ABSTRACT
PURPOSE: Glucosamine sulfate (GS) is a naturally occurring sugar that possesses some immunosuppressive effects in vitro and in vivo, but its mechanism is unknown. We investigated whether GS could modulate the proinflammatory cytokine-induced expression of the gene for intercellular adhesion molecule (ICAM)-1, an inflammatory protein in human retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used as a model to determine the effects of GS on the expression of the ICAM-1 gene upregulated by TNF-alpha or IFN-gamma, by Western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The activation and nuclear translocation of the nuclear factors NF-kappaB and STAT1 were evaluated by immunocytochemistry, Western blot analysis, and electrophoretic mobility shift assay (EMSA). RESULTS: Both TNF-alpha and IFN-gamma increased the expression of ICAM-1 at the mRNA and protein levels in a time- and dose-dependent manner in ARPE-19 cells. GS effectively downregulated the TNF-alpha- or IFN-gamma-induced expression of ICAM-1 in the protein and mRNA level in a dose-dependent manner. GS further inhibited the nuclear translocation of p65 proteins in TNF-alpha and phosphorylated STAT1 in IFN-gamma-stimulated ARPE-19 cells. CONCLUSIONS: GS inhibits the expression of the ICAM-1 gene in ARPE-19 cell stimulated with TNF-alpha or IFN-gamma through blockade of NF-kappaB subunit p65 and nuclear translocation of STAT1. This study has demonstrated a potentially important property of GS in reducing ICAM-1 mediated inflammatory mechanisms in the eye.
Subject(s)
Glucosamine/pharmacology , Intercellular Adhesion Molecule-1/biosynthesis , Interferon-gamma/antagonists & inhibitors , Pigment Epithelium of Eye/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Blotting, Western , Cell Culture Techniques , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Fluorescent Antibody Technique, Indirect , Humans , Intercellular Adhesion Molecule-1/genetics , Interferon-gamma/pharmacology , NF-kappa B/metabolism , Phosphorylation , Pigment Epithelium of Eye/drug effects , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , STAT1 Transcription Factor/metabolism , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-RegulationABSTRACT
PURPOSE: The aim of this study was to evaluate the expression of vascular endothelial growth factor (VEGF) in response to retinoic acid (RA) in human retinal pigment epithelial cells. METHODS: Expression of VEGF in human ARPE-19 cells was determined by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). mRNA stability was assessed after the administration of actinomycin D. The induction of the VEGF gene by various RAs was also determined by semiquantitative RT-PCR. RESULTS: All-trans retinoic acid (atRA) time-dependently increased VEGF mRNA levels. The effect of atRA was dose-dependent in a range between 10(-7) M and 10(-6) M. Treatment with actinomycin D revealed that atRA induces the VEGF gene at the transcriptional level. Of the various RAs tested, atRA was the most potent inducer of the VEGF gene. CONCLUSIONS: We demonstrated that atRA stimulates the induction of the VEGF gene in ARPE-19 cells, suggesting a novel pathway for the development of age-related macular degeneration.
