Wolfgang F. Vogel

Discoidin Domain Receptor 1 Tyrosine Kinase Has an Essential Role in Mammary Gland Development

ABSTRACT 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.

The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair

Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain receptors (DDRs) are a novel class of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinase (MMP) production, but little is known about their expression and function in the vasculature. We posited a critical role for the DDRs controlling smooth muscle cell migration and proliferation and thus repair following arterial injury. Smooth muscle cells were isolated from the aortas of mice with a targeted deletion of the DDR1 gene (DDR1-null) and studied in culture using models that mimic critical steps in neointimal thickening. Our studies suggest that DDR1 plays an important role in regulating attachment to collagen, chemotaxis, proliferation, and MMP production in smooth muscle cells. Following mechanical injury to the carotid arteries, cross-sectional area of the neointima was significantly lower in DDR1-null mice than in wild-type mice. There was also a significant decrease in collagen deposition in the injured arteries of the DDR1-null mice. Our results support the hypothesis that DDR1 plays an important role as a collagen receptor, mediating intimal thickening after vascular injury.

PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation.

Stimulation of the prolactin receptor (PRLR), a member of the cytokine/growth hormone receptor family, results in activation of the associated Jak2 tyrosine kinase and downstream signaling pathways. We report that PTP1D, a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 (SH2) domains, physically associates with the PRLR‐Jak2 complex and is tyrosine‐phosphorylated upon stimulation with prolactin. The formation of the trimeric PRLR‐Jak2‐PTP1D complex is critical for transmission of a lactogenic signal, while PTP1D phosphorylation is necessary, but not sufficient. The dominant negative inhibitory effect of a phosphatase‐deficient mutant on expression of a beta‐casein promoter‐controlled reporter gene is evidence for an essential role of fully functional PTP1D in the regulation of milk protein gene transcription.

Tyrosine Kinase Activity of Discoidin Domain Receptor 1 Is Necessary for Smooth Muscle Cell Migration and Matrix Metalloproteinase Expression

Smooth muscle cell (SMC) interactions with collagen mediate cell migration during the pathogenesis of atherosclerosis and restenosis. Discoidin domain receptors (DDRs) have been identified as novel collagen receptors. We used aortic SMCs from wild-type and DDR1−/− mice to evaluate the function of the DDR1 in regulating migration. DDR1−/− SMCs exhibited impaired attachment to and migration toward a type I collagen substrate. Matrix metalloproteinase-2 (MMP-2) and MMP-9 activities were concomitantly reduced in these cells. Transfection of a full-length cDNA for DDR1b rescued these deficits, whereas kinase-dead mutants of DDR1 restored attachment but not migration and MMP production. These results suggest that active DDR1 kinase is a central mediator of SMC migration.

Expression and mutation analysis of the discoidin domain receptors 1 and 2 in non-small cell lung carcinoma

The discoidin domain receptors, (DDR)1 and DDR2, have been linked to numerous human cancers. We sought to determine expression levels of DDRs in human lung cancer, investigate prognostic determinates, and determine the prevalence of recently reported mutations in these receptor tyrosine kinases. Tumour samples from 146 non-small cell lung carcinoma (NSCLC) patients were analysed for relative expression of DDR1 and DDR2 using quantitative real-time PCR (qRT-PCR). An additional 23 matched tumour and normal tissues were tested for differential expression of DDR1 and DDR2, and previously reported somatic mutations. Discoidin domain receptor 1 was found to be significantly upregulated by 2.15-fold (P=0.0005) and DDR2 significantly downregulated to an equivalent extent (P=0.0001) in tumour vs normal lung tissue. Discoidin domain receptor 2 expression was not predictive for patient survival; however, DDR1 expression was significantly associated with overall (hazard ratio (HR) 0.43, 95% CI=0.22–0.83, P=0.014) and disease-free survival (HR=0.56, 95% CI=0.33–0.94, P=0.029). Multivariate analysis revealed DDR1 is an independent favourable predictor for prognosis independent of tumour differentiation, stage, histology, and patient age. However, contrary to previous work, we did not observe DDR mutations. We conclude that whereas altered expression of DDRs may contribute to malignant progression of NSCLC, it is unlikely that this results from mutations in the DDR1 and DDR2 genes that we investigated.

Identification of two novel, kinase-deficient variants of discoidin domain receptor 1: differential expression in human colon cancer cell lines

Discoidin domain receptor 1 belongs to a subfamily of tyrosine kinase receptors activated by various types of collagen. Alternative splicing in the juxtamembrane region of the receptor results in the insertion of 37 amino acids. Here we used primer flanking the juxtamembrane region to search for additional isoforms. From human colon carcinoma Colo 206F cells, we identified two novel isoforms, DDR1d and DDR1e. In the DDR1d sequence, exon 11 and 12 are deleted, which results in a frame‐shift mutation and premature termination of translation. DDR1e arises from an alternative usage of a splice acceptor site in exon 10 and deletion of exon 11 and12. Both new isoforms are predicted to be membrane‐anchored, but kinase‐deficient, receptors. Transcripts for DDR1d and DDR1e were present to various degrees in a panel of human colon cancer cell lines. Using antibodies to several different receptor epitopes, we found correlating expression of DDR1d and DDR1e protein with the expected molecular weight of 68 and 95 kDa in colon carcinoma cell lines. Despite the abundant expression of truncated DDR1d in Colo 206F cells, stimulation with collagen induced full‐length receptor phosphorylation, thereby suggesting a lack of dominant‐negative inhibition by DDR1d. Exogenous overexpression of DDR1d in transient transfection assays supported a nondominant negative mechanism of signal modulation.

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.

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 &agr;1(I) and &agr;1(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.

Ligand-induced shedding of discoidin domain receptor 1

Tyrosine kinases belonging to the discoidin domain receptor (DDR) family are activated upon stimulation with various types of collagen. In response to collagen treatment, immunoprecipitation of DDR1 with an antibody specific to the juxtamembrane region results in co‐purification of a previously unrecognized tyrosine phosphorylated protein of 62 kDa molecular weight. Here, this protein is identified as C‐terminal cleavage product of the full‐length DDR1 receptor and a DDR1‐specific shedding enzyme postulated. Shedding of DDR1 can be partially blocked by the furin inhibitor decanoyl‐RVKR‐chloromethylketone and completely inhibited by the hydroxamate‐based inhibitor batimastat. The characteristic of the DDR1 sheddase to be blocked by batimastat suggests that it belongs to the membrane‐bound matrix metalloproteinase or disintegrin and metalloproteinase family of proteases.

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.