Mechanism Of Gαi1 Regulating MAP4-induced Epidermal Cell Migration Through Akt/mTORC Pathway Under Hypoxia

Background and Objectives:Wound healing and tissue repair have always been one of the hotspots and difficulties in medical research.Wounds are also the most common diseases encountered in clinical plastic surgery.To promote the repair of skin injury is the main task of our treatment.With the penetration of high-tech such as biotechnology and molecular biology,researchs on wounds repair has entered a new phase.Although some progress has been made in basic research and treatment of various types of wounds,many problems still remain unresolved.The natural healing process after skin injury mainly includes granulation tissue formation and re-epithelialization of the wound.During the re-epithelialization of the wound,the migration of epidermal cells is a key step in wound healing.The dynamic process includes four steps:1.extension,2.adhesion,3.contraction,4.dissociation,involving the interaction of epidermal cells with extracellular matrix,skeleton reorganization,asymmetric division and proliferation（Asymmetric）Divisions)etc.The process is complex and its specific mechanism is still unknown,which is the focus of wound healing research in the world.We explore the regulatory molecules and mechanisms which affects epidermal cell migration after skin injury,it can further elucidate the mechanism of wound healing and it plays an important role in promoting skin wound healing after effective intervention,providing new methods and strategies in various types of wound healing and tissue repair clinically.Current research argues that the blood vessels will rupture and the related cells are under high oxygen consumption after the skin tissues were injuried,and it will make the wound microenvironment in low oxygen（Hypoxia）,or a lack of oxygen（Anoxia）status and futher promote the edge and basal cells to secrete a variety of cytokines,including transforming growth factor（TGF）,cutin cell growth factor（KGF）,vascular endothelial growth factor（VEGF）,platelet-derived growth factor（PDGF）and so on.These cytokines play an active role in promoting cell migration and the healing of the wound.Reseachers have found that hypoxic（or anoxia）condition can promote the migration of wound epidermal cells and plays a positive role in promoting wound healing^[6].However,the mechanism of hypoxia promoting the migration of epidermal cells and re-epithelialization of the wound remains remains unclear.Cell migration is a complex cytological behavior,and each step of migration contains changes in cytoskeleton,especially in microtubule dynamics,which requires a series of intracellular signal transduction and regulation to precisely regulate microtubule dynamics.Studies have shown that cytoskeletal structures such as microtubules are destroyed in hypoxic cells such as tumors.Hypoxia can increase VEGF expression and is regulated by microtubule regulatory protein Stathmin.Hypoxic environment can affect cytoskeletal changes and promote the migration of vascular endothelial cells,playing an important role in angiogenesis^[9,10].With the support of the national natural science foundation of China（project number:81101436）,we have studied the effect of hypoxia condition to the cell migration of epidermal cells（HaCaT cells）,the results show that the cytoskeleton of epidermal cells（HaCaT cells）in the low oxygen（1%oxygen concentration）has been changed in a short period of time,and the cell migration ability was promoted.These results suggest that the hypoxic environment after skin injury can promote epidermal cell migration by regulating microtubule dynamics.Microtubule associated proteins（microtubule-associated proteins,MAPs）which are associated with microtubule dynamics stability and dimeric Kinesin,Stathmin family which are associated with microtubule depolymerization can change the stable microtubule/depolymerization balance,playing a variety of biological functions including cell migration in hypoxia under stress stimulation and other pathophysiological conditions.Our study also found that the expression of the major microtuble-associate protein 4（MAP4）in non-neural cells changed in the hypoxic environment,and this change trend was consistent with the changes in cytoskeletal status and cell migration.It was shown that hypoxic state can regulate MAP4,which further affects microtubule kinetics and epidermal cell migration.In previous studies,it has been confirmed that MAP4 can affect the migration ability of HaCaT cells by regulating the expression of tctex-1 in a hypoxic environment,but the specific signaling molecule that controls MAP4 to regulate the migration of epidermal cells remains unclear.Hypoxia,as an extracellular signal,is transmitted to the cell through the pathway of"cell membrane--intracellular signal--MAP4--cytoskeleton change and migration ability change".The process begins by sending signals into the cell via cell membrane receptors.As an important membrane protein receptor family,G Protein Coupled receptor（G Protein Coupled Receptors,GPCRs）can perceive oxygen tension,hormones and neurotransmitters and chemokines etc.to participate in a variety of intracellular signal transduction process.After external stimulation,GPCRs will undergo conformational changes,exchanging GTP with GBP（GDP）on G protein,and activating G protein subunit to participate in the next signal transmission process.According to the functional characteristics of G subunit,it is divided into Gαs,Gαi,Gαq and Gα12.Studies have shown that Gαi1 and Gαi3 proteins may be involved in cell migration,which has been confirmed in recent tumor cell studiesTo sum up,this study aims to simulate the local hypoxic microenvironment after skin injury,and observe the effect of Gαi1 on cell migration by regulating the expression of microtubule dynamics of human immortalized epidermal cells（HaCaT cells）in hypoxic environment in vitro experiments,and further to study the specific mechanism.On the basis of previous studies,we conduct a retrospective and descriptive analysis of the factors affecting the curative effect of wound surgery by collecting clinical data,and conclude the specific factors affecting the curative effect of wound surgery in clinical practice.We further explore the specific molecules and mechanisms which influence wound healing key steps.By establishing a HaCaT cells related to hypoxia model,using gene chip technology,In vitro cell scratch test,live cell workstation test,protein immunoblot（western blot,WB）technology and immunofluorescence staining（immumofluorescence,IF）and other experimental technology and methods to systematically observe the expression changes of MAP4 in HaCaT cells and the changes of cell microtubule dynamics,the influence on cell migration ability,as well as the relationship between Gα_i1 and MAP4,and to explore whether they affect the migration ability of HaCaT cells through Akt/mTORC signaling pathway under hypoxic conditions.Methods:Part I systematically observing and confirming the hypothesis that hypoxic microenvironment promotes epidermal cell migration1.Establishing the epidermal cells of low oxygen treatment model and the system of cell culture methods:the immortalized epidermal cell lines（HaCaT cells）were cultured in oxygen and hypoxic condition respectively,the method of low oxygen training was to put the cells into American Thermo hypoxic training incubator（1%oxygen and 5%carbon dioxide,94%nitrogen）,which was a more simple,efficient and reliable method than using gas tank of oxygen device and cells grow well.2.In vitro cell scratch test and living cells workstation are used to observe and analyze the epidermal cells’ability to migrate in hypoxia conditions:putting HaCaT cells in hypoxia training incubator（1%oxygen and 5%carbon dioxide,94%nitrogen）for 24 h hypoxic culture,using two kinds of cell migration in vitro experimental model（In vitro cell scratch test and living cells workstation）system to observe HaCaT cells’ability to migrate in the low oxygen environment.All the experimental data obtained were expressed as mean standard deviation（x±s）.SPSS12.0 statistical software was used for statistical analysis.The statistical method was univariate anova,and the comparison between groups was t-test.P<0.05 was considered as significant difference,while P>0.05 was considered as no statistical significance.Part II using gene chip technology to detect the differences in transcriptome expression between epidermal cells treated with hypoxia and normoxia to futher find out the major genes and molecular signaling pathways that affect epidermal cell migration by regulating MAP4 in a hypoxic microenvironment1.All groups of HaCaT cells were put into low-oxygen incubators for 24h respectively,and RNA of each group was collected which was then sent to a specialized biotechnology company to detect the expression differences of transcriptome between cells using gene chip technology.2.GO and Pathway analysis were carried out on the gene expression differences of various transcriptome between the different groups cells detected by gene chip technology to find out the major genes and molecular signaling pathways that affect the epidermal cell migration through the regulation of MAP4 in the hypoxic microenvironment.Part III systematically observing the expression changes and interactions of key proteins Gα_i1 and MAP4 in HaCaT cells under hypoxic microenvironment,and the effects on cell migration.1.Setting 4 different hypoxic cell culture time points:0.5h,1h,3h and 24h,WB experiment was used to observe the expression of Gα_i1 and MAP4 in HaCaT cells.2.The company constructed Gα_i1 low expression vector,transfecting HaCaT cells,and cultured and screened stable Gα_i1 low expression HaCaT cell model.The cryopreserved MAP4 cells with low expression of HaCaT in the pre-resuscitation study were recovered.It was divided into three groups including normal group,the Gα_i1 low expression group and the MAP4 low expression group.The interaction between Gα_i1 and MAP4 in the HaCaT cells was observed by WB experiment under the condition of low oxygen culture.The time point of cell hypoxia culture was the same as above.At the same time,cell migration was observed by In vitro cell scratch test and living cells workstation.Part IV systematicly observing of Gα_i1 regulation of epidermal cell migration under hypoxic conditions of MAP4 expression by Akt/mTORC signaling pathway1.The company constructed Gαi1 high expression vector and Gαi3 high/low expression vector,transfected HaCaT cells,and cultured and screened Gαi1 high expression vector HaCaT cells and Gαi3 high/low expression vector HaCaT cells model.The cells were divided into three groups,including the normal group,the Gαi1 high/low expression group and Gαi3 high/low expression group,the cells hypoxia cluturing time was same as above.By In vitro cell scratch test,Transwell cell migration and living cells workstation to observe the migration ability of all cells in different groups under hypoxic conditions.In addition,the proliferation ability of primary cell culture was observed and analyzed after Gαi1/3 was interfered.2.The normal group,Gαi1 low expression group and high expression group were set,and the cell hypoxia culture time was the same as above.The expression of Akt/mTORC signaling pathway-related protein in the hypoxic microenvironment was analyzed by WB assay.After further treatment with PI3K/Akt/mTORC signaling pathway inhibitors,WB assay and In vitro cell scratch test were used to analyze the expression of major proteins in PI3K/Akt/mTORC signaling pathway in hypoxic microenvironment.Changes in cell migration.Results:1.The hypoxic culture model and scientific culture method of human immortalized epidermal cells（HaCaT cells）were re-improved and successfully established.2.The results of In vitro cell scratch test and living cells workstation showed that the migration ability of HaCaT cells were enhanced under hypoxic conditions（1%oxygen,5%carbon dioxide,94%nitrogen）（P<0.01）.3.We have detected and analyzed the different expression of each transcriptome in epidermal cells after hypoxia treatment by using gene chip technology,and have found out the major genes and molecular signaling pathways affecting epidermal cell migration in hypoxic microenvironment:G proteinαsubunit,MAP4 and Akt/mTORC signaling pathways.4.After HaCaT cells were clutured with hypoxia,the results of WB showed that the expression of Gαi1 and MAP4 in HaCaT cells increased transiently in the early stage（0.5h-1h）,with the prolongation of hypoxia treatment time（3h-24h）,the expression of both Gαi1 and MAP4 will decrease rapidly,and the trend is similar（P<0.01）.5.The high and low expression vectors of Gαi1/3 were successfully established and transfected into HaCaT cells,and the stable Gαi1 high/low expression HaCaT cell model was established.The results of WB assay showed that the expression of interfering Gαi1would affect the expression of MAP4 and Tctex-1 in HaCaT cells.Conversely,interfering the expression of MAP4 also decreased the expression of Gαi1 and Tctex-1,and there was an interaction relationship between them.Further experiments on In vitro cell scratch test and living cells workstation showed that HaCaT cells were significantly inhibited after normal culture and hypoxia treatment by interfering MAP4 expression.Conversely,HaCaT cells’migration ability will also be suppressed after interference with MAP4 expression.6.After HaCaT cells were cultured in hypoxia condition for 24h,the expression of Akt,GSK-3β,S6,4E-BP1 in the Akt/mTORC signaling pathway in HaCaT cells of the Gαi1interference group was decreased compared with the normal HaCaT cells,and the cells migration ability was significantly decreased.After further inhibiting the Akt/mTORC signaling pathway by the inhibitor,it was found that the expression of Tctex-1 in HaCaT cells was also decreased,and the cell migration ability was significantly decreased.Conclusions:Hypoxia microenvironment can regulate the expression of Gαi1 in HaCaT cells,which can enhance the migration ability of cells.The mechanism may be that Gαi1 can regulate the expression of MAP4 through Akt/mTORC signaling pathway,which in turn affects the expression of Tctex-1 and changes the cellular microtubule dynamics to jointly affect the migration of epidermal cells.