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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.  相似文献   

2.
Discoidin domain receptor 2 (DDR2) is an unusual receptor tyrosine kinase in that its ligand is fibrillar collagen rather than a growth factor-like peptide. We examined signal transduction pathways of DDR2. Here we show that DDR2 is also unusual in that it requires Src activity to be maximally tyrosine-phosphorylated, and that Src activity also promotes association of DDR2 with Shc. The interaction with Shc involves a portion of Shc not previously implicated in interaction with receptor tyrosine kinases. These results identify Src kinase and the adaptor protein Shc as key signaling intermediates in DDR2 signal transduction. Furthermore, Src is required for DDR2-mediated transactivation of the matrix metalloproteinase-2 promoter. The data support a model in which Src and the DDR2 receptor cooperate in a regulated fashion to direct the phosphorylation of both the receptor and its targets.  相似文献   

3.
During malignant invasion tumor cells establish contact with extracellular matrix proteins, including fibrillar collagen. In addition to providing a physical barrier against invasion, fibrillar collagen also restricts cell proliferation. It has been assumed that the growth regulatory activity of fibrillar collagen is the result of an indirect restrictive effect on cell spreading and cytoskeletal organization. Here we provide evidence for a direct inhibitory effect of fibrillar collagen on proliferation of human melanoma and fibrosarcoma cells that involves activation of the tyrosine kinase discoidin domain receptor 2 and is independent of effects on cell spreading. Cells plated in the presence of fibrillar collagen were growth arrested in the G0/G1 phase of the cell cycle. However treatment with the tyrosine kinase inhibitor genistein, down-regulation of discoidin domain receptor 2, or collagen deglycosylation that prevents discoidin domain receptor 2 activation allowed cells to enter the cell cycle in the presence of fibrillar collagen without a requirement for spreading and actin organization. Our data provide evidence for a novel direct mechanism by which cell contact with fibrillar collagen restricts proliferation.  相似文献   

4.
The anti-transplant rejection drug cyclosporin A (CsA) causes loss of collagen homeostasis in rapidly remodeling connective tissues, such as human gingiva. As a result of CsA treatment, collagen degradation by fibroblasts is inhibited, which leads to a net increase of tissue collagen and gingival overgrowth. Since fibrillar collagen is the primary ligand for the discoidin domain receptor 1 (DDR1), we hypothesized that CsA perturbs DDR1-associated functions that affect collagen homeostasis. For these experiments, human fibroblasts obtained from gingival explants or mouse 3T3 fibroblasts (wild type, over-expressing DDR1 or DDR1 knockdown) or mouse GD25 cells (expressing DDR1 but null for β1 integrin), were treated with vehicle (dimethyl sulfoxide) or with CsA. The effect of CsA on cell binding to collagen was examined by flow cytometry; cell-mediated collagen remodeling was analyzed with contraction, compaction and migration assays. We found that CsA inhibited cell binding to collagen, internalization of collagen, contraction of collagen gels and cell migration over collagen in a DDR1-dependent manner. CsA also enhanced collagen compaction around cell extensions. Treatment with CsA strongly reduced surface levels of β1 integrins in wild type and DDR1 over-expressing 3T3 cells but did not affect β1 integrin activation or focal adhesion formation. We conclude that CsA inhibition of collagen remodeling is mediated through its effects on both DDR1 and cell surface levels of the β1 integrin.  相似文献   

5.
Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers.  相似文献   

6.
Discoidin domain receptor 1 (DDR1) is a widely expressed tyrosine kinase receptor which binds to and gets activated by collagens including collagen type 1. Little is understood about the interaction of DDR1 with collagen and its possible functional implications. Here, we elucidate the binding pattern of the DDR1 extracellular domain (ECD) to collagen type 1 and its impact on collagen fibrillogenesis. Our in vitro assays utilized DDR1-Fc fusion proteins, which contain only the ECD of DDR1. Using surface plasmon resonance, we confirmed that further oligomerization of DDR1-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Single-molecule imaging by means of atomic force microscopy revealed that DDR1 oligomers bound at overlapping or adjacent collagen molecules and were nearly absent on isolated collagen molecules. Interaction of DDR1 oligomers with collagen was found to modulate collagen fibrillogenesis both in vitro and in cell-based assays. Collagen fibers formed in the presence of DDR1 had a larger average diameter, were more cross-linked and lacked the native banded structure. The presence of DDR1 ECD resulted in "locking" of collagen molecules in an incomplete fibrillar state both in vitro and on surfaces of cells overexpressing DDR1. Our results signify an important functional role of the DDR1 ECD, which occurs naturally in kinase-dead isoforms of DDR1 and as a shedded soluble protein. The modulation of collagen fibrillogenesis by the DDR1 ECD elucidates a novel mechanism of collagen regulation by DDR1.  相似文献   

7.
Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor tyrosine kinases, DDR1 and DDR2. Here, we used a recombinant fusion protein between the extracellular domain of DDR1 and alkaline phosphatase to detect specific receptor binding sites during mouse development. Major sites of DDR1-binding activity, indicative of ligand expression, were found in skeletal bones, the skin, and the urogenital tract. Ligand expression in the uterus during implantation and in the mammary gland during pregnancy colocalized with the expression of the DDR1 receptor. The generation of DDR1-null mice by gene targeting yielded homozygous mutant animals that were viable but smaller in size than control littermates. The majority of mutant females were unable to bear offspring due to a lack of proper blastocyst implantation into the uterine wall. When implantation did occur, the mutant females were unable to lactate. Histological analysis showed that the alveolar epithelium failed to secrete milk proteins into the lumen of the mammary gland. The lactational defect appears to be caused by hyperproliferation and abnormal branching of mammary ducts. These results suggest that DDR1 is a key mediator of the stromal-epithelial interaction during ductal morphogenesis in the mammary gland.  相似文献   

8.
9.
Under physiological buffer conditions (17 mM Pi, pH 6.3), the endogenous lectin of Dictyostelium discoideum, discoidin I, binds to two types of receptors on the surface of glutaraldehyde-fixed, wild-type (NC-4) D. discoideum cells. We have designated these two types of receptors the carbohydrate or C sites and the ionic or I sites. Binding to the C sites is saturable with respect to discoidin I and is inhibited by hapten sugars (such as N-acetyl-d-galactosamine), but not by increasing buffer ionic strength with NaCl or polyelectrolytes. The number of C sites increases about 4-fold during the first 8.5 h of suspension differentiation, reaching a capacity for about 2–104 discoidin I tetramers per cell. The binding activity of the C sites is reduced about 50% by sequential NaIO4 oxidation/NaBH4 reduction of the fixed cells, but it is not reduced by CHCl3-CH3OH extraction of the fixed cells. In marked contrast, binding to the I sites appears nonsaturable with respect to discoidin I, and it is inhibited by increasing buffer ionic strength with NaCl or polyelectrolytes (such as poly-l-glutamic acid or heparin), but not by hapten sugars. The I sites are present on both vegetative and differentiated fixed cells and can bind more than 106 discoidin I tetramers per cell. The binding activity of the I sites on fixed cells is not reduced by sequential NaIO4 oxidation/NaBH4 reduction, but is reduced 70 to 90% by CHCl3-CH3OH extraction. The data suggest that the I sites represent ionic lipids that bind discoidin I electrostatically.  相似文献   

10.
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12.
Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.  相似文献   

13.
14.
ABSTRACT

The preservation of tissue and organ architecture and function depends on tightly regulated interactions of cells with the extracellular matrix (ECM). These interactions are maintained in a dynamic equilibrium that balances intracellular, myosin-generated tension with extracellular resistance conferred by the mechanical properties of the extracellular matrix. Disturbances of this equilibrium can lead to the development of fibrotic lesions that are associated with a wide repertoire of high prevalence diseases including obstructive cardiovascular diseases, muscular dystrophy and cancer. Mechanotransduction is the process by which mechanical cues are converted into biochemical signals. At the core of mechanotransduction are sensory systems, which are frequently located at sites of cell-ECM and cell-cell contacts. As integrins (cell-ECM junctions) and cadherins (cell-cell contacts) have been extensively studied, we focus here on the properties of the discoidin domain receptor 1 (DDR1), a tyrosine kinase that mediates cell adhesion to collagen. DDR1 expression is positively associated with fibrotic lesions of heart, kidney, liver, lung and perivascular tissues. As the most common end-point of all fibrotic disorders is dysregulated collagen remodeling, we consider here the mechanical signaling functions of DDR1 in processing of fibrillar collagen that lead to tissue fibrosis.  相似文献   

15.
We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis.  相似文献   

16.
Differentiation of preadipocytes into functional adipocytes depends on early proliferative events (mitotic clonal expansion) and extracellular matrix interactions. We report that discoidin domain receptor (DDR) 2, a novel adhesion receptor, is expressed in 3T3-L1 preadipocytes and is downregulated during the early phase of adipogenesis. DDR2 overexpression (DDR2-L1 preadipocytes) reduced subconfluent proliferation by 56% (p<0.001) and insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by 34% (p<0.05). The mitotic clonal expansion phase of differentiating confluent DDR2-L1 preadipocytes was impaired by approximately 25% (p<0.05). Although induction of peroxisome proliferator-activated receptor gamma, fatty acid synthase, and adiponectin was not altered, the resulting adipocytes were 55% larger (p<0.05), and contained 66% more triacylglycerol (p<0.01). The induction of CCAAT/enhancer binding protein alpha was reduced by 37% (p<0.05), correlating with a similar reduction in insulin-stimulated IRS-1 tyrosine phosphorylation and glucose transport in DDR2-L1 adipocytes (decreases of 22% and 27%, respectively; p<0.05 for both). Our data show that DDR2 is expressed in adipose cells and that its overexpression leads to insulin resistance.  相似文献   

17.
18.
Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators. Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.  相似文献   

19.
Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation.  相似文献   

20.
The extracellular matrix (ECM) acts as a critical factor during morphogenesis. Because the organization of the ECM directly influences the structure of tissues and organs, a determination of the way that ECM organization is regulated should help to clarify morphogenesis. We have analyzed the assembly of Del1, an ECM protein produced by endothelial cells in embryos, in the ECM. Del1 consists of three epidermal growth factor repeats (E1–E3) at its N-terminus and two discoidin domains (C1, C2) at its C-terminus. Experiments with various deletion mutants of Del1 have revealed that fragments containing the C-terminus of C1, which has a lectin-like structure, direct deposition in the ECM. The efficiency of deposition varies according to the presence of other domains in Del1. A fragment containing E3 and C1 has the strongest deposition activity, whereas fragments containing C2, which is highly homologous to C1, have low deposition activity. Digestion of ECM with hyaluronidase from bovine testis releases Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. In vivo gene transfer experiments have shown that fusion with the deposition domain of Del1 dramatically alters the distribution of exogenous proteins in mice. Thus, the extent of Del1 deposition may modify the organization of the ECM.  相似文献   

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