The effect of integrin a1b1 on Smad2/3 and phosphoSmad2/3 expression in murine chondrocytes

Abstract

Background: Chondrocytes, the cells of cartilage, maintain and repair the extracellularmatrix by secreting and degrading proteins such as collagen type II andaggrecan.1 Integrins are transmembrane adhesion proteins that cells use to both bindto and sense the extracellular matrix. Chondrocytes express many different types ofintegrin molecules and this study focuses on the collagen II and VI receptor integrina1b1. Transforming growth factor-beta (TGF-b) stimulates chondrocytes to repaircartilage through a number of intracellular pathways of which the Smad2/3 pathwayis a known major fibrotic pathway.2 An excess build-up of bony tissue leads to theformation of fibrotic osteoarthritis (OA) in integrin a1-null mice at an earlier age andto a more severe extent than wild type controls.3 Based on these data we hypothesizethat integrin a1b1 controls the sensitivity of chondrocytes to TGF-b. When integrina1b1 is missing chondrocytes are oversensitive to TGF-b, possibly through Smad2/3pathway up regulation. The objective of this experiment is to measure the proteinlevels of Smad2/3 and phosphoSmad2/3 in wild type and integrin a1-null murinechondrocytes. We expect up regulation of phosphoSmad2/3 in integrin a1-null micecompared to wild-type controls.3Methods: Enzymatically isolated chondrocytes from 4-6 month old mice weresonicated and the released protein concentrated through centrifugal filtration. Proteinwas measured through Western-blotting analysis.Results: Integrin a1-null murine chondrocytes contain Smad2.Discussion: Smad2 has been shown in wild-type chondrocytes, but not previously inintegrin a1-null chondrocytes.4 However, Smad2/3 and up-regulated phosphoSmad2/3have been found in integrin a1-null renal medulla cells.2 With further development oftechniques, Smad3 and phosphoSmad2/3 will be measured in wild-type and integrina1-null murine chondrocytes and thus the pathway for fibrotic OA in integrin a1-nullmice can be further determined.