RESUMEN
PURPOSE: Calcium signaling is an important intracellular pathway. Increased intracellular calcium is associated with cytokine regulation and inflammatory signals secretion. The purpose of this study is to understand the molecular mechanisms by which calcium signaling controls IL-8 activation in human RPE cells. METHODS: Fluorescence-based calcium imaging and different mutants of IL-8 plasmids were used in this study. The IL-8 promoter activation, gene expression, and secretion were detected by using luciferase reporter assay, quantitative real-time PCR (Q-PCR), and ELISA, respectively. In addition, pharmacological inhibitors and small interfering RNA (siRNA) were applied to clarify the mechanisms of IL-8 activation. RESULTS: Our study reported that intracellular calcium mobilization activated IL-8 gene expression and secretion. Application of pharmacological inhibitor BAY 11-7082, siRNA, and plasmids of the nuclear factor κ light chain enhancer of activated B cells (NF-κB) binding site, we identified that NF-κB is the main transcription factor involved in intracellular calcium mobilization-mediated IL-8 activation in human RPE cells. CONCLUSIONS: Collectively, our findings highlight the important role of intracellular calcium mobilization in the activation of IL-8. These findings may be helpful for the clinical applications in the age-related macular degeneration (AMD) prevention and treatment.
Asunto(s)
Calcio/metabolismo , Regulación de la Expresión Génica , Interleucina-8/genética , Degeneración Macular/metabolismo , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Western Blotting , Señalización del Calcio , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-8/biosíntesis , Degeneración Macular/patología , ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer cells. EGFR-mediated signaling involves inflammatory gene expression including cyclooxygenase (COX)-2 and interleukin (IL)-8, and is associated with cancer pathogenesis. In a search of phytochemicals with anti-inflammatory activity, the COX-2 and IL-8 inhibitory activities of some marine compounds were examined. After screening these compounds 11-episinulariolide acetate (1) from soft coral exhibited the most potent activity. Reverse-transcription PCR; western blotting; ELISA and luciferase assays were used to test the effect of compound 1 on EGF-stimulated expressions of COX-2 and IL-8 in A431 human epidermoid carcinoma cells. After exposure to 10 µM of compound 1, expression levels of COX-2 and IL-8 were reduced. In addition; intracellular Ca²âº increase and Ca²âº-dependent transcription factor activation were blocked by compound 1. Thus, compound 1 can potentially serve as a lead compound for targeting Ca²âº signaling-dependent inflammatory diseases.
