Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelialcells is nuclear factor-kappa B dependent

Background. Endotoxin is an important factor in the development of acute re nal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the mo st potent triggers for renal cells to produce monocyte chemoattractant prot ein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in r esponse to LPS is incomplete. Methods. Transcriptional regulation of MCP-1 was investigated in rat proxim al tubule cells (PTCs) in primary culture and was exposed to LPS using elec tromobility shift assay and supershift analysis for nuclear factor-kappa B (NF-kappa B) and Western blot for the NF-kappa B inhibitory protein I kappa B. To prove the role for NF-kappa B, activator protein (AP-1), and sequenc e-specific transcription factor (Sp1), mutation and deletion analysis was p erformed using a 3.5 kb fragment of rat MCP-1 5'-flanking region inserted i nto a luciferase reporter construct transfected into tubular epithelial cel l line (NRK-52E). Results. LPS increased NF-kappa B in a dose- and time-dependent manner, whi ch paralleled that of MCP-1 mRNA expression. I kappa B alpha decreased with in 30 minutes of LPS treatment, but returned to basal levels by two hours. I kappa B beta levels were depressed within one hour and remained low throu ghout the culture period after LPS stimulation. The activity of the transfe cted 5'-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-kappa B binding sites, located in the enhancer region of the 5'-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 a nd Sp1 sites, located in the proximal promoter region of MCP-1, reduced bas al luciferase activity in unstimulated cells, but had no effect on LPS-stim ulated luciferase activity. Conclusions. These studies prove that NF-kappa B is critical for LPS-induce d MCP-1 transcription, and AP-1 and Sp1 are essential for basal expression of MCP-1 in rat tubule cells. The species-specific nature of transcriptiona l regulation of MCP-1 has important implications for the delineation of tre atment to prevent endotoxin-mediated renal injury.