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

Abstract

BackgroundIn 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).ResultsWe 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.ConclusionsIn 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.

Glycolaldehyde-derived advanced glycation end products (glycol-AGEs)-induced vascular smooth muscle cell dysfunction is regulated by the AGES-receptor (RAGE) axis in endothelium

“…These factors have important functions in the regulation of the inflammatory and proliferative responses in VSMCs, which are a major component of blood vessels (Chistiakov et al 2015). In addition, VSMCs showed increased proliferation and production of fibronectin owing to AGEs-induced inflammatory cytokine production (Kume et al 2000;Sakata et al 2000;Lee et al 2012). In our experiments, glycol-AGEs were found to increase the expressions of RAGE, MCP-1, IL-6, and PAI-1 in VSMCs for 24 h when cells were cocultured with HUVECs and THP-1 cells ( Figure 5).…”

Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells

“…MAPKs are important intracellular signalings and play critical roles in regulating inflammatory responses (12). LPS has been shown to mediate p42/p44 MAPK, p38 MAPK, and JNK1/2 activation (10,29). Moreover, p42/p44 MAPK has been shown to participate in the regulation of a large variety of processes, including cell adhesion, cell migration, cell survival, differentiation, metabolism, proliferation, and transcription (26).…”

“…c-Src has been shown to regulate ICAM-1 expression in various cell types (9,16). In addition, LPS can mediate c-Src activation and then cause the inflammatory responses (7,10). Previous studies indicated that c-Src regulates platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) transactivation (13), which further promotes inflammatory responses.…”

Hypoxic Trophoblast HMGB1 Induces Endothelial Cell Hyperpermeability via the TRL-4/Caveolin-1 Pathway

“…Immunoprecipitation assay was performed as described previously (43). HUVEC lysates or kidney lysates of C57BL/6 mice containing 200 mg of total protein were incubated with rabbit anti-CAV-1 (Abcam; 1/100 v/w) or rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4˚C on a rotating device, followed by the addition of Protein A-G PLUS Agarose (Santa Cruz Biotechnology) overnight under the same conditions.…”