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Osamu Yamanaka, Yong Yuan, Yujin Zhang, Chia-Yang Liu, Shizuya Saika, Winston W. Kao; Lumican Binds TGF-β type I receptor and Stimulates the Phosphorylation of ERK and Smads in Cultured Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1946.
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© ARVO (1962-2015); The Authors (2016-present)
Lumican, a member of Small Leucine-rich Proteoglycan family, plays an important role in corneal wound healing. We reported that lumican binds TGFβ type I receptor (TBR1) by two-hybrid analysis (ARVO2005). To elucidate the biological function of lumican, we examined the signaling of lumican administered to human telomerase immortalized corneal epithelial cells (HTCE) cells and 293 cells.
Recombinant GST-lumican (glutathione S-tranferase-lumican fusion protein) was isolated from E. coli transfected with pGEX-2T-Lum plasmid, and subsequently purified using glutathione-Sepharose affinity column. Closure of scratch wound with confluent HTCE cultures was used to examine the wound healing process in the presence or absence of recombinant GST-lumican, in vitro. Immune fluorescence staining and western blot analysis with phosphorylation of ERK1/2, p38MAPK, Smad2 and Smad3 and expression of TBR1 were performed. To investigate the binding of GST-lumican to TBR1, 293 cells transfected with TBR1 plasmid were incubated with GST-lumican at 4°C for 1 h then further incubated at 37°C for 0, 5, 15, 30 and 60 min. Internalization of lumican/TBR1 complex was visualized by double immune fluorescence staining with anti-lumican and TBR1 antibodies.
In the HTCE scratch wound model, expression of TBR1 and activation of Smad2 and Smad3 via phosphorylation were observed in 5 min of adding GST-lumican and in 30 min phosphorylation of ERK1/2 was observed. Phospho-p38MAPK expression was not detected up to 1 hr in the presence or absence of GST-lumican. Co-localization of GST-lumican with Tbr1 was observed diffusely in 293 cells after 4°C incubation, at 15 min further 37°C incubation limited the immunolocalization of GST-lumican and TBR1 to around the nuclei not whole cell surface. At 30 min, immunoreactivity of them was not observed.
Our data showed that lumican exert its effect on cell proliferation and healing of scratched wound of HTCE cells via its binding to TBR1.
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