Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-κB pathway

Kou-Gi Shyu,Bao-Wei Wang,Chiu-Mei Lin,Hang Chang

doi:10.1186/1423-0127-17-15

Kou-Gi Shyu, Bao-Wei Wang + Show 2 more

Open Access

https://doi.org/10.1186/1423-0127-17-15

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Abstract

BackgroundToll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes.MethodsNeonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected.ResultsCyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-α antibody, and p38α MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-κB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-κB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-α antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-α and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-α antibody, or p38α siRNA 30 min before Ang II and TNF-α stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-α.ConclusionsCyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-κB pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

Highlights

Toll-like receptors (TLRs) are pattern recognition receptors that play an important role in the induction of innate immunity by recognition of exogenous pathogen-associated molecular patterns and endogenous ligands [1]
Cyclic stretch enhances toll-like receptor 4 protein and mRNA expression in cultured cardiomyocytes To test the effect of cyclic stretch on the Toll-like receptor 4 (TLR4) expression, 10% and 20% of cyclic stretch were used
When cardiomyocytes were stretched at 10% elongation, the levels of TLR4 protein slightly increased after stretch for 2 h and did not increase significantly as compared to control cells without stretch from 2 to 24 h (Fig. 1A and 1B)

Summary

Introduction

Toll-like receptors (TLRs) are pattern recognition receptors that play an important role in the induction of innate immunity by recognition of exogenous pathogen-associated molecular patterns and endogenous ligands [1]. The cyclic strain system subjects cultured cells to repetitive stretching-relaxation at rates comparable to dynamic stretch overload in vivo This system has been applied widely to study the molecular mechanisms of gene expression and signal transduction in many cell types [15,16,17]. We sought to investigate whether stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes

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Conclusion