RESUMO
PURPOSE: Complement activation is implicated in the pathogenesis of age-related macular degeneration (AMD). Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) are present in eyes of individuals with AMD. Herein, we investigated the effect of complement activation on induction of ApoE accumulation in human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were primed with a complement-fixing antibody followed by treatment with C1q-depleted (C1q-Dep) human serum to elicit alternative pathway complement activation. Controls included anti-C5 antibody-treated serum and heat-inactivated C1q-Dep. Total protein was determined on RPE cell extracts, conditioned media, and extracellular matrix (ECM) by Western blot. ApoE and MAC colocalization was assessed on cultured RPE cells and human eyes by immunofluorescent stain. ApoE mRNA expression was evaluated by quantitative PCR (qPCR). RESULTS: Complement challenge upregulated cell-associated ApoE, but not apolipoprotein A1. ApoE accumulation was blocked by anti-C5 antibody and enhanced by repetitive complement challenge. ApoE mRNA levels were not affected by complement challenge. ApoE was frequently colocalized with MAC in complement-treated cells and drusen from human eyes. ApoE was released into complement-treated conditioned media after a single complement challenge and accumulated on ECM after repetitive complement challenge. CONCLUSIONS: Complement challenge induces time-dependent ApoE accumulation in RPE cells. An understanding of the mechanisms by which complement affects RPE ApoE accumulation may help to better explain drusen composition, and provide insights into potential therapeutic targets.
Assuntos
Apolipoproteínas E/genética , Ativação do Complemento/fisiologia , Regulação da Expressão Gênica , Degeneração Macular/genética , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Apolipoproteínas E/biossíntese , Western Blotting , Células Cultivadas , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Espectrometria de Massas , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/patologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-ß (TGF-ß) induces the expression of CYR61 in PSCs through canonical TGF-ß-ALK5-Smad2/3 signaling. Activation of TGF-ß signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-ß-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.
