Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kappaB dependent.
BACKGROUND
Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-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 response to LPS is incomplete.


METHODS
Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-kappaB (NF-kappaB) and Western blot for the NF-kappaB inhibitory protein IkappaB. To prove the role for NF-kappaB, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5'-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E).


RESULTS
LPS increased NF-kappaB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IkappaBalpha decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IkappaBbeta levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5'-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-kappaB 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 and Sp1 sites, located in the proximal promoter region of MCP-1, reduced basal luciferase activity in unstimulated cells, but had no effect on LPS-stimulated luciferase activity.


CONCLUSIONS
These studies prove that NF-kappaB is critical for LPS-induced 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 transcriptional regulation of MCP-1 has important implications for the delineation of treatment to prevent endotoxin-mediated renal injury.
Wang
Y
Y
Department of Renal Medicine and Storr Liver Unit, The University of Sydney at Westmead Hospital, Sydney, New South Wales, Australia.
Rangan
G K
GK
Goodwin
B
B
Tay
Y C
YC
Harris
D C
DC
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D007684:Kidney Tubules / Q000187:drug effects* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D016329:Sp1 Transcription Factor / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology
D018932:Chemokine CCL2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1