| Summary: | 博士 === 國防醫學院 === 醫學科學研究所 === 99 === The purpose of this study is to investigate the effects and mechanisms of glucosamine (GlcN) on the proliferation, migration and epithelial-mesenchymal transition of retinal pigment epithelial cells in response to epidermal growth factor (EGF) and transforming growth factor (TGF-β).
Cell proliferation was measured in the human retinal pigment epithelial cell line (ARPE-19) cells with the 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio] -1,3-benzene disulfonate (WST-1) assay and cell counting. The results were confirmed in human donor cells with the carboxyfluorescein diacetate succinimidyl ester cell proliferation assay (CFSE) cell proliferation assay. In ARPE-19 cells, cell-cycle progression was determined by flow cytometry; the protein levels of cell cycle regulators and heat shock proteins were measured by western blotting; the levels and branching of N-glycans were assessed using the L-Phaseolus vulgaris agglutinin lectin-binding assay; and the modulation of N-glycans on EGF receptor (EGFR) was examined by western blotting. We also evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signaling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed.
We found that GlcN inhibited retinal pigment epithelium (RPE) proliferation in a dose-dependent manner. During cell-cycle progression induced by EGF, GlcN caused delays at the G1–S and G2–M transitions without affecting cell viability. GlcN modulated the level and branching of N-glycans on EGFR, suppressed phosphorylation of EGFR, and reduced phosphorylation of extracellular signal-regulated kinases, serine/threonine protein kinase, and the signal transducer and activator of transcription 3 (STAT3). GlcN had only minor effects on the expression of Hsp90, Grp78, and transcription factor CHOP/GADD 153 markers of nonspecific stress in the endoplasmic reticulum. GlcN also reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus EGF or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). TGF-β2 also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. PDGF and CTGF stimulated cell migration; and TGF-β2 stimulated wound closure, contraction of collagen, and changes in cell morphology. GlcN countered all these TGF-β stimulated effects.
In conclusion, treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. GlcN could inhibit the EGF and TGF-β signaling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR. Further researches into the role of GlcN as a potential agent for the prevention and treatment of RPE-mediated ocular proliferative or fibrotic disorders, such as proliferative vitreoretinopathy, are warranted.
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