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An extracellular matrix-specific microarray allowed the identification of target genes downstream of discoidin domain
Elena Faraci1, Maresa Eck, Bernhard Gerstmayer
1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont, Canada M5S 1A8.
Abstract:
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.
Insights
Discoidin domain receptors (DDR1 and DDR2) regulate cell-matrix communication. This study identified key genes, like P-selectin glycoprotein ligand, altered by DDR activation, revealing insights into collagen-receptor signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Discoidin domain receptors (DDR1 and DDR2) are collagen-activated tyrosine kinases crucial for cell-matrix communication.
- The downstream target genes and physiological roles of activated DDRs remain largely uncharacterized.
Purpose of the Study:
- To develop a novel method for dissecting signaling pathways initiated by extracellular matrix (ECM) receptors.
- To identify genes regulated by DDR1 and DDR2 activation.
Main Methods:
- Utilized a doxycycline-inducible repression system (tet-off) to generate cell lines overexpressing DDR1 or DDR2.
- Employed ECM-specific microarrays for gene expression analysis in human and mouse cells.
- Analyzed changes in genes encoding ECM proteins and ECM-interacting factors.
Main Results:
- Approximately 10% of studied genes showed significant ( > twofold) up- or down-regulation upon DDR overexpression.
- Upregulation of P-selectin glycoprotein ligand was a common downstream event for both DDR1 and DDR2.
- Agrin, syndecan-1, and alpha3 integrin were identified as key repressed target genes.
Conclusions:
- Developed a valuable ECM-specific microarray approach to study collagen-receptor signaling.
- Identified specific genes regulated by DDR activation, providing insights into their physiological significance.
- Highlighted P-selectin glycoprotein ligand as a key downstream effector of DDR signaling.