We had earlier reported that collagen
We had earlier reported that collagen fibers with intact native banded structure were occasionally observed in the kinase-deficient, membrane-anchored DDR2 ECD (DDR2/-KD) samples; however, in our DDR1/ECD and DDR2/ECD samples, observation of native banded structure of collagen was far more infrequent. D-periodicity of collagen fibers from native cultures was measured at 61±5 nm, which is in agreement with previous studies by us and others. Previously, we found that the membrane-anchored DDR2/-KD inhibited lateral fiber growth, compared to native cultures. While fiber diameter measurements for the first week of culture for DDR1/ECD and DDR2/ECD gave results similar to those of DDR2/-KD samples (25.0–28.9 nm), fibers in DDR2/-KD cultures exhibited lateral growth of around 10 nm over the 3 weeks of culture observed. In contrast, a sustained inhibition of lateral growth of collagen fibers was observed by DDR ECD ll-37 resulting in average collagen fiber diameters between 20 and 30 nm throughout a 4-week period. Together, our results show that soluble DDR2 ECD inhibits collagen fibrillogenesis in the ECM consistent with membrane-anchored DDR2, albeit with a slightly higher potential. We speculate that this stronger inhibition of collagen fiber structure and lateral diameter is due to the soluble DDR ECD being distributed throughout the ECM and thus having more ability to affect collagen fiber formation even in ECM regions away from the pericellular regions.
We found that both DDR1 and DDR2 ECD increased matrix mineralization as compared to native cells, with the effect of DDR2 ECD being more prominent. Both soluble (DDR2/ECD) and membrane-bound DDR2 ECD (DDR2/-KD), when compared to wild-type cells, induced larger mineral deposits. In this regard, a recent study has reported abnormal calcification arising due to mutations in the DDR2 gene in spondylo-meta-epiphyseal dysplasia (SMED) in humans. It is interesting to note that all the mutations reported were found in the DDR2 intracellular domain and not in its ECD. Although the expression levels of DDR2 were not reported in this study, it is likely that expression of DDR2 ECD present in the full-length mutated receptor in SMED cases along with impaired signaling of the mutated receptor may lead to increased calcification. Matrix mineralization in both DDR1 and DDR2 knockout mice have not been reported in detail; however, in DDR1 knockout mice, reduced bone calcification was described in the fibula bone. Our observations suggest the importance of evaluating matrix mineralization with respect to expression of both the full-length DDR receptors and their isoforms containing the ECDs. Since the collagen type I binding site for decorin is in close proximity to that of DDR2, further investigations are needed to understand if binding of DDR2 ECD to collagen type I promotes crystal formation by interfering with decorin binding. It is interesting to compare the effect of DDRs on collagen fibrillogenesis and matrix mineralization to those of decorin. Both DDR ECDs and decorin inhibit collagen fibrillogenesis and result in reduction of collagen fiber diameters. In contrast, while DDR ECDs enhance matrix mineralization, decorin is found to be an inhibitor of collagen calcification. No reports elucidating the ultrastructure of native ECM in DDR1 or DDR2 knockout mice have yet been made.
We conclude that expression of both membrane-bound and soluble DDR1 and DDR2 ECDs can alter the morphology of endogenous collagen fibers, thus perturbing the overall ECM structure. We speculate that such perturbations, if observed in vivo, may significantly alter the integrity and biomechanical properties of resulting tissues. Further studies need to be addressed to elucidate which DDR1 and DDR2 isoforms are modulated in pathological states in vivo and how their expression alters ECM morphology and tissue biomechanics.
Materials and Methods