title: Inhibition of human scleral fibroblast cell attachment to collagen type I by TGFBIp.
abstract: OBJECTIVE
  Transforming growth factor beta-induced protein (TGFBIp; 68 kDa) is a secreted extracellular matrix (ECM) protein that has been demonstrated to regulate cell attachment in a variety of cell types. The sclera synthesizes and secretes TGFBIp, which may function to facilitate scleral ECM remodeling events associated with myopia development. Here the authors report that human scleral fibroblasts (HSFs) express TGFBI and that its protein product, TGFBIp, mediates an effect on cell attachment.
  
  
  METHODS
  TGFBI/TGFBIp expression was evaluated by RT-PCR and immunoblot of HSF lysates and culture supernatants. The effect of rTGFBIp (50 microg/mL) on cell attachment to collagen type I was determined with the use of fluid-phase cell attachment assays in HSFs, human foreskin fibroblasts (HFFs), and human corneal stroma fibroblasts (HCFs). Binding assays using biotinylated rTGFBIp were used to assess TGFBIp binding to the HSF surface. Flow cytometry and immunocytochemistry were used to determine both alphavbeta3 and alphavbeta5 expression and localization to the HSF cell surface.
  
  
  RESULTS
  HSFs expressed TGFBI and secreted TGFBIp (approximately 833 ng/h). rTGFBIp significantly decreased (25 microg/mL; P <or= 0.05) HSF attachment to collagen type I, whereas rTGFBIp did not significantly affect cell attachment of HFFs (P = 0.50) or HCFs (P = 0.24) to collagen compared with BSA. Integrins alphavbeta3 and alphavbeta5 were detected on the cell surface, and both anti-alphavbeta3 and anti-alphavbeta5 functionally blocked rTGFBIp binding to HSFs.
  
  
  CONCLUSIONS
  TGFBIp plays an inhibitory role in HSF attachment to collagen type I in vitro through interactions with alphavbeta3 and alphavbeta5 integrin receptors. These results suggest that TGFBIp may modulate scleral cell-matrix interactions in vivo, thereby affecting scleral viscoelasticity.
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
authors.forename: Lilian
authors.initials: L
authors.lastname: Shelton
authors.forename: Jody A Summers
authors.initials: JA
authors.lastname: Rada
mesh_terms: D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology; D016326:Extracellular Matrix Proteins / Q000494:pharmacology* / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D052816:Foreskin / Q000166:cytology; D015870:Gene Expression; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008297:Male; D019030:Receptors, Vitronectin / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D000099222:betaIG-H3 Protein
chemical_list: D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; D039302:Integrin alphaVbeta3; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C064660:integrin alphaVbeta5