Altered Signaling by Discoidin Domain Receptor 2 Exposed to Abnormal Collagens

Objectives: Discoidin domain receptor 2 (Ddr2), a receptor tyrosine kinase selectively activated by type I fibrillar collagen of bone, signals through both ERK1/2 and p38 MAP kinases to stimulate cell growth and differentiation. Ddr2 plays a major role in craniofacial and skeletal development. Previous worked showed that abnormalities in collagen such as nonenzymatic glycation can reduce its ability to stimulate Ddr2. The purpose of this study is to investigate the impacts of various collagen modifications on Ddr2 activation and MAP kinase signaling.
Methods: Here we examined Ddr2 activation by chemically and genetically modified collagens. Initially, MC3T3 osteoblast cells were treated with increasing concentrations of type I collagen (0, 5, 10, 20, 100 or 200 mg/mL) for either 3 hours for protein experiments, or for both 24 hours and 48 hours for gene expression analysis. An appropriate amount of collagen was selected and cells were treated with non-modified collagen, chemically glycated collagen or collagen purified from tail tendons of Brittle mice, which contain a Gly349Cys mutation in the Col1a1 gene. Denatured collagen served as a negative control. Western Blotting and qPCR were used to measure protein and RNA expression levels, respectively. Markers such as p-Ddr2, Ddr2, Ddr1, MMP13, and MMP14 were quantified in order to access activation in the presence or absence of collagen.
Results: Gene expression results showed a dose-response activation of Ddr2, Ddr1, MMP13, and MMP14, with a greater increased observed at 48 hours, compared to 24 hours. Differences in Ddr2 activation profiles between different collagens will be reported and the implications for Ddr2 function will be discussed.
Conclusions: Our results suggest that modifications to collagen can impact Ddr2 activation and potentially contribute to skeletal and related abnormalities associated with various diseases including diabetes and osteogenesis imperfecta.