Regulation of collagen fibrillogenesis by cell-surface expression of kinase dead DDR2

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.     

Getting a grip on adhesion: Cadherin switching and collagen signaling

Epithelial-mesenchymal transition (EMT) is a developmental biological process that is hijacked during tumor progression. Cadherin switching, which disrupts adherens junctions and alters cadherin-associated signaling pathways, is common during EMT. In many tumors, substantial extracellular matrix (ECM) is deposited. Collagen is the most abundant ECM constituent and it mediates specific signaling pathways by binding to integrins and discoidin domain receptors (DDRs). The interaction of the collagen receptors results in activation of signaling pathways that promote tumor progression including an induction of the cadherin switching. DDR inhibitors have demonstrated anticancer therapeutic efficacy preclinically by inhibiting the collagen signaling. Understanding how collagen signaling impacts cellular processes including EMT and cadherin switching is of great interest especially given the strong interest in stromal targeted therapies for desmoplastic cancers.     

DDR1 and DDR2 physical interaction leads to signaling interconnection but with possible distinct functions

ABSTRACT

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are members of the tyrosine kinase receptors activated after binding with collagen. DDRs are implicated in numerous physiological and pathological functions such as proliferation, adhesion and migration. Little is known about the expression of the two receptors in normal and cancer cells and most of studies focus only on one receptor. Western blot analysis of DDR1 and DDR2 expression in different tumor cell lines shows an absence of high co-expression of the two receptors suggesting a deleterious effect of their presence at high amount. To study the consequences of high DDR1 and DDR2 co-expression in cells, we over-express the two receptors in HEK 293T cells and compare biological effects to HEK cells over-expressing DDR1 or DDR2. To distinguish between the intracellular dependent and independent activities of the two receptors we over-express an intracellular truncated dominant-negative DDR1 or DDR2 protein (DDR1DN and DDR2DN). No major differences of Erk or Jak2 activation are found after collagen I stimulation, nevertheless Erk activation is higher in cells co-expressing DDR1 and DDR2. DDR1 increases cell proliferation but co-expression of DDR1 and DDR2 is inhibitory. DDR1 but not DDR2 is implicated in cell adhesion to a collagen I matrix. DDR1, and DDR1 and DDR2 co-expression inhibit cell migration. Moreover a DDR1/DDR2 physical interaction is found by co-immunoprecipitation assays. Taken together, our results show a deleterious effect of high co-expression of DDR1 and DDR2 and a physical interaction between the two receptors.     

Discoidin domain receptor 2 inhibits fibrillogenesis of collagen type 1

Discoidin domain receptors (DDR1 and DDR2) are widely expressed cell-surface receptors, which bind to and are activated by collagens, including collagen type 1. Activation of DDRs and the resulting downstream signaling is known to regulate the extracellular matrix. However, little is known about how DDRs interact with collagen and its direct impact on collagen regulation. Here, we have established that by binding to collagen, the extracellular domain (ECD) of DDR2 inhibits collagen fibrillogenesis and alters the morphology of collagen type 1 fibers. Our in vitro assays utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. Using surface plasmon resonance, we confirmed that further oligomerization of DDR2-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Collagen turbidity measurements and biochemical assays indicated that DDR2 delays the formation of collagen fibrils. Atomic force microscopy of soluble collagen revealed that a predominately monomeric state of collagen was present with DDR2, while control solutions had an abundance of polymeric collagen. Transmission electron microscopy of collagen fibers, showed that the native periodic banded structure of collagen fibers was weakened and nearly absent in the presence of DDR2. Further, using a cell-based assay we demonstrate that overexpression of full length DDR2 inhibits fibrillogenesis of collagen type 1. Our results demonstrate a novel and important functional role of the DDR2 ECD that may contribute to collagen regulation via modulation of fibrillogenesis.     

Sensing extracellular matrix: an update on discoidin domain receptor function

Discoidin Domain Receptors (DDRs) have recently emerged as non-integrin-type receptors for collagen. The two mammalian gene products Discoidin Domain Receptor 1 and -2 constitute a subfamily of tyrosine kinase receptors that are selectively expressed in a number of different cell types and organs. Upon collagen activation, DDRs regulate cell adhesion, proliferation and extracellular matrix remodeling. Here we review the various signaling pathways and cellular responses evoked by activated DDRs. Additionally, we give an overview of the more recent advances in understanding the role of DDRs in various human diseases, in particular during tumor progression, atherosclerosis, inflammation and tissue fibrosis. Furthermore, we discuss potential roles of genes homologous to mammalian DDRs identified in flies, worms and sponges. We show that the structural organization of these DDR-related genes is highly conserved throughout evolution suggesting that invertebrate DDRs may also function as receptors for collagen. By highlighting current questions about these unusual collagen receptors, we hope to attract new research on DDRs from a variety of different fields.     

Molecular analysis of collagen binding by the human discoidin domain receptors,DDR1 and DDR2. Identification of collagen binding sites in DDR2

The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.     

Discoidin domain receptors in disease

Discoidin domain receptors, DDR1 and DDR2, lie at the intersection of two large receptor families, namely the extracellular matrix and tyrosine kinase receptors. As such, DDRs are uniquely positioned to function as sensors for extracellular matrix and to regulate a wide range of cell functions from migration and proliferation to cytokine secretion and extracellular matrix homeostasis/remodeling. While activation of DDRs by extracellular matrix collagens is required for normal development and tissue homeostasis, aberrant activation of these receptors following injury or in disease is detrimental. The availability of mice lacking DDRs has enabled us to identify key roles played by these receptors in disease initiation and progression. DDR1 promotes inflammation in atherosclerosis, lung fibrosis and kidney injury, while DDR2 contributes to osteoarthritis. Furthermore, both DDRs have been implicated in cancer progression. Yet the mechanisms whereby DDRs contribute to disease progression are poorly understood. In this review we highlight the mechanisms whereby DDRs regulate two important processes, namely inflammation and tissue fibrosis. In addition, we discuss the challenges of targeting DDRs in disease. Selective targeting of these receptors requires understanding of how they interact with and are activated by extracellular matrix, and whether their cellular function is dependent on or independent of receptor kinase activity.     

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

Discoidin domain receptor 1 interactions with myosin motors contribute to collagen remodeling and tissue fibrosis

Discoidin Domain Receptor (DDR) genes and their homologues have been identified in sponges, worms and flies. These genes code for proteins that are implicated in cell adhesion to matrix proteins. DDRs are now recognized as playing central regulatory roles in several high prevalence human diseases, including invasive cancers, atherosclerosis, and organ fibrosis. While the mechanisms by which DDRs contribute to these diseases are just now being delineated, one of the common themes involves cell adhesion to collagen and the assembly and organization of collagen fibers in the extracellular matrix. In mammals, the multi-functional roles of DDRs in promoting cell adhesion to collagen fibers and in mediating collagen-dependent signaling, suggest that DDRs contribute to multiple pathways of extracellular matrix remodeling, which are centrally important processes in health and disease. In this review we consider that interactions of the cytoplasmic domains of DDR1 with cytoskeletal motor proteins may contribute to matrix remodeling by promoting collagen fiber alignment and compaction. Poorly controlled collagen remodeling with excessive compaction of matrix proteins is a hallmark of fibrotic lesions in many organs and tissues that are affected by infectious, traumatic or chemical-mediated injury. An improved understanding of the mechanisms by which DDRs mediate collagen remodeling and collagen-dependent signaling could suggest new drug targets for treatment of fibrotic diseases.     

Silencing of Discoidin Domain Receptor-1 (DDR1) Concurrently Inhibits Multiple Steps of Metastasis Cascade in Gastric Cancer

Accumulating evidence suggests that a unique set of receptor tyrosine kinases, known as discoidin domain receptors (DDRs), plays a role in cancer progression by interacting with the surrounding collagen matrix. In this study, we investigated the expression and role of DDR1 in human gastric cancer metastasis. Proliferation, migration, invasion, and tube formation assays were conducted in DDR1-expressing MKN74 gastric cancer cells and corresponding DDR1-silenced cells. The effects of DDR1 on tumor growth and metastasis were examined in orthotopically implanted and experimental liver metastasis models in nude mice. The expression of DDR1 in surgical specimens was analyzed by immunohistochemistry. DDR1 was expressed in human gastric cancer cell lines, and its expression in human gastric tumors was associated with poor prognosis. Among seven gastric cancer cell lines, MKN74 expressed the highest levels of DDR1. DDR1-silenced MKN74 cells showed unaltered proliferation activity. In contrast, migration, invasion, and tube formation were significantly reduced. When examined in an orthotopic nude mouse model, DDR1-silenced implanted tumors significantly reduced angiogenesis and lymphangiogenesis, thereby leading to reductions in lymph node metastasis and liver metastasis. In a model of experimental liver metastasis, DDR1-silenced cells almost completely inhibited liver colonization and metastasis. DDR1 deficiency led to reduced expression of the genes encoding vascular endothelial growth factor (VEGF)-A, VEGF-C, and platelet-derived growth factor-B. These results suggest that DDR1 is involved in gastric cancer tumor progression and that silencing of DDR1 inhibits multiple steps of the gastric cancer metastasis process.     

Multitasking discoidin domain receptors are involved in several and specific hallmarks of cancer

ABSTRACT

Discoidin domain receptors, DDR1 and DDR2, are two members of collagen receptor family that belong to tyrosine kinase receptor subgroup. Unlike other matrix receptor-like integrins, these collagen receptors have not been extensively studied. However, more and more studies are focusing on their involvement in cancer. These two receptors are present in several subcellular localizations such as intercellular junction or along type I collagen fibers. Consequently, they are involved in multiple cellular functions, for instance, cell cohesion, proliferation, adhesion, migration and invasion. Furthermore, various signaling pathways are associated with these multiple functions. In this review, we highlight and characterize hallmarks of cancer in which DDRs play crucial roles. We discuss recent data from studies that demonstrate the involvement of DDRs in tumor proliferation, cancer mutations, drug resistance, inflammation, neo-angiogenesis and metastasis. DDRs could be potential targets in cancer and we conclude this review by discussing the different ways to inhibits them.     

Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation

The discoidin domain receptors (DDRs) are collagen binding receptor tyrosine kinases that play important roles in cell migration, invasion and adhesion. Crosstalk between growth factor signaling and components of the extracellular matrix are drivers of cellular function but the integrated signaling networks downstream of such crosstalk events have not been extensively characterized. In this report, we have employed mass spectrometry-based quantitative phosphotyrosine analysis to identify crosstalk between DDR2 and the insulin receptor. Our phosphoproteomic analysis reveals a cluster of phosphorylation sites in which collagen and insulin cooperate to enhance phosphotyrosine levels. Importantly, Y740 on the DDR2 catalytic loop was found in this cluster indicating that insulin acts to promote collagen I signaling by increasing the activity of DDR2. Furthermore, we identify two additional migration associated proteins that are candidate substrates downstream of DDR2 activation. Our data suggests that insulin promotes collagen I signaling through the upregulation of DDR2 phosphorylation which may have important consequences in DDR2 function in health and disease.     

Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions

Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor- and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.     

Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links,Insertions, or Deletions in the Extracellular Juxtamembrane Region: MECHANISTIC IMPLICATIONS*

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.     

Collagen recognition and transmembrane signalling by discoidin domain receptors

The discoidin domain receptors, DDR1 and DDR2, are two closely related receptor tyrosine kinases that are activated by triple-helical collagen in a slow and sustained manner. The DDRs have important roles in embryo development and their dysregulation is associated with human diseases, such as fibrosis, arthritis and cancer. The extracellular region of DDRs consists of a collagen-binding discoidin (DS) domain and a DS-like domain. The transmembrane region mediates the ligand-independent dimerisation of DDRs and is connected to the tyrosine kinase domain by an unusually long juxtamembrane domain. The major DDR binding site in fibrillar collagens is a GVMGFO motif (O is hydroxyproline), which is recognised by an amphiphilic trench at the top of the DS domain. How collagen binding leads to DDR activation is not understood. GVMGFO-containing triple-helical peptides activate DDRs with the characteristic slow kinetics, suggesting that the supramolecular structure of collagen is not required. Activation can be blocked allosterically by monoclonal antibodies that bind to the DS-like domain. Thus, collagen most likely causes a conformational change within the DDR dimer, which may lead to the formation of larger DDR clusters. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays

Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.