UPA-Induced EGFR Phosphorylation Is Mediated By TEM8

Recently, targeted disruption of the tumor vasculature has become highlyconcerned in tumor biology and therapeutics. Tumor vascularization provides oxygenand nutrients to tumor occurrence and development. Meanwhile, tumor gain thepotential migration ability through the blood vessels, and thus be able to mitigate itshypoxia condition caused by unlimited proliferation. While angiogenesis is thegermination of existing vasculature caused by the generation of pro-angiogenicproteins and new vascular networks formed by endothelial progenitor cells incirculation. If angiogenesis was inhibited or changed, the tumor could not get thesupply of nutrients from the newborn vasculature, then these tumor tissue will die ofstarvation. Thus, targeting tumor angiogenesis is particularly important for tumortherapy. Moreover, these vascular networks would be affected by the tumormicroenvironment and resulting in altered expression of various proteins, whichmight develop a new factor that could disrupt or block the growth of tumorvasculature. Multiple molecular experiments have found some related overexpressedproteins, among which tumor endothelial marker8(TEM8) is a special one that isoverexpressed in a variety of tumor vasculature.TEM8, one of transmembrane protein type Ⅰ, is highly conserved amongspecies and high-expressed in embryonic cells and the tumor vasculature. It couldpromote tumor-associated angiogenesis and act as the receptor of PA (anthrax toxinprotective antigen) thus mediates anthrax toxin into host cells. However, its exactphysiological function still remains unclear. Some studies have found that TEM8promote tumor cell migration and adhesion, and play a key role in tumor angiogenesis.In vitro studies found that TEM8is important to a variety of endothelial cells, it couldbind extracellular matrix proteins in the extracellular region as well as anthrax toxinprotective antigen. Clinical studies have shown that TEM8overexpressed in tumorendothelial cells but expressed irregularly and far below the tumor endothelium innormal adult tissues. These findings indicated that high expression of TEM8in maturecells is due to abnormal vasculature formation, such as tumor vasculature, whichmeans TEM8could be a candidate target of the therapy of tumor endothelial tissue.TEM8has three natural mutants, among which IsoformⅠwith14potential phosphorylation sites and proline-rich C-terminal cytoplasmic region is consist of563amino acid residues including320residues of extracellular region,23residues oftransmembrane domain and221residues of intracellular region. The extracellularregion of TEM8is primarily formed by a metal ion binding site (MIDAS) with thevWA domain and it is the region of extracellular protein-protein interactions. Besides,it is high homology with integrin domain Ⅰthus indicating that it shares similarphysiological function with integrin. In addition, it is reported that uPA can promotecell adhesion, migration and multiplication by combining integrin, EGFR, etc.In our previous study, an antibody-like molecule TEM8-Fc (a recombinantprotein that comprised the extracellular vWA domain of TEM8and the Fc protein ofhuman IgG1) has been constructed and expressed and it could inhibit angiogenesisand the growth of various tumor cell migration and angiogenesis, but its anti-tumormechanism remains unclear. In former experiments, a new TEM8physiologicalligand–uPA was got by co-immunoprecipitation, it was proved to have a certaininteraction with TEM8and a synergistic anti-tumor effect. Therefore, this studyconceived uPA and TEM8and EGFR interaction which could promote EGFRphosphorylation and active downstream signaling pathways, and made TEM8as ananti-tumor target.In order to verify the interactions among TEM8, uPA, and EGFR, and TEM8iscapable of binding uPA, EGFR to achieve their corresponding physiological functions,TEM8-Fc protein was prepared and detected by ELISA, gene silencing,immunoprecipitation, flow cytometry. Combined with previous laboratoryexperiments, the interaction of uPA and TEM8have been confirmed. Flow cytometryanalysis showed that the wild-type CHO-K1is capable of binding to PE-labeled uPAwhile TEM8-deficient CHO-Pr230could not bind uPA, indicating that uPA binding tothe surface of CHO cells depends on the expression of TEM8. The results of genesilencing exhibits that the TEM8protein silencing greatly reduces uPA bindingcapacity in HepG2and CHO cells, which means TEM8could be considered as antheruPA receptor except uPAR. Immunoprecipitation experiments confirm a certaininteraction between TEM8and uPA and such interaction rely on a dose-response.Non-competitive inhibition ELISA of purified TEM8-Fc protein and uPA implies thatuPA and TEM8showed strong binding force. Meanwhile, under the stimulation ofuPA, immunofluorescence experiments show that colocalization phenomenon occursin TEM8and EGFR，which indicate that uPA promote the interaction between TEM8and EGFR.Based on experiments above, following study was focused on EGFRphosphorylation, detected the phosphorylation and phosphorylation level of EGFRunder the stimulation of EGF and uPA. According to phosphorylation of chips results,uPA enables the up-regulate of phosphorylation levels of phosphorylation sites Y1173and Y845in EGFR. To understand the function of TEM8in phosphorylation of EGFRunder the stimulation of uPA, study firstly focuses on the phosphorylation of EGFRunder the stimulation of EGF, uPA in TEM8, EGFR high expression HepG2cell line.Integrated the phosphorylation level of phosphorylation sites Y845, Y992, Y1045,Y1068, Y1086, Y1173in EGFR, the phosphorylation of WB shows that uPA increasethe phosphorylation level of Y1173, Y845in EGFR particularly a significant increasein Y845phosphorylation site, indicating uPA can enhance phosphorylation of EGFR.Secondly, TEM8, EGFR over-expressing cell lines were built, low EGFR and TEM8expression HEK293F cells were selected and TEM8/EGFR coexpression cell linesTEM8-EGFR/HEK293F was got, by comparing the EGFR phosphorylation level ofTEM8-EGFR/HEK293F and EGFR/HEK293F cell lines, the function of TEM8inEGFR phosphorylation under the stimulation of uPA can be detected.In the study, we found that TEM8can get into the nucleus under the stimulationof uPA, EGF, that means such stimulation contributes to the entry of TEM8proteinsinto the nucleus. To explore whether enter the nucleus is a normal physiologicalfunction of TEM8, and such phenomenon causes what physiological changes, wedesigned following experiments. First, to ensure the entry of TEM8,Immunofluorescence layer sweep experiments have been done and the results showthat10min under the EGF stimulation or15min under the uPA stimulation TEM8canbe detected in nucleus. For further confirmation, nucleocytoplasmic separation hasbeen done in TEM8high expression HepG2cells and it shows that EGF, uPAstimulation can significantly increase the expression level of TEM8in the nucleuswhile a corresponding decrease of TEM8expression was found in the cytoplasm,which can be concluded that TEM8indeed enter the nucleus after stimulation.These study indicate that TEM8as an integrin-like protein could interact withuPA and EGFR and thereby regulating the phosphorylation of EGFR, connect uPAwith EGFR, and as a new uPA receptor mediates the regulation of uPA to thephosphorylation of EGFR.