title: The P2X(7) receptor regulates proteoglycan expression in the corneal stroma.
abstract: OBJECTIVE
  Previously, the authors demonstrated that the lack of the P2X(7) receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X(7) receptor on components of the corneal stroma extracellular matrix.
  
  
  METHODS
  Unwounded corneas from P2X(7) knockout mice (P2X(7) (-/-)) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma.
  
  
  RESULTS
  P2X(7) (-/-) mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X(7) (-/-) mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X(7) (-/-) compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X(7) (-/-) mice, indicating that there are more heparan sulfate proteoglycans in the latter.
  
  
  CONCLUSIONS
  Our results indicate that loss of P2X(7) alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma.
authors.affiliation: ECI Biotech, Worcester, MA, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Mankus
authors.forename: Cheryl
authors.initials: C
authors.lastname: Chi
authors.forename: Celeste
authors.initials: C
authors.lastname: Rich
authors.forename: Ruiyi
authors.initials: R
authors.lastname: Ren
authors.forename: Vickery
authors.initials: V
authors.lastname: Trinkaus-Randall
mesh_terms: D000818:Animals; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D058575:Decorin / Q000378:metabolism; D024022:Fibrillar Collagens / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation; D006025:Glycosaminoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D007632:Keratan Sulfate / Q000378:metabolism; D051379:Mice; D011509:Proteoglycans / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D058486:Receptors, Purinergic P2X7 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D058575:Decorin; D024022:Fibrillar Collagens; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D007632:Keratan Sulfate; D011509:Proteoglycans; D012333:RNA, Messenger; D058486:Receptors, Purinergic P2X7; C013786:A73025