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  • Collagen compaction and alignment are

    2019-12-09

    Collagen compaction and alignment are generated by cell-mediated contractile forces (Stopak and Harris, 1982). The transmission of actomyosin forces to collagen has been mainly attributed to integrins and the activation MRS 2179 tetrasodium salt of the Rho-ROCK pathway (Clark et al., 2007, Kural and Billiar, 2013, Provenzano et al., 2008). DDR1 overexpression enhances collagen compaction (Staudinger et al., 2013) and regulates collagen deposition and tissue architecture in the mouse auditory system (Meyer zum Gottesberge et al., 2008). The DDR1-NMIIA complex is involved in cell adhesion and cell migration on collagen (Huang et al., 2009) and in maintaining tension and elasticity in the inner ear (Meyer Zum Gottesberge and Hansen, 2014). We found that collagen alignment was markedly reduced in mechanically loaded periodontal ligament of DDR1 KO mice, in cultured cells with DDR1 knockdown, and in cells expressing DDR1d, a kinase domain-deficient truncation isoform. We also found that the C-terminal kinase domain of DDR1 is required for the binding to NMIIA and for the force-transmission involved in collagen tractional remodeling, even in cells that lack β1-integrin adhesions. DDR1 can regulate adhesion to collagen either independently of integrin function (Kamohara et al., 2001) or by modifying integrin activation (Staudinger et al., 2013, Xu et al., 2012), and at least some of the downstream pathways stimulated by DDR1 intersect with integrin-activated signaling systems (Valiathan et al., 2012). We identified a collagen adhesion complex that is spatially separated from classical β1-integrin/vinculin-containing focal adhesions, as previously described (Vogel et al., 2000). DDR1-positive adhesions were enriched with NMIIA and were involved in the alignment of collagen fibrils independent of integrin function or presence. Furthermore, our in vitro data show that the complex comprises NMIIA filaments and an interaction involving the coiled-coil domain of NMIIA rods (amino MRS 2179 tetrasodium salt [aa] residues 1,339–1,891) with the C-terminal kinase domain of DDR1 (aa residues 437–876). Phosphorylation of the MLC regulates NMIIA assembly into filaments (Kimura et al., 1996). We found that NMIIA filament assembly mediated by MLCK was required for DDR1 association with NMIIA and force transmission to collagen, which is important for ECM remodeling. Our findings indicate that the C-terminal kinase domain of DDR1 is important for force transmission from NMIIA to the ECM and suggest that MLCK activity is important for NMIIA filament binding to DDR1. Upon binding to collagen, DDR1 undergoes slow and sustained auto-phosphorylation (Fu et al., 2014, Vogel et al., 1997), but it is not understood how DDR1 binding to collagen leads to activation of the DDR1 kinase domain. One proposed mechanism involves collagen-induced DDR1 clustering (Mihai et al., 2009), because DDR1 oligomerization is required for high-affinity binding to collagen (Yeung et al., 2013). Work using cells expressing DDR1d, a kinase-deficient isoform, supports the involvement of the kinase domain in regulating receptor oligomerization, auto-phosphorylation, and clustering at the cell surface (Fu et al., 2014). We found that in cells binding to fibrillar collagen, activated DDR1 was mainly oligomeric, supporting the notion that receptor oligomerization precedes receptor activation (Yeung et al., 2013). Furthermore, we found that cells expressing the kinase-depleted DDR1 (DDR1d), cells expressing kinase-dead DDR1 (DDR1e), or DDR1 OE cells cultured on non-activating fibronectin substrates all formed clusters. However, cells that expressed kinase-active DDR1 exhibited large increases of cluster size, and only cells expressing full-length kinase-active DDR1 could mechanically remodel collagen. In these cells, collagen mechanical remodeling was strongly inhibited by nilotinib. In contrast, cells expressing the kinase-depleted DDR1d or the kinase-dead DDR1e showed low levels of collagen mechanical remodeling and nilotinib had no significant effect. Thus, DDR1 kinase activity is required for collagen compaction and alignment. Altogether, our results indicate that DDR1 receptor clustering induces DDR1 activation, which reinforces DDR1 binding to collagen, most likely by increasing receptor clustering. Furthermore, DDR1 activation enhances the association of DDR1 with NMIIA, which optimizes the transmission of myosin-dependent contractile forces to collagen.