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  • The fibrillogenesis process of collagen is understood

    2020-11-17

    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 SB202190 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. Multiple protein species are known to naturally exist for the transmembrane receptors DDR1 and DDR2. Five splice variants have been characterized for DDR1 (“a” through “e”). The d and e isoforms lack the intracellular kinase domain of DDR1. The splicing of DDR1 to various extents has been reported in human ovarian cancer, breast cancer, and fetal brain and has thus far been best characterized in colon cancer cell lines. In normal and diseased arteries of nonhuman primates, three isoforms (a, b, and d) have been detected; these isoforms are differently expressed in advanced atherosclerotic lesions and have been detected in normal human lung tissue and cultured human smooth muscle cells (SMCs). Although splice variants for DDR2 have not yet been characterized, there is ample evidence to support they exist. In one report, Northern blot analysis using a cDNA probe corresponding to the ECD of DDR2 has revealed multiple mRNA species in melanoma carcinoma and virus-transformed normal embryonic lung cell lines. In a separate study, a major 10-kb transcript and a minor 4.5-kb transcript for DDR2 were detected in human and rat heart and other tissues using Northern analysis, along with additional weak bands at 0.8, 3.6, 2.4, and 1.7 kb. In mouse and rat heart tissue, a DDR2 probe hybridized to multiple RNAs of varying lengths (∼4 and 7 kb). At least two transcripts for DDR2 (9.5 and 4.5 kb) and several protein species (130, 90, 50, and 45 kDa) have been found in cultured human SMCs using antibodies against DDR2 ECD. Besides alternate splicing of DDRs, shedding of the DDR1 ECD as a soluble protein in the ECM is another naturally occurring phenomenon reported for several mammalian cells.13, 14 While no direct evidence for DDR2 ECD shedding exists, the Western blots with DDR2 antibodies on SMCs transiently transfected with full-length DDR2 show several smaller molecular species (90, 50, and 45 kDa) besides the full-length 130-kDa receptor, suggesting the likelihood of shedding of DDR2 ECD. Our current investigations highlight the relevance of further characterizing DDR2 isoforms and understanding the different functional roles of DDR1 and DDR2 protein variants.