Abstract
Purpose::
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.
Methods::
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.
Results::
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.
Conclusions::
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.
Keywords: cornea: basic science • proteoglycans/glycosaminoglycans • transcription factors