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  • br Materials and Methods br

    2019-12-13


    Materials and Methods
    Acknowledgements
    Introduction Collagen fibrillogenesis, the assembly of collagen fibers, is a critical process in the development, maturation, and repair of mammalian tissue. Alterations in the structure and amount of deposited collagen fibers can greatly alter the integrity of the whole tissue. Even a single point mutation in collagen type I can severely compromise the strength of cortical bone tissue leading to osteogenesis imperfecta. Liproxstatin-1 Further, the interaction between collagen-binding proteins and collagen molecules during fibrillogenesis can promote significant alterations in the resulting collagen fiber structure and subsequent extracellular matrix (ECM) remodeling.2, 3 For example, soluble collagen-binding proteins such as decorin, biglycan, fibronectin, and vitronectin are thought to play a significant role in the process of collagen fibrillogenesis and bone mineralization due to their interaction with collagen molecules. The collagen-binding membrane proteins discoidin domain receptors (DDR1 and DDR2) are transmembrane receptors belonging to the family of receptor tyrosine kinases and have been studied for ECM remodeling in atherosclerosis,5, 6, 7 osteoarthritis,8, 9, 10 and several malignancies. It is well established that activation of the DDR1 and DDR2 kinase domain up-regulates the Liproxstatin-1 of various matrix metalloproteinases5, 11 and alters the biosynthesis of collagen. The extracellular domain (ECD) of DDRs is known to be necessary and sufficient for its interaction with collagen. Besides the full-length receptor, the DDR1 ECD is also found in five distinct isoforms and as a shed protein in the ECM.13, 14 Several protein and mRNA15, 16, 17 species consisting of the DDR2 ECD have also been observed in vivo. However, the functional roles of these ECDs of DDRs lacking their kinase domain are not well understood. We had previously elucidated that DDR1 ECD and DDR2 ECD inhibit collagen fibrillogenesis in vitro when used as purified proteins. Further, we have recently demonstrated that the DDR2 ECD when anchored on the cell surface preserves the capacity to inhibit collagen fibrillogenesis independent of its kinase activity. It is therefore likely that the expression of soluble ECD of DDRs by cells such as those found in the shedding of DDR1 ECD may play an important role in matrix remodeling. The fibrillogenesis process of collagen is understood to initiate in the extracellular space near the plasma membrane where secretory vesicles form regions of deep invagination. However, it is not clear how and when collagen-binding proteins interact with collagen molecules during fibrillogenesis or to what extent membrane-bound versus soluble collagen-binding proteins affect the collagen fibrillogenesis process by cells. In this study, we seek to elucidate the alterations in collagen fibrillogenesis arising due to soluble DDR1 and DDR2 ECDs secreted by the cells and compare the results with our previous findings utilizing the kinase-deficient, membrane-bound DDR2 ECD (DDR2/-KD). Similar to our previous study, we created stably transfected mouse osteoblast cell lines to express DDR1/ECD or DDR2/ECD as a soluble protein. We utilized a number of ultrastructural and biochemical analyses to elucidate how alterations in the collagen matrix, due to DDR ECDs, affects collagen fibrillogenesis and matrix mineralization.
    Results
    Discussion In this study, we elucidate how cell-secreted, soluble ECDs of DDR1 and DDR2 inhibit collagen fibrillogenesis and enhance matrix mineralization for ECM endogenously generated by the cells. Using similar experimental approaches, in our earlier work we had demonstrated that kinase-deficient and membrane-anchored DDR2 ECD (DDR2/-KD) also inhibits collagen fibrillogenesis. Such an inhibition of collagen fibrillogenesis by DDR1 and DDR2 ECD was originally observed by us using purified proteins in vitro. Thus, our current investigations along with our previous studies2, 18, 19 enable us to compare the role of membrane-bound versus soluble proteins (DDR-ECD) in regulating collagen fibrillogenesis.