Another notable finding in this study is that
Another notable finding in this study is that Cbl-b-mediated ubiquitination accelerates the degradation of phosphorylated DDR2. Cbl family has been documented to facilitate RTK degradation either in proteasome through K48-linked poly-ubiquitination or in lysosome through mono-, multiple mono- and K63-linked poly-ubiquitination , , , , . In this study, although we could not distinguish whether DDR2 is multimono- or poly-ubiquitinated by Cbl-b, we showed that ubiquitinated DDR2 can be stabilized by proteasome inhibitor MG-132, rather than a lysosome inhibitor, NH4Cl. This suggests that the proteasome pathway is involved in processing ubiquitinated DDR2. However, because proteasome inhibitors might affect lysosome function by interfering with ubiquitin recycling or lysosome proteins turnover , we cannot completely rule out the possibility of endocytic trafficking and lysosome degradation of DDR2.
Our present study showed that Cbl-b promotes DDR2 ubiquitination and attenuates collagen II-induced MMP-13 Cy5 azide in pre-osteoblasts and synovial fibroblasts, respectively. Previously, we and others demonstrated that collagen II–DDR2–MMP-13 axis may contribute to articular cartilage destruction in arthritis , . These observations collectively suggest that Cbl-b abundance may impact DDR2-related pathogenesis. Indeed, we documented that Cbl-b−/− mice exhibit synovial hyperplasia and erosion of cartilage in a collagen-induced autoimmune arthritis model . Although such phenotype could be partially explained by aberrant immune response  and increased osteoclasts activity  caused by Cbl-b deficiency, our current study can provide additional mechanism from the aspect of synovial fibroblasts. Thus, the appropriate Cbl-b level contributes to appropriate DDR2 signaling and the functional consequence.
Acknowledgments The authors would like to thank Dr. Yuan Shao and Chris Elly (La Jolla Institute for Allergy and Immunology) for providing the constructs of Cbl, Cbl-b and Cbl-b WA and Dr. Haining Huang (La Jolla Institute for Allergy and Immunology) for technique assistances. This work has been supported by Chinese National Key Basic Research & Development Program (2010CB529705), grants from National Natural Science Foundation of China (30972723, 30800492 and 30901335) and China Scholarship (20063037).
Introduction The extracellular matrix (ECM), the insoluble material in tissues which provides mechanical integrity and specific ligand-mediated signaling to cells, is an inherently complex material that can present multiple kinds of cues including mechanical and topographic features, and multiple adhesive ligands on the same molecule. The various combinations of different cues can likely result in a continuum of cellular phenotypes , , . In vitro, Type I collagen is an important engineering material for use as scaffolding for tissue engineering applications , . Hence, an understanding of how the various cues presented by collagen matrices to cells influence cell behavior is likely to be important for the rational design of scaffoldings that can be used for biomedical applications . Type I collagen self-associates after secretion from cells to form supramolecular fibrils. It is a major component of the large vessels and is intimately associated with vascular smooth muscle cells (vSMC). Type 1 collagen interacts with cells primarily through beta1 integrins, including integrins alpha1/beta1 and alpha2/beta1. However Type I collagen is also recognized by discoidin domain receptors (DDRs), of which there are two isoforms, DDR1 and DDR2 , . DDR2 appears to have greater specificity for fibril forming collagens including Type I collagen . Focal adhesion kinase (FAK) is a major tyrosine kinase localized to focal adhesions, and has been implicated in mechanosensitive cell responses such as migration and proliferation , , , , . Exposure of a number of cell types to collagen has been shown to down-regulate the levels of cellular FAK , , but the mechanism by which the downregulation occurs is unknown. In this report, we systematically altered different properties of collagen matrices including the extent fibril formation, mechanical stiffness of the fibrils, and the presence of carbohydrate residues on collagen, to perturb the extracellular cues that cells sense. Using these different modifications of a Type I collagen matrix, we examined how cellular FAK levels are influenced by specific cues provided by the collagen ECM.