Migration inhibition of mammary epithelial cells by Syk is blocked in the presence of DDR1 receptors.

The non-receptor tyrosine kinase Syk is a well-characterized hematopoietic signal transducer, which is also expressed in non-hematopoietic cells. In epithelial cells, the function of Syk is not wholly known. It interacts with the receptor tyrosine kinase DDR1 and is frequently lost from metastatic mammary tumors. Here, using genetic tracing, we demonstrate Syk expression in murine mammary epithelium, myoepithelium and skin epithelium, but not in intestinal or lung epithelia. Investigating possible functions of Syk, we found a substantial suppression of cell mobility that depended on Syk kinase activity in trans-well migration and wounding assays. Co-expression of DDR1 resulted in constitutive interaction and strong activation of Syk kinase. Most importantly, Syk-mediated migration inhibition was blocked in the presence of DDR1, while conversely DDR1 knockdown restored migration inhibition. Our study identifies Syk as a potent inhibitor of epithelial migration and describes a first functional consequence of the interaction with the collagen receptor DDR1.

Expression of the tyrosine kinase discoidin domain receptor 1 (DDR1) in human central nervous system myelin.

During development of the mouse brain, the protein kinase discoidin domain receptor 1 (DDR1) is present prenatally in neurons of the proliferative areas, and postnatally, DDR1 expression is no longer detected in neurons, but a spatial-temporal expression pattern in oligodendrocytes that overlaps with the dynamics of the myelination process is detected. Notably, oligodendrocytic DDR1 expression is upregulated in mice during experimentally induced remyelination. Recently, we demonstrated that DDR1 expression is high in human brain and that there is an association between the gene and schizophrenia in a case-control study. However, data regarding expression of DDR1 in the human brain are scarce. Here, we describe the expression pattern of DDR1 in the human adult cerebral cortex. Using several immunohistological techniques and in situ hybridization, we identified DDR1 in the following: a) myelin, b) capillary endothelial cells in the gray as well as white matter, and c) in the soma of some oligodendrocytes and astrocytes in the white matter. The most important overall finding in this study was that DDR1 is present in myelin and is expressed by oligodendrocyte cells. We detected the presence of DDR1 mRNA and protein in myelin and observed that DDR1 co-localized with the classical myelin basic protein (MBP). Moreover, we found a strong positive correlation between expression levels of DDR1 and two myelin-associated genes, myelin-associated glycoprotein (MAG) and oligodendrocyte transcription factor 2 (OLIG2). These observations suggest that DDR1 could be an important constituent of myelin. Because defects in myelination are linked to several mental disorders such as schizophrenia, the function of DDR1 in the process of myelination warrants further investigation.

Loss of collagen-receptor DDR1 delays renal fibrosis in hereditary type IV collagen disease.

Alport syndrome is a hereditary type IV collagen disease leading to progressive renal fibrosis, hearing loss and ocular changes. End stage renal failure usually develops during adolescence. COL4A3-/- mice serve as an animal model for progressive renal scarring in Alport syndrome. The present study evaluates the role of Discoidin Domain Receptor 1 (DDR1) in cell-matrix interaction involved in pathogenesis of Alport syndrome including renal inflammation and fibrosis. DDR1/COL4A3 Double-knockouts were compared to COL4A3-/- mice with 50% or 100% expression of DDR1, wildtype controls and to DDR1-/- COL4A3+/+ controls for over 6years. Double-knockouts lived 47% longer, mice with 50% DDR1 lived 29% longer and showed improved renal function (reduction in proteinuria and blood urea nitrogen) compared to animals with 100% DDR1 expression. Loss of DDR1 reduced proinflammatory, profibrotic cells via signaling of TGFbeta, CTGF, NFkappaB and IL-6 and decreased deposition of extracellular matrix. Immunogold-staining and in-situ hybridisation identified podocytes as major players in DDR1-mediated fibrosis and inflammation within the kidney. In summary, glomerular epithelial cells (podocytes) express DDR1. Loss of DDR1-expression in the kidney delayed renal fibrosis and inflammation in hereditary type IV collagen disease. This supports our hypothesis that podocyte-matrix interaction via collagen receptors plays an important part in progression of renal fibrosis in Alport disease. The blockade of collagen-receptor DDR1 might serve as an important new therapeutic concept in progressive fibrotic and inflammatory diseases in the future.

Wnt-5a-induced phosphorylation of DARPP-32 inhibits breast cancer cell migration in a CREB-dependent manner.

Tumor cell migration plays a central role in the process of cancer metastasis. We recently identified dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) as an antimigratory phosphoprotein in breast cancer cells. Here we link this effect of DARPP-32 to Wnt-5a signaling by demonstrating that recombinant Wnt-5a triggers cAMP elevation at the plasma membrane and Thr34-DARPP-32 phosphorylation in MCF-7 cells. In agreement, both protein kinase A (PKA) inhibitors and siRNA-mediated knockdown of Frizzled-3 receptor or Galpha(s) expression abolished Wnt-5a-induced phosphorylation of DARPP-32. Furthermore, Wnt-5a induced DARPP-32-dependent inhibition of MCF-7 cell migration. Phospho-Thr-34-DARPP-32 interacted with protein phosphatase-1 (PP1) and potentiated the Wnt-5a-mediated phosphorylation of CREB, a well-known PP1 substrate, but had no effect on CREB phosphorylation by itself. Moreover, inhibition of the Wnt-5a/DARPP-32/CREB pathway, by expression of dominant negative CREB (DN-CREB), diminished the antimigratory effect of Wnt-5a-induced phospho-Thr-34-DARPP-32. Phalloidin-staining revealed that that the presence of phospho-Thr-34-DARPP-32 in MCF-7 cells results in reduced filopodia formation. In accordance, the activity of the Rho GTPase Cdc42, known to be crucial for filopodia formation, was reduced in MCF-7 cells expressing phospho-Thr-34-DARPP-32. The effects of DARPP-32 on cell migration and filopodia formation could be reversed in T47D breast cancer cells that were depleted of their endogenous DARPP-32 by siRNA targeting. Consequently, Wnt-5a activates a Frizzled-3/Galpha(s)/cAMP/PKA signaling pathway that triggers a DARPP-32- and CREB-dependent antimigratory response in breast cancer cells, representing a novel mechanism whereby Wnt-5a can inhibit breast cancer cell migration.

The collagen receptor DDR1 regulates cell spreading and motility by associating with myosin IIA.

The spreading and migration of cells on adhesive substrates is regulated by the counterbalance of contractile and protrusive forces. Non-muscle myosin IIA, an ubiquitously expressed contractile protein and enzyme, is implicated in the regulation of cell spreading and directional migration in response to various stimuli. Here we show that discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor activated by type I collagen, associates with the non-muscle myosin IIA heavy chain (NMHC-IIA) upon ligand stimulation. An association was also indicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform lacking the kinase domain. DDR1 was important for assembly of NMHC-IIA into filaments on cells plated on collagen. DDR1 expression inhibited cell spreading over collagen but promoted cell migration. By contrast, blockade of non-muscle myosin II activity by blebbistatin enhanced cell spreading but inhibited migration over collagen. We propose that myosin and DDR1 impact cell spreading and migration by regulating adhesive contacts with collagen.

Discoidin domain receptor 1 (ddr1) deletion decreases atherosclerosis by accelerating matrix accumulation and reducing inflammation in low-density lipoprotein receptor-deficient mice.

Collagens are abundant within the atherosclerotic plaque, where they contribute to lesion volume and mechanical stability and influence cell signaling. The discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds to collagen, is expressed in blood vessels, but evidence for a functional role during atherogenesis is incomplete. In the present study, we generated Ddr1(+/+);Ldlr(-/-) and Ddr1(-/-);Ldlr(-/-) mice and fed them an atherogenic diet for 12 or 24 weeks. Targeted deletion of Ddr1 resulted in a 50% to 60% reduction in atherosclerotic lesion area in the descending aorta at both 12 and 24 weeks. Ddr1(-/-);Ldlr(-/-) plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1(+/+);Ldlr(-/-) plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1(-/-) smooth muscle cells in vitro, revealed increased mRNA levels for procollagen alpha1(I) and alpha1(III) and tropoelastin, suggesting an enhancement of matrix synthesis in the absence of DDR1. Furthermore, whereas plaque smooth muscle cell content was unchanged, Ddr1(-/-);Ldlr(-/-) plaques had a 49% decrease in macrophage content at 12 weeks, with a concomitant reduction of in situ gelatinolytic activity. Moreover, mRNA expression of both monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 was reduced in vivo, and Ddr1(-/-);Ldlr(-/-) macrophages demonstrated impaired matrix metalloproteinase expression in vitro. These data suggest novel roles for DDR1 in macrophage recruitment and invasion during atherogenesis. In conclusion, our data support a role for DDR1 in the regulation of both inflammation and fibrosis early in plaque development. Deletion of DDR1 attenuated atherogenesis and resulted in the formation of matrix-rich plaques.

Inner ear defects and hearing loss in mice lacking the collagen receptor DDR1.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor that is activated by native collagen. The physiological functions of DDR1 include matrix homeostasis and cell growth, adhesion, branching, and migration, but the specific role of DDR1 in the development and function of the inner ear has not been analyzed. Here, we show that deletion of the DDR1 gene in mouse is associated with a severe decrease in auditory function and substantial structural alterations in the inner ear. Immunohistochemical analysis demonstrated DDR1 expression in several locations in the cochlea, mostly associated with basement membrane and fibrillar collagens; in particular in basal cells of the stria vascularis, type III fibrocytes, and cells lining the basilar membrane of the organ of Corti. In the stria vascularis, loss of DDR1 function resulted in altered morphology of the basal cells and accumulation of electron-dense matrix within the strial epithelial layer in conjunction with a focal and progressive deterioration of strial cells. Cell types in proximity to the basilar membrane, such as Claudius', inner and outer sulcus cells, also showed marked ultrastructural alterations. Changes in the organ of Corti, such as deterioration of the supporting cells, specifically the outer hair cells, Deiters', Hensen's and bordering cells, are likely to interfere with mechanical properties of the organ and may be responsible for the hearing loss observed in DDR1-null mice. These findings may also have relevance to the role of DDR1 in other disease processes, for example, those affecting the kidney.

Identification of disulfide-linked dimers of the receptor tyrosine kinase DDR1.

Discoidin domain receptor 1 (DDR1) is a transmembrane receptor tyrosine kinase activated by triple-helical collagen. So far six different isoforms of DDR1 have been described. Aberrant expression and signaling of DDR1 have been implicated in several human diseases linked to accelerated matrix degradation and remodeling, including tumor invasion, atherosclerosis, and lung fibrosis. Here we show that DDR1 exists as a disulfide-linked dimer in transfected as well as endogenously expressing cells. This dimer formation occurred irrespective of its kinase domain, as dimers were also found for the truncated DDR1d isoform. A deletion analysis of the extracellular domain showed that DDR1 mutants lacking the stalk region failed to form dimers, whereas deletion of the discoidin domain did not prevent dimerization. Point mutagenesis within the stalk region suggested that cysteines 303 and 348 are necessary for dimerization, collagen binding, and activation of kinase function. The identification of DDR1 dimers provides new insights into the molecular structure of receptor tyrosine kinases and suggests distinct signaling mechanisms of each receptor subfamily.

Flat-panel detector-based volume computed tomography: a novel 3D imaging technique to monitor osteolytic bone lesions in a mouse tumor metastasis model.

Skeletal metastasis is an important cause of mortality in patients with breast cancer. Hence, animal models, in combination with various imaging techniques, are in high demand for preclinical assessment of novel therapies. We evaluated the applicability of flat-panel volume computed tomography (fpVCT) to noninvasive detection of osteolytic bone metastases that develop in severe immunodeficient mice after intracardial injection of MDA-MB-231 breast cancer cells. A single fpVCT scan at 200-microm isotropic resolution was employed to detect osteolysis within the entire skeleton. Osteolytic lesions identified by fpVCT correlated with Faxitron X-ray analysis and were subsequently confirmed by histopathological examination. Isotropic three-dimensional image data sets obtained by fpVCT were the basis for the precise visualization of the extent of the lesion within the cortical bone and for the measurement of bone loss. Furthermore, fpVCT imaging allows continuous monitoring of growth kinetics for each metastatic site and visualization of lesions in more complex regions of the skeleton, such as the skull. Our findings suggest that fpVCT is a powerful tool that can be used to monitor the occurrence and progression of osteolytic lesions in vivo and can be further developed to monitor responses to antimetastatic therapies over the course of the disease.

Semi-automatic classification of skeletal morphology in genetically altered mice using flat-panel volume computed tomography.

Rapid progress in exploring the human and mouse genome has resulted in the generation of a multitude of mouse models to study gene functions in their biological context. However, effective screening methods that allow rapid noninvasive phenotyping of transgenic and knockout mice are still lacking. To identify murine models with bone alterations in vivo, we used flat-panel volume computed tomography (fpVCT) for high-resolution 3-D imaging and developed an algorithm with a computational intelligence system. First, we tested the accuracy and reliability of this approach by imaging discoidin domain receptor 2- (DDR2-) deficient mice, which display distinct skull abnormalities as shown by comparative landmark-based analysis. High-contrast fpVCT data of the skull with 200 microm isotropic resolution and 8-s scan time allowed segmentation and computation of significant shape features as well as visualization of morphological differences. The application of a trained artificial neuronal network to these datasets permitted a semi-automatic and highly accurate phenotype classification of DDR2-deficient compared to C57BL/6 wild-type mice. Even heterozygous DDR2 mice with only subtle phenotypic alterations were correctly determined by fpVCT imaging and identified as a new class. In addition, we successfully applied the algorithm to classify knockout mice lacking the DDR1 gene with no apparent skull deformities. Thus, this new method seems to be a potential tool to identify novel mouse phenotypes with skull changes from transgenic and knockout mice on the basis of random mutagenesis as well as from genetic models. However for this purpose, new neuronal networks have to be created and trained. In summary, the combination of fpVCT images with artificial neuronal networks provides a reliable, novel method for rapid, cost-effective, and noninvasive primary screening tool to detect skeletal phenotypes in mice.

Discoidin domain receptor 1 null mice are protected against hypertension-induced renal disease.

A frequent complication of hypertension is the development of chronic renal failure. This pathology usually is initiated by inflammatory events and is characterized by the abnormal accumulation of collagens within the renal tissue. The purpose of this study was to investigate the role of discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor that displays tyrosine-kinase activity, in the development of renal fibrosis. To this end, hypertension was induced with angiotensin in mice that were genetically deficient of DDR1 and in wild-type controls. After 4 or 6 wk of angiotensin II administration, wild-type mice developed hypertension that was associated with perivascular inflammation, glomerular sclerosis, and proteinuria. Systolic pressure increase was similar in the DDR1-deficient mice, but the histologic lesions of glomerular fibrosis and inflammation were significantly blunted and proteinuria was markedly prevented. Immunostaining for lymphocytes, macrophages, and collagens I and IV was prominent in the renal cortex of wild-type mice but substantially reduced in DDR1 null mice. In separate experiments, renal cortical slices of DDR1 null mice showed a blunted response of chemokines to LPS that was accompanied by a considerable protection against the LPS-induced mortality. These results indicate the importance of DDR1 in mediating inflammation and fibrosis. Use of DDR1 inhibitors could provide a completely novel therapeutic approach against diseases that have these combined pathologies.

Phosphorylation of DARPP-32 regulates breast cancer cell migration downstream of the receptor tyrosine kinase DDR1.

Cell migration plays a central role in processes such as development, wound healing and cancer metastasis. Here we describe a novel interaction between DDR1, a receptor tyrosine kinase activated by collagen, and the phosphoprotein DARPP-32 in mammary epithelial cells. DARPP-32 expression was readily detected in non-transformed mammary cell lines, but was strongly reduced or even absent in breast tumor cell lines, such as MCF7. Transfection of MCF7 cells with DARPP-32 resulted in severely impaired cell migration, while DARPP-32 transfection into the DDR1-deficient breast cancer cell line MDA-MB-231 did not alter migration. Co-expression of both DDR1 and DARPP-32 in MDA-MB-231 cells inhibited migration, thereby supporting a critical role of the DDR1/DARPP-32 complex in motility. Mutational substitution of the phosphorylation sites Thr-34 or Thr-75 on DARPP-32 revealed that phosphorylation of Thr-34 is necessary for the ability of DARPP-32 to impair breast tumor cell migration. Thus, DARPP-32 signaling downstream of DDR1 is a potential new target for effective anti-metastatic breast cancer therapy.

Discoidin domain receptor 1-deficient mice are resistant to bleomycin-induced lung fibrosis.

RATIONALE: Discoidin domain receptor 1 (DDR1) is a tyrosine kinase activated by native collagens. Based on previous findings showing increased DDR1 expression in bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis, we hypothesized that DDR1 mediates disease progression after lung injury. OBJECTIVES: To investigate the inflammatory and fibrotic responses of DDR1 knockout and wild-type mice to bleomycin-induced lung injury. METHODS: Age- and sex-matched DDR1 knockout and wild-type C57BL/6 mice received a single intratracheal instillation of 2 U/kg bleomycin or saline, respectively. After 2 wk, lung inflammation and fibrosis were assessed using immunohistochemistry, real-time polymerase chain reaction, TUNEL assay, ELISA, fluorescence-activated cell sorting, and Western blot analysis. MEASUREMENTS AND MAIN RESULTS: Compared with wild-type animals, DDR1-null mice were largely protected against bleomycin-induced injury. Bleomycin-induced increases in collagen protein levels and tenascin-C mRNA levels were abrogated in knockout animals. Furthermore, myofibroblast expansion and apoptosis were much lower in these animals compared with their wild-type counterparts. Absence of inflammation in knockout mice was confirmed by lavage cell count and a cytokine ELISA. Western blot analysis of injured lung tissue revealed that DDR1-null mice failed to respond to the bleomycin insult with p38 MAPK activation, which was readily observed in wild-type mice. CONCLUSIONS: DDR1 expression is a prerequisite for the development of lung inflammation and fibrosis. Blockade of DDR1 may therefore be a novel therapeutic intervention in patients with pulmonary fibrosis.

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.

DDR1 signaling is essential to sustain Stat5 function during lactogenesis.

Postnatal development of the mammary gland is achieved by an interplay of endocrine and extracellular matrix-derived signals. Despite intense research, a comprehensive understanding of the temporal and spatial coordination of these hormonal and basement membrane stimuli is still lacking. Here, we address the role of the collagen-receptor DDR1 in integrating extracellular matrix-derived signaling with the lactogenic pathway initiated by the prolactin receptor. We found that stimulation of DDR1-overexpressing mammary epithelial HC11 cells with collagen and prolactin resulted in stronger and more sustained induction of Stat5 phosphorylation as compared to control cells. Enhanced Stat5 activity in HC11-DDR1 cells correlated with increased beta-casein gene expression. In contrast, cells derived from DDR1-null mice showed reduced Stat5 activation upon lactogenic stimulation and completely failed to induce beta-casein expression. The cell-autonomous role of DDR1 in controlling ductal branching and alveologenesis prior to the onset of lactogenesis was corroborated by mammary tissue transplantation experiments. Our results show that aside from hormone- and cytokine receptors, DDR1 signaling establishes a third matrix-derived pathway vital to maintain mammary gland function.

Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1.

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.

Expression and signaling activity of Wnt-5a/discoidin domain receptor-1 and Syk plays distinct but decisive roles in breast cancer patient survival.

PURPOSE: The loss of Wnt-5a, a G-protein-coupled receptor ligand, or Syk, an intracellular kinase, has in separate studies been associated with poor prognosis of breast cancer patients. Both proteins are involved in cell adhesion, a key event in epithelial cancer metastasis. Here, we have investigated whether Syk is part of the Wnt-5a/discoidin domain receptor-1 (DDR1) signaling pathway and if a signaling interaction of these proteins is important for breast cancer-specific survival. EXPERIMENTAL DESIGN: The signaling interactions between Wnt-5a/DDR1 and Syk were addressed in mammary cell lines. Their mRNA and protein levels and the respective clinical correlates were investigated in 94 cases of primary breast cancer. RESULTS: The expression of Wnt-5a and Syk correlated in four of five tumor cell lines. However, despite a constitutive association between Syk and the Wnt-5a-dependent adhesion receptor DDR1, we found no evidence of a Wnt-5a/DDR1-mediated activation of Syk. Instead, beta(1) integrins initiate the adhesion-induced activation of Syk. In tumors from breast cancer patients, the protein expression of Wnt-5a and Syk were differently regulated at the translational and transcriptional level, respectively. Analysis of breast cancer-specific survival revealed that the presence of Wnt-5a and Syk in primary tumors has good predictive value for a favorable outcome. Intriguingly, a simultaneous loss of both proteins did not reduce survival more than loss of either. CONCLUSIONS: Despite the difference in regulation of Wnt-5a and Syk protein expression and their lack of signaling interaction, our clinical data indicate that a favorable prognosis in breast cancer requires the expression and signaling activity of both.

DDR1-deficient mice show localized subepithelial GBM thickening with focal loss of slit diaphragms and proteinuria.

BACKGROUND: Type IV collagen in basement membranes is a ligand for the receptor tyrosine kinase discoidin domain receptor 1 (DDR1). DDR1 is expressed in renal cells and regulates cell adhesion and proliferation ex vivo. The interaction between type IV collagen and cell surface receptors is believed important for normal renal function as well as significant in chronic renal diseases and we therefore analyzed mice with a targeted deletion of DDR1. METHODS: Homozygous DDR1 knockout mice were compared to heterozygous and wild-type animals. The quantitative and qualitative amount of proteinuria was measured by urine-microelectrophoresis. Structural changes of the kidneys were determined by immunohistochemistry, light microscopy, and electron microscopy. RESULTS: Compared to heterozygous littermates, adult DDR1 knockout mice showed a selective middle- to high-molecular proteinuria of up to 0.3 g/L and urinary acanthocytes. There was no evidence of uremia with no change in serum urea in the first 9 months of age. Little apparent change in renal morphology was detected using light microscopy. However, electron microscopy showed a localized, subepithelial, mushroom-like isodense thickening of the glomerular basement membrane (GBM). Within these areas, a focal loss of the podocytic slit diaphragms occurred. CONCLUSION: The loss of cell-matrix communication in DDR1-deficient podocytes appears to result in excess synthesis of basement membrane proteins leading to disturbed anchorage of foot processes and disruption of the slit diaphragm. Our data suggest that the interaction between type IV collagen and DDR1 plays an important role in maintaining the structural integrity of the GBM.

Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor.

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.

An extracellular matrix-specific microarray allowed the identification of target genes downstream of discoidin domain receptors.

The two discoidin domain receptors, DDR1 and DDR2, are tyrosine kinases that are activated by collagen and are essential regulators of cell-matrix communication. However, the target genes downstream of activated DDRs and their physiological significance are largely unknown. Here, we describe a novel method to dissect signaling pathways induced by extracellular matrix (ECM) receptors. Using the doxycycline-inducible repression system (tet-off), we generated human fibrosarcoma and mouse fibroblast cell lines over-expressing DDR1 or DDR2. These cell lines were employed for gene expression analysis using microarrays specific for human and mouse genes coding for ECM proteins or ECM-interacting factors. We found that approximately 10% of the genes studied were up- or down-regulated more than twofold in response to signals generated by over-expressing DDRs. A common event downstream of DDR1 and DDR2 in human and mouse cells was the up-regulation of P-selectin glycoprotein ligand. Key target genes repressed upon DDR activation were agrin, syndecan-1 and alpha3 integrin. ECM-specific microarrays were found a valuable tool to dissect gene expression changes induced by collagen-receptor signaling pathways.