Targeting of focal adhesion kinase by small interfering RNAs reduces chondrocyte redifferentiation capacity in alginate beads culture with type II collagen.
Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell-extracellular matrix (ECM) interactions, and chondrocyte-type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte-type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte-specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p-FAK(Y397), and p-ERK1/2. In contrast, expression levels of p-FAK(Y397) and p-ERK1/2, but not p-Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF-beta1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK-siRNA) inhibited mRNA expression of type II collagen and SOX-6 compared to the control. These FAK-siRNA-transfected cells could not recover type II collagen even in the presence of TGF-beta1 or type II collagen in alginate beads culture. We also found that FAK-siRNA-transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation.
Kim
Yun Hee
YH
Brain Korea 21 Project for Medical Sciences and Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, South Korea.
Lee
Jin Woo
JW
D000328:Adult; D000464:Alginates / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D019902:Chondrocytes / Q000166:cytology* / Q000201:enzymology*; D024043:Collagen Type II / Q000378:metabolism*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D020723:Glucuronic Acid / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D008875:Middle Aged; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000378:metabolism*; D051785:Smad Proteins / Q000378:metabolism; D013552:Swine
D000464:Alginates; D024043:Collagen Type II; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D034741:RNA, Small Interfering; D051785:Smad Proteins; D020723:Glucuronic Acid; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases