Widely expressed mammalian adhesion receptors for fibrillar
Widely expressed mammalian adhesion receptors for fibrillar collagens include the α2β1- and α11β1-integrins and the discoidin domain receptors (DDRs), DDR1 and DDR2 (Leitinger, 2011). Integrins and DDRs bind distinct and separate motifs in native fibrillar collagen (Curat et al., 2001, Vogel et al., 1997). The functional significance of collagen interactions with integrins is well defined (Leitinger, 2011), and a link between integrin expression, cell contraction, collagen realignment, and ECM stiffness has been established (Levental et al., 2009, Paszek et al., 2005, Provenzano et al., 2008, Yeung et al., 2005). However, the impact of collagen-DDR1 interactions on collagen remodeling is not understood. DDR1 is a six-domain, trans-membrane receptor tyrosine kinase that, because of alternative splicing, is expressed as five variants (Leitinger, 2011). Upon binding to collagen, DDR1 undergoes auto-phosphorylation, a process that occurs over minutes to hours (Vogel et al., 1997). DDR1 is involved in the regulation of cell adhesion, migration, proliferation, and differentiation (Leitinger, 2011, Vogel et al., 2006) and may influence collagen degradation by regulating the activity of metalloproteinases (Ferri et al., 2004, Hou et al., 2001). Genetic disruption of DDR1 is associated with fibrotic conditions of kidney (Flamant et al., 2006), liver (Song et al., 2011), lung (Avivi-Green et al., 2006), and perivascular tissues (Franco et al., 2010). High levels of DDR1 Fmoc-Tyr(tBu)-OH have been detected in several human cancers, including lesions of breast (Johnson et al., 1993), ovary (Laval et al., 1994), and lung (Ford et al., 2007), suggesting the potential importance of DDR1 in collagen remodeling of the tumor stroma.
DDR1 associates with non-muscle myosin IIA (NMIIA) during cell migration and spreading on collagen (Huang et al., 2009), but it is not known whether this association is important for DDR1-dependent collagen mechanical remodeling. Here we considered that DDR1 mediates ECM remodeling by focusing cell-generated contraction forces on collagen fibrils. We show in vivo that mechanical tension is associated with enhanced DDR1 expression and alignment of collagen fibers. In cultured fibroblasts, collagen-induced DDR1 activation strengthened adhesion to collagen, increased the association of DDR1 through its C-terminal domain with NMIIA filaments, and enhanced collagen tractional remodeling. Collectively, the data show that DDR1 mediates tractional remodeling of collagen through its interaction with NMIIA.
Discussion DDR1 expression and function are linked to fibrotic disorders such as atherosclerosis, arthritis, and many types of cancer (Avivi-Green et al., 2006, Flamant et al., 2006, Ford et al., 2007, Toy et al., 2015, Valiathan et al., 2012). The hallmark of the ECM in fibrotic lesions is the anisotropic arrangement of aligned collagen fibers (Fang et al., 2014, Malik et al., 2015). Because DDR1 binds collagen and associates with NMIIA in migrating cells (Huang et al., 2009), we hypothesized that DDR1 mediates tractional collagen remodeling by transmitting cell-generated forces. Our main findings are that in vivo, the development of well-aligned collagen fibers is associated with DDR1 expression. In cultured cells, adhesion to fibrillar collagen induced initial DDR1 clustering that promoted DDR1 activation and its association with NMIIA. DDR1 activation reinforced DDR1 binding to collagen by increasing receptor clustering and enhanced the association of DDR1 with NMIIA filaments. Collectively, these processes optimize the transmission of myosin contractile forces to collagen. Moreover, DDR1 mechanical signaling is evidently influenced by substrate compliance. The expression of DDR1 is enhanced in wound-healing keloids, which has been attributed to keloid fibroblasts; treatment with DDR1 antisense blocks collagen production (Jiang et al., 2009). Furthermore, DDR1 null mice show reduced collagen deposition in injured carotid arteries (Hou et al., 2001) and in injured kidney (Borza et al., 2016) compared with DDR1 wild-type mice. These data point to the involvement of DDR1 in wound healing, but its specific role is not defined. We found that in mechanically restrained wounds, both DDR1 expression and DDR1 phosphorylation (Y792) were greater than in unsplinted wounds. Mechanical tension in wound healing prolongs the remodeling and contraction phases and contributes to increased scar formation (Cremers et al., 2015, Hinz et al., 2001). Mechanically restrained wounds exhibited increased expression of DDR1, correlating with enhanced α-SMA and more oriented collagen fibrils, features that characterize higher levels of tension in fibrotic lesions (Klingberg et al., 2013). Well-aligned collagen fibers can also contribute to localized tissue stiffening; in turn, tissue stiffness perpetuates the differentiation of local fibroblasts into myofibroblasts. After attachment to stiff substrates, myofibroblasts maintain their phenotype, even when re-plated on soft matrices (Wells, 2013). Collectively, our data show that DDR1 expression is temporally and spatially linked to mechanical tension in the ECM, possibly by enhancing collagen alignment and consequent tissue stiffening.