrosine kinase transmembrane receptors,including the ErbB2,ErbB3,and ErbB4 receptors. Alternative splicing of the NRG1 gene results in transcripts encoding 3 major isoforms.Typeâ… NRG1,also known as heregulin(HRG),a 44kD glycoprotein,is expressed predominantly in neural tissue,respiratory epithelium,and endocardium. Through interaction with ErbB receptors,heregulin can promote the survival of epithelial cells,neuron,glial cells,cardiomyocytes,and other cell types.HRG can also induced the expression of VEGF and stimulate angiogenesis.The aim of this study was to detect the expression of HRG and ErbB receptors in MSCs,and investigate whether HRG can promote the survival of MSCs.Firstly,we detected the expression of HRG and ErbB receptors in MSCs.In MSCs transplantation,MSCs undergo Ischemia surrounding,so we also tested the changes of HRG and ErbB receptors under serum deprivation and hypoxia(SD/hypoxia) conditions,which are both components of ischemia in vivo.Secondly,after Knowing the expression of ErbB receptors in MSCs,we tested the effects of heregulin on the apoptosis of MSCs induced by SD/hypoxia.Partâ… Expression of Heregulin and ErbB receptors in mesenchymal stem cellsMethods:According to the density difference of cell,MSCs are separated by gradient centrifugation on Ficoll-Hypaque(1.077 g/ml)from bone marrow of male SpragueDawley rats.After isolation,MSCs were cultured and passaged,Surface molecules marker of passages 3 MSCs were examined with Flow cytometry analyses.The cells passaged 3 were used in all experiments.The expression of ErbB receptors and Heregulin was tested under normal and SD/hypoxia conditions by using regular RT-PCR,quantitative Real-Time PCR,immunofluroescence,and western blot Analysis.To test the secretion of HRG,the conditioned medium of MSCs was collected and concentrated,and then Analyzed by Western Blot.Results:1.The shape and characteristic of MSCsPassages 3 MSCs were fusiform and fibroblast-like adherent cells,there were almost no contaminating cells under light microscope,MSCs were shown as a homogeneous cell population.Surface molecules marker of passages 3 MSCs were examined using Flow cytometry analyses.Most adherent cells expressed adhesion molecules CD44 and CD90.In contrast,the majority of MSCs were negative for hemopoietic cells marker CD45,which conform to the characteristics of MSCs.2.The mRNA expression of ErbB receptorsBy using either regular RT-PCR or quantitative Real-Time PCR,three ErbB receptors mRNA were detectable in MSCs,although the mRNA expression level of ErbB4 was lower than that of ErbB3,and the mRNA expression level of ErbB3 was lower than that of ErbB2.All three ErbB receptors mRNA expression were significantly down-regulated by SD/hypoxia.By immunofluroescence technique,the expression of ErbB4 receptor was detected at a low level.3.The expression and secretion of HRGHRG was expressed either at mRNA or protein level in MSCs,and SD/hypoxia could increase the expression of HRG,SD/hypoxia more than 12 hours could induce the secretion of HRG.Partâ…¡Heregulin protects mesenchymal stem cells from serum deprivation and hypoxia-induced apoptosisMethods:The cells were washed with DMEM twice and placed in DMEM,then Tyrphostin AG1478 10uM(ErbB3,ErbB4 receptors inhibitor),AG825 10uM(ErbB2 receptor inhibitor),Wortmannin 0.2uM(PI3K inhibitor)and HRG(5-100ng/mL)were added according to the need of tests.After 1 hour,the cells were incubated in a modular incubator chamber and infused with mixed gas(95%N₂ and 5%CO₂)at 37℃until a specific time-point.O₂ concentration is lower than 0.5%.Apoptosis was detected using the TUNEL and Hoechst test.The changes of PI3K/Akt pathway,MAPK(ERK, JNK,P38)pathway,Bcl-2,Bax and Caspase3 were tested by Western blot analysis.Results:1.HRG protected against apoptosis induced by SD/hypoxiaSD/hypoxia caused a significant increase in apoptosis compared with control group,for 18 hours(22.96±0.97%vs 2.89±0.62%,p＜0.01),for 12 hours (14.41±1.61%vs 2.89±0.62%,p＜0.01).For choosing an appropriate concentration of HRG,we detected the effects of different concentrations of HRG on the apoptosis induced by SD/hypoxia for 18 hours.We found HRG could decrease the apoptosis induced by SD/hypoxia,and the effects were dose-depended from 10-50ng/ml. 50ng/ml could achieve the maximal protective effects.The effects of HRG on the apoptosis induced by SD/hypoxia for 18 hours or 12 hours were similar,a significant decrease from 22.96±0.97%to 12.85±0.91%in 18 hours and from 14.41±1.61%to 8.59±0.54%in 12 hours(p＜0.01).AG1478 and AG825 could block the anti-apoptosis effects of HRG2.HRG induced the phosphorylation of Akt and ERKSD/hypoxia significantly inhibited the phosphorylation of Akt(Ser473)and significantly activated the phosphorylation of ERK(Tyr204)in comparison with control group(p＜0.01).HRG significantly increased the phosphorylation of Akt and ERK,but combination with AG1478 or AG825,these effects were disappeared. However,the phosphorylation of JNK(Thr183/Tyr185)and p38(Thr180/Tyr182) was not affected by SD/hypoxia or HRG3.HRG increased the Bcl-2/Bax ratio and decreased the caspase3 activation SD/hypoxia significantly decreased the Bcl-2/Bax ratio(p＜0.01)and significantly activated the caspase3(p＜0.01).but treatment with HRG significantly increased the Bcl-2/Bax ratio and significantly inhibited the activation of caspase3 induced by SD/hypoxia.These effects of HRG could be blocked by Wortmannin, indicating that PI3K/Akt pathway take critical roles in the effects of HRG against MSCs apoptosis induced by SD/hypoxia.Conclusions:MSCs can express all three ErbB receptors and HRG,and SD/hypoxia significantly decrease the mRNA expression of ErbB receptors,significantly increase the expression of HRG and activate the secretion of HRG.HRG can decrease the apoptosis of MSCs induced by SD/hypoxia through the activation of PI3K/Akt and ERK pathway,and the increase of Bcl-2/Bax ratio and the inhibition of caspase3 activation.