Experimental Study On The Mechanisms Of DDR1 In The Regulation Of Carvernous Sinus Invasion By Pituitary Adenomas

BackgroundPituitary adenomas is a benign brain tumor which usually lead to severely health problems.Some of them could invade surrounding structures,especially cavernous sinus. The molecular mechanisms that dictate this local invasive behavior of Pas remain poorly understood by now.Few theory and mechanism can explain the discrepancy between biology characteristics and histologic characteristics in invasive pituitary adenomas. Special microenvironment in cavernous sinus is a possibly important factor that contribute to invasion.DDR1,a cell surface receptor,can receive signal from tumor microenvironment,induce autophosphorylation and upregrate expression of MMP-2/9, promote the invasiveness of pituitary adenomas.Thereby,DDR1 may play an impotant role between pituitary adenomas cells and tumor microenvironment.In the present investigation we expressed and purified soluble protein NDDR1,which have a capability to block DDR1 with competition inhibition effection,by eukaryotic expression system and genetic engineering.We used MRI finding as criterion to assess the clinical invasiveness of the pituitary adenomas,Based on Immunohistochemistry founding of large pituitary adenomas sample,NDDR1 and nitotinbib was used to inhibit DDR1 in pituitary adenornas primary cultured cells.Our study aimed to investigate the effects of DDR1 on the regulation of expression of MMP-2 and MMP-9 in pituitary adenomas,and to explore its potential role and mechanism of action of carvernous sinus invasion by pituitary adenomas.Part one:Expression of DDR1 and MMP-2/9 in human pituitary adenomas and their significaneesObjective:To determine the expression of DDR1 and MMP-2/9 in pituitary adenomas and their relationships with biological behaviour of pituitary adenomas;to explore the significances between DDR1 and MMP-2/9.Methods:All adenomas samples were divided into invasion group and non-invasion group according to MRI finding or divided into functional adenomas group and non-functional adenomas group on the basis of clinic manifestation and results of Immunohistochemistry.Cellular localization of DDR1 was detected by confocal microscopy observaion,Immunohistochemistry was used to confirm the expression of DDR1 protein and MMP-2/9 protein.Semiquantitative analysis of DDR1 protein and MMP-2/9 protein was evaluated by Western blot;the expressions of DDR1 mRNA and MMP-2/9 mRNA were also analysised by the method of reverse transcriptase poly merase chin reaction(RT-PCR).Results:1.DDR1 immunoreactivity was located in cellular membrane of pituitary adenomas cells.2.The expression of DDR1 protein and mRNA in invasive pituitary adenomas was significantly higher than in noninvasive pituitary adenomas(P＜0.01);the expression of DDR1 in functional pituitary adenomas was significantly higher than in non-functional pituitary adenomas(P＜0.01);3.There was no significant difference of MMP-2/9 protein and mRNA between functional and non-functional pituitary adenomas(P＞0.05).In invasive pituitary adenomas,there were more expression of MMP-2/9 protein and mRNA than in corresponding noninvasive adenomas(P＜0.01).4.A positive correlation existed between expression of DDR1 and MMP-2(r=0.857,P＜0.01) in functional pituitary adenomas,either between DDR1 and MMP-9(r=0.813,P＜0.01).Conclusions:DDR1 may play an important role in the invasiveness of pituitary adenomas. DDR1 could increase the invasiveness of pituitary adenomas by up-regulating expression of MMP-2/9.Part two:Express and purify of competition inhibition protein(NDDR1) and identify the biologic activity of NDDR1Objective:To express and purify soluble protein(NDDR1) which have a capability to block DDR1 with competition inhibition effection,to identify the biologic activity of NDDR1 and determine the dose-effect relationship between NDDR1 and pituitary adenomas primary cultured cells.Motheds:Extracellular domain of DDR1 was amplify by PCR and an eukaryotic expression plasmid pcDNA3.1-NDDR1 was constructed.Then the plasmid was transiently transfected into 293 cells,purified the expressed protein NDDR1.Competitive blinding inhibition assay by ELISA was used to demonstrate the biologic activity of NDDR1 and determine the optimum dose blocking DDR1 activity of pituitary adenomas primary cultured cells.Results:1.3mg/L protein of NDDR1 expressed and purified by mammalian express system was obtained.2.Competitive blinding inhibition assay demonstrated that expressed NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary culture cells.3.2μg/L of NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary cultured cells.Conclusions:Soluble protein NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary cultured cells with a competitive inhibition effection.Part three:Activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 in pituitary adenomas primary cultured cellsObjective:To determine the collagen subtypes that induces the signal pathway of DDR1 in pituitary adenomas primary cultured cells.To investigate autophosphorylation of DDR1 after DDR1 signal was started.To investigate regulation on MMP-2 and MMP-9 of DDR1. To demonstrate the activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 in cavernous sinus invasion by pituirary adenomas.Motheds:The MMP-2/9 level of the supernatants of primary cultured human pituitary adenoma cells was detectd by ELISA after stimulated by different collage subtypes. Autophosphorylation of DDR1 was evaluated by immuno-precipitation after blocked by NDDR1 and nilotinib.Gelatine zymography was used to detected MMP-2/9 activity and protein expression after DDR1 signal pathway was stimulated or blocked.An invasion model was established by Transwell chambers and cell invasion was observed with microscope after intervention by NDDR1 and nilotinib.Results:Nine cases of functional pituitary adenomas primary cultured cells were obtaind successully.1.CollagenⅠwas the main collagen subtypes that induces the signal pathway of primary cultures cell of pituitary adenomas.2.Autophosphorylation of DDR1 was significant higher after stimulated by collagenⅠ(P＜0.05),NDDR1 and nilotinib could inhibit autophosphorylation of DDR1.3.CollagenⅠstimulation increased the activity and protein expression of MMP-2/9(P＜0.05) and this effection could be blocked by NDDR1 and nilotinb.4.Invasiveness of primary cultured cells decreased after intervention by NDDR1 and nilotinib.Conclusions:CollagenⅠcould increase the invasiveness of pituitary adenomas by inducing autophosphorylation of DDR1 and up-regulating expression of MMP-2/9. blocking DDR1 could decrease the invasiveness of adenomas efficiently.Our results demonstrated that activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 maybe an important factor and mechanism of action in cavernous sinus invasion by pituitary adenomas.Prominent deposition of collagenⅠin cavernous sinus could be one of pathology foundation in cavernous sinus invasion by pituirary adenomas.