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M. K. Gandhari, J. Doyle, L. Roberts, S. Chakravarti; Lumican Expression is Regulated by NF-B in LPS-Mediated Innate Immune Response. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1497.
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Lipopolysaccharide (LPS), an endotoxin in the cell wall of gram-negative bacteria, induces aninnate immune response and inflammation that is mediated by nuclear translocation of the NF-ΚB transcription factor and up-regulation of pro-inflammatory genes. We have shown previously that mice deficient in the ECM proteoglycan lumican have impaired LPS-mediated induction of pro-inflammatory cytokines and healing of LPS-keratitis. Here we investigated whether the lumican gene (Lum) is directlyregulated by NF-ΚB in response to LPS.
Mouse embryonic fibroblasts (MEF) were treated with LPS (10ng/ml) for 0, 15, 30, 60, 120 and 240 minutes. NF-ΚB activation was assessed by Western blot analysis of nuclear and cytoplasmic extracts. Quantitative RT-PCR was used to assess the expression of lumican at each of these time-points.
NF-ΚB was detectable in the cytoplasmic fractions at all time points, with a marked drop at 60 minutes after LPS treatment. NF-ΚB increased in the nuclear fraction 30 minutes after LPS exposure, with maximal increase at 240 minutes. Lum mRNA expression also increased 30 minutes after LPS treatment.
LPS-mediated induction of Lum follows the same time frame as NF-ΚB activation by LPS, implying that Lum may be directlyregulated by NF-ΚB. Chromatin immunoprecipitations will be performed to test whether the p65 subunit of NF-ΚB binds to the two NF-ΚB consensus-binding sites in the Lum promoter to regulate gene expression.
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