The Function And Mechanism Of ?1 Integrin And CD146 In Galectin-3 Induced Vascular Endothelial Cell Migration

Caner carcinoma,as one of the public health porblem in the world,is a great harm to the human health.Angiogenesis plays a central role in the tumor development.Galectin-3,abbreviated as Gal-3,the only chimera member of galectin family,has shown robust bioactivities,especially in promoting angiogenesis.To date,the mechanism of angiogenesis that Gal-3-induced was unclear,although few receptors have been reported.Endothelial cell migration is a key step in angiogenesis.In the current study,we used human epidermal microvascular endothelial cells(HMEC-1)to study the influence of Gal-3 to the cell migration in the aspects of functional receptors and associated mechanism.Results and innovation points were as follows:Firstly,the effect of Gal-3 and different truncated proteins to the migration of HMEC-1 was examed by transwell assay.Our study showed for the first time that Gal-3(1-250 aa)promoted HMEC-1 migration in a carbohydrate-dependent manner,and the C-terminal domain of Gal-3,i.e carbohydrate recognition domain(111-250aa,CRD)also obviously stimulated the cells migration in a same manner.However,the N-terminal domain has no influence to the HMEC-1 migration.Further studies showed that the CRD-mediated cell migration was dose-dependent.Additionally,both full-length Gal-3 and its CRD immobilized onto the transwell insert triggered cell migration,similarly to their counterparts in solution in the transwell chamber.Secondly,we confirmed the?1 integrin as the functional receptor of Gal-3-and CRD-induced HMEC-1 migration for the first time.Using immunoprecipitation assay combined with mass analysis,we found that?1 integrin has a high abundant reach to85%and the match peptides number was 17.Furthermore,using pull down assay,we confirmed that Gal-3 and CRD interacted with?1 integrin not only in the cell lysate,but also on the cell surface.Knocking down the expression of?1 integrin,the migration of HMEC-1 induced by Gal-3 and CRD was significantly decreased by nearly 60%.The same result was also obtained when using?1 integrin functional blocking antibody TDM29,which inhibited Gal-3-and CRD-mediated cell migration about 50%.Thridly,we screened the?integrin subunit that complexed with?1 integrin to mediate the Gal-3-and CRD-induced HMEC-1 cell migration in three aspects:1)knocking down the expression of?1 integrin decreased the?1,?2,?5 and?6 intergrin protein expression,and altered the glycosylation statue of?3 and?4;2)these six?subunits interacted with Gal-3 not only in the cell lysis,but also on the cell surface;3)The functional blocking antibodies of these?integrin subunits were used.Results showed that?2,?4,?5 and?6 functional blocking antibodies inhibited the Gal-3-triggered HMEC-1 migration,however,?2,?4 and?6 functional blocking antibodies inhibited the migration that CRD-induced.Moreover,the?2?1 integrin blocking antibody BHA2.1 significantly inhibited both Gal-3-and CRD-induced cell migration.We further studied the downstream signaling pathway,results showed that the FAK inhibitor PF573228 inhibited both Gal-3-and CRD-induced cell migration.Additionally,knocking down the expression of?1 integrin obviously attenuated the phosphorylation of FAK stimulated by Gal-3 and CRD.Finally,we also found the other glycoprotein,i.e CD146 that has a relatively high abundance(31.2%)and peptides match(15).Knocking down the expression of CD146,the migration of Gal-3-induced was significantly reduced.Furthermore,using pull down assay,we found that Gal-3 directly interacted with CD146 in a carbohydrate-dependent manner.Additionally,the binding affinity between Gal-3and CD146 was confirmed by biolayer interferometry,giving an apparent dissociation constant(K_D)of 1.1?M.Further studies showed that CD146 was a dimer in solution in a Domain 5-independent manner when assayed with native-PAGE and gel filtration.Using CD146 extracellular truncated mutants,we found that Gal-3 interacted with CD146 mainly dependent upon the extracellular Domain 5.In addition,we identified4 N-glycosylation sites,mapping in the 56,418,449,544 and 9 O-glycosylation sites,which are located in the 104,242,249,283,298,299,349,413,557 about CD146ecto domain analysed with mass spectrometry.In summary,we studied the effect of Gal-3-and CRD-induced HMEC-1 cell migration and related mechanism and identified that Gal-3 and CRD promoted cell migration through binding to and activating?1 integrin for the first time.Moreover,we also for the first time confirmed that CD146 was a functional partner to mediate the Gal-3-induced cell migration and the interaction between CD146 glycosylation and Gal-3.These results will provide theoretical basis for elucidating the mechanism of Gal-3 promoting tumor angiogenesis and developing drug targeting Gal-3.