Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kB dependent
| Autor: | Bryan Goodwin, Yuet.-Ching. Tay, Yang Wang, Yiping Wang, Gopala K. Rangan, David Harris |
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| Rok vydání: | 2000 |
| Předmět: |
Lipopolysaccharides
Male medicine.medical_specialty renal injury Sp1 Transcription Factor Molecular Sequence Data monocyte chemoattractant protein-1 activator protein-1 tubule cells 030204 cardiovascular system & hematology Biology 03 medical and health sciences 0302 clinical medicine Internal medicine Gene expression medicine Animals Luciferase NF-kB Rats Wistar sequence specific transcription factor Promoter Regions Genetic Cells Cultured Chemokine CCL2 030304 developmental biology Regulation of gene expression 0303 health sciences Sp1 transcription factor Kidney Base Sequence NF-kappa B Epithelial Cells Transfection NFKB1 Molecular biology Rats Transcription Factor AP-1 Kidney Tubules medicine.anatomical_structure Endocrinology Gene Expression Regulation Nephrology Cell culture |
| Zdroj: | Kidney International. 57:2011-2022 |
| ISSN: | 0085-2538 |
| DOI: | 10.1046/j.1523-1755.2000.00051.x |
| Popis: | Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kB 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-kB (NF-kB) and Western blot for the NF-kB inhibitory protein IkB. To prove the role for NF-kB, 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-kB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IkBα decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IkBα 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-kB 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-kB 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. |
| Databáze: | OpenAIRE |
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