Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
Our data showed that lumican exert its effect on cell proliferation and healing of scratched wound of HTCE cells via its binding to TBR1.
Keywords: extracellular matrix • receptors • signal transduction