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Open Access Research

Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

I-Ta Lee1, Ruey-Horng Shih2, Chih-Chung Lin1, Jung-Tsan Chen2 and Chuen-Mao Yang2*

Author Affiliations

1 Department of Anesthetics, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan

2 Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan

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Cell Communication and Signaling 2012, 10:33  doi:10.1186/1478-811X-10-33

Published: 15 November 2012

Abstract

Background

In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria) can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1), which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs).

Results

We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88), Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2), and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], c-Src (PP1), p38 MAPK (SB202190), and p300 (GR343). LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody.

Conclusions

In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in renal diseases.

Keywords:
Lipopolysaccharide; Vascular cell adhesion molecule-1; Toll-like receptors; Reactive oxygen species; NADPH oxidase