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  • br Materials and methods br Results br Discussion DDR played


    Materials and methods
    Discussion DDR2 played a role in cell motile behavior, but the relationship between motility and collagen activation of DDR2 tyrosine kinase was unclear. Studies with smooth muscle cells from DDR1−/− mice demonstrated a defect in cell attachment to collagen and a loss of cell chemotaxis towards soluble collagen, but only the latter required a functional DDR-kinase domain [15]. Investigators reported that DDR1 was a negative regulator of cell spreading [16], which could be attributed to DDR1 activation of myosin mitomycin [29] or inhibition of α2β1 function [28]. Yet others reported that DDR2 was required for fibroblast migration through matrigel, but the chemotactic stimulus for migration was serum not soluble collagen [18]. Finally, rather than interacting with collagen, the role of DDR1 in cell–cell interaction [30] and collective cell migration in organotypic collagen culture models [17] was found to depend on DDR1 interaction with E-cadherin. We found that silencing DDR2 inhibited BR5 cells spreading and migration regardless whether cells were interacting with fibronectin or collagen-coated 2D surfaces. However, cells attached to either surface did not show DDR2 tyrosine kinase activation unless soluble collagen was added to the medium. The requirement for DDR2 in spreading and migration appeared to be independent of adhesion ligand and collagen-stimulated DDR2 activation but dependent of activation of pFak. Inhibition of cell migration by silencing DDR2 also was observed for human fibroblasts migrating in nested collagen matrices, but collagen matrix contraction by these cells was not inhibited indicating the specificity of the DDR2 silencing effect. Our new observations support previous work comparing cell migration and collagen matrix contraction, which showed that different growth factors and regulatory mechanisms control migration and contraction in 3D collagen matrices [23]. Decreased spreading and formation of mitomycin stress fibers by DDR2 silenced cells could reflect a link between DDR2 activation and of focal adhesion kinase. Other studies have suggested that DDR1 signaling affects myosin function activation [29] or inhibition [17], [29] depending on cell type and experimental assay under investigation. The current findings should be distinguished from work on DDR function in cell behavior is analyzed with and without soluble collagen in the medium. While it is well established that soluble collagen can activate DDR tyrosine kinase [4], [5] and that activation depends on a triple helical binding domain within individual collagen molecules [6]. Indeed, in fibrous connective tissue, it seems unlikely that significant soluble collagen would be present in the normal tissue environment [31]. Regulation of collagen fibrillogenesis does not require the DDR tyrosine kinase domain [13], [14]. The relative ability of collagen fragments and fibrillar structures as activators of DDRs and downstream signally pathways have yet to be studied, will be critical to understanding DDR2 functions depend on collagen-stimulated tyrosine kinase signaling.
    Acknowledgments We are grateful to Drs. Frederick Grinnell, Michael White, Mathew Petroll and William Snell for their many insights and suggestions regarding this work in UT Southwestern Medical Center in Dallas Texas. This research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) (to M.L.H.H.) and National Institutes of Health Grant (NIH)GM31321 (to F.G).
    Introduction Liver fibrosis is a common chronic disease and is related to excessive intrahepatic deposition of extracellular matrix (ECM). Hepatic stellate cell (HSC) is the main source cell of ECM in the liver (1), and its activation is the key factor for liver fibrosis 2, 3, 4. Discoidin domain receptors (DDR) are a new subgroup of the receptor tyrosine kinase family recently discovered 5, 6. DDR can be activated by collagen type I and III of ECM 7, 8, 9, 10. Previous studies showed that the activated HSCs express DDR2 mRNA 11, 12, suggesting that DDR2 may further activate HSC by combining with collagenous fibers, thus participating in the development of liver fibrosis. Little is known about the relationship between DDR2 and liver fibrosis. The purpose of this study was to investigate the role of DDR2 in the pathogenesis of liver fibrosis in a rat model.