Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation.
Ontology highlight
ABSTRACT:
SUBMITTER: Iwai LK
PROVIDER: S-EPMC3725701 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The fetal isoform A of the insulin receptor (IR-A) is frequently overexpressed in a variety of malignancies including breast cancer. IR overexpression has a recognized role in cancer progression and resistance to anticancer therapies. In particular, IR-A has a peculiar mitogenic potential and is activated not only by insulin but also by IGF-2. Previously, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.We now evaluated whether DDR1 is able to exert a role in breast cancer biology by functionally cross-talking with IR. In MCF-7 human breast cancer cells, IR and DDR1 co-immunoprecipitated and co-localized after insulin or IGF-2 stimulation. In a panel of breast cancer cells, DDR1 knockdown by specific siRNAs markedly inhibited IR downstream signaling as well as proliferation, migration and colony formation in response to insulin and IGF-2. These effects were accompanied by reduction of IR protein and mRNA expression, which involved both transcriptional and post-transcriptional effects. DDR1 overexpression elicited opposite effects. Bioinformatics analysis of public domain databases showed that IR and DDR1 co-expression significantly correlates with several clinically relevant histopathological and molecular features of human breast carcinomas.These findings demonstrate that, in human breast cancer cells, DDR1 regulates IR expression and ligand dependent biological actions. This novel functional crosstalk is likely clinically relevant and may become a new molecular target in breast cancer.
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Project description:To evaluate the association between promoter DNA methylation and discoidin domain receptor 1 (DDR1) gene expression in men with nonobstructive azoospermia (NOA).Comparing fibroblasts cultured from testicular biopsies using a high resolution Infinium 450K methylation array.Basic research laboratory.Men with NOA (n = 16) and with normal spermatogenesis (n = 5).None.Bisulfite clonal sequencing for validation and quantification of CpG methylation of DDR1; gene expression analysis of DDR1 with quantitative polymerase chain reaction and immunohistochemistry to validate the array results at mRNA and protein levels.We validated promoter methylation, mRNA and protein levels of the CpG sites identified from array results. Differentially methylated CpG sites (?20K) were identified using an F-test in the NOA samples. We identified 20 genes with >30% difference in DNA methylation within the promoter region of men with NOA and fertile controls. Of the aberrantly methylated genes, 10 were hypomethylated and 10 were hypermethylated genes. From the top 10 hypermethylated genes, six genes (MRI1, DCAF12L1, TMEM95, CECR2, DDR1, and NPHS2) were selected for validation because they were shown to be expressed in the testis. Of the six genes expressed in the fibroblasts cultured from testis, DDR1 showed an abnormal gene expression pattern. Three patients (19%) out of the 16 men with NOA for whom gene expression data were available had lower DDR1 expression levels (1.8x fold decrease) than the fertile men, whereas four (25%) men had higher expression levels (2x fold increase) of DDR1 compared with the levels in fertile men. Quantitative analysis by bisulfite clonal sequencing showed that one of the CpG sites (cg13329862) of DDR1 promoter was hypermethylated in NOA patients compared with fertile controls (53% vs. 15%). Immunohistochemical analysis suggests presence of DDR1 within cytoplasm of germ cells and peritubular connective tissue (in men with hypospermatogenesis) and decreased expression of the protein in men with Sertoli-cell only syndrome.Abnormal gene expression of DDR1 is associated with NOA. The functional relevance of aberrant methylation of DDR1 to expression of DDR1 in men with NOA warrants further investigation.
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Project description:Discoidin domain receptor 1 (Drd1) is a collagen-binding membrane protein, but its role in osteoblasts during osteogenesis remains undefined. We generated inducible osteoblast-specific Ddr1 knockout (OKOΔDdr1) mice; their stature at birth, body weight and body length were significantly decreased compared with those of control Ddr1f/f-4OHT mice. We hypothesize that Ddr1 regulates osteogenesis of osteoblasts. Micro-CT showed that compared to 4-week-old Ddr1f/f-4OHT mice, OKOΔDdr1 mice presented significant decreases in cancellous bone volume and trabecular number and significant increases in trabecular separation. The cortical bone volume was decreased in OKOΔDdr1 mice, resulting in decreased mechanical properties of femurs compared with those of Ddr1f/f-4OHT mice. In femurs of 4-week-old OKOΔDdr1 mice, H&E staining showed fewer osteocytes and decreased cortical bone thickness than Ddr1f/f-4OHT. Osteoblast differentiation markers, including BMP2, Runx2, alkaline phosphatase (ALP), Col-I and OC, were decreased compared with those of control mice. Ddr1 knockdown in osteoblasts resulted in decreased mineralization, ALP activity, phosphorylated p38 and protein levels of BMP2, Runx2, ALP, Col-I and OC during osteogenesis. Overexpression and knockdown of Ddr1 in osteoblasts demonstrated that DDR1 mediates the expression and activity of Runx2 and the downstream osteogenesis markers during osteogenesis through regulation of p38 phosphorylation.
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Project description:The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors that are members of the receptor tyrosine kinase family. Both DDRs bind a number of different collagen types and play important roles in embryo development. Dysregulated DDR function is associated with progression of various human diseases, including fibrosis, arthritis, and cancer. By interacting with key components of the extracellular matrix and displaying distinct activation kinetics, the DDRs form a unique subfamily of receptor tyrosine kinases. DDR-facilitated cellular functions include cell migration, cell survival, proliferation, and differentiation, as well as remodeling of extracellular matrices. This review summarizes the current knowledge of DDR-ligand interactions, DDR-initiated signal pathways and the molecular mechanisms that regulate receptor function. Also discussed are the roles of DDRs in development and disease progression.
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