Yonsei Med J.  2011 Jul;52(4):610-615. 10.3349/ymj.2011.52.4.610.

Toll-Like Receptor 4 Signaling is Involved in IgA-Stimulated Mesangial Cell Activation

• Lim BJ
• Lee D
• Hong SW
• Jeong HJ

• KMID: 1727993
• DOI: http://doi.org/10.3349/ymj.2011.52.4.610

Abstract

PURPOSE
Deposition of polymeric IgA1 in the kidney mesangium is the hallmark of IgA nephropathy, but the molecular mechanisms of IgA-mediated mesangial responses and inflammatory injuries remain poorly understood. We hypothesize that Toll-like receptor 4 (TLR4) is involved in IgA-induced mesangial cell activation.
MATERIALS AND METHODS
Mouse mesangial cells were stimulated with lipopolysaccharide (LPS) (1 microg/mL), IgA (20 microg/mL), or both, and TLR4 expression was measured by real time RT-PCR and Western blot. Intracellular responses to LPS or IgA were assessed by Western blot for ERK1/2, JNK, p38 MAP kinases (MAPKs), Ikappa-Balpha degradation and fibronectin secretion. MCP-1 secretion was assessed by ELISA. Small interfering RNA (siRNA) of TLR4 was used to confirm that the effects were caused by TLR4 activity.
RESULTS
LPS- or IgA-treatment upregulated the levels of TLR4 mRNA and protein in cultured MMC at 24 h. LPS and IgA induced rapid phosphorylation of MAPKs, but degradation of Ikappa-Balpha was observed only in LPS-treated MMC. LPS, but not IgA, induced increased secretion of MCP-1 and fibronectin at 24 h or 48 h. Combined LPS and IgA treatment did not cause additional increases in TLR4 mRNA and protein levels or Ikappa-Balpha degradation, and MCP-1 and fibronectin secretions were less than with LPS alone. LPS- or IgA-induced TLR4 protein levels and MAPK activation were inhibited by transfection with TLR4 siRNA.
CONCLUSION
These results indicate that the activation of MAPKs and MCP-1 secretion are mediated by TLR4, at least in part, in IgA-treated mesangial cells. TLR4 is involved in mesangial cell injury by induction of pro-inflammatory cytokines in IgA nephropathy.

Keyword

IgA nephropathy; mesangial cell; cytokine; toll-like receptor

MeSH Terms

Animals
Chemokine CCL2/secretion
Enzyme-Linked Immunosorbent Assay
Extracellular Signal-Regulated MAP Kinases/metabolism
Fibronectins/secretion
Glomerulonephritis, IGA/*metabolism
I-kappa B Proteins/metabolism
Mesangial Cells/*metabolism/secretion
Mice
Mice, Transgenic
Phosphorylation
RNA Interference
RNA, Messenger/metabolism
*Signal Transduction
Toll-Like Receptor 4/antagonists & inhibitors/genetics/*metabolism

Figure

• 

  Fig. 1 TLR4 mRNA level was upregulated by LPS at 12 hours and 24 hours, and by IgA at 24 hours. Combined LPS and IgA stimulation did not increase the TLR4 mRNA level. *p<0.05 vs. 24 hours control, †p<0.05 vs. 12 hours control, ‡p<0.05 vs. 12 hours LPS. TLR4, Toll-like receptor 4; mRNA, messenger ribonucleic acid; LPS, lipopolysaccharide.

• 

  Fig. 2 TLR4 protein expression was increased by LPS or IgA stimulation at 24 hours and 48 hours. However, combined stimulation of LPS and IgA did not show an additive effect. *p<0.05 vs. 48 hours control, †p<0.05 vs. 24 hours control. TLR4, Toll-like receptor 4; mRNA, messenger ribonucleic acid; LPS, lipopolysaccharide.

• 

  Fig. 3 LPS, IgA, and combined LPS and IgA treatment induced rapid phosphorylation of ERK1/2, JNK, and p38 MAPKs at 5 minutes. LPS, lipopolysaccharide; MAPKS, MAP kinases.

• 

  Fig. 4 Iκ-Bα degradation was observed at 60 minutes in LPS-treated MMC, but not in IgA- and combined LPS and IgA-treated MMC. *p<0.05 vs. control. LPS, lipopolysaccharide; MMC, mouse mesangial cells.

• 

  Fig. 5 MCP-1 secretion in the culture media increased at 48 hours in LPS-treated MMC, but was significantly lower after treatment with combined LPS and IgA compared to LPS alone. *p<0.05 vs. 48 hours control, †p<0.05 vs. 48 hours LPS, ‡p<0.05 vs. 48 hours IgA. LPS, lipopolysaccharide; MMC, mouse mesangial cells.

• 

  Fig. 6 Fibronectin secretion in culture media increased at 24 hours and 48 hours in LPS-treated MMC, but did not increase after treatment with IgA alone or combined LPS and IgA. *p<0.05 vs. 24 hours control, †p<0.05 vs. 48 hours control. LPS, lipopolysaccharide; MMC, mouse mesangial cells.

• 

  Fig. 7 LPS- or IgA-induced TLR4 protein expression and MAPK activation were inhibited by transfection with TLR4 siRNA. LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; MAPK, MAP kinase; siRNA, small interfering ribonucleic acid.

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