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Serum amyloid A1 isoforms display different efficacy at Toll-like receptor 2 and formyl peptide receptor 2.

ABSTRACT: Serum amyloid A (SAA) is a major acute-phase protein and a precursor of amyloid A, the deposit of which leads to amyloidosis. Different alleles exist in SAA1, a predominant form of the human SAA gene family. Emerging evidence has shown correlations between these alleles and diseases including familiar Mediterranean fever and amyloidosis. However, it remains unclear how the proteins encoded by these SAA1 alleles act differently. Here we report the characterization of proteins encoded by SAA1.1, SAA1.3, and SAA1.5, in comparison to that encoded by SAA2.2, for their preference of the SAA receptors including formyl peptide receptor 2 (FPR2) and Toll-like receptor 2 (TLR2). SAA1.1 was more efficacious than SAA1.3 and SAA1.5 but equally efficacious to SAA2.2 in calcium mobilization and chemotaxis assays, which measure the activation of the G protein-coupled FPR2. In agreement with this, SAA1.1 and SAA2.2 induced more robust phosphorylation of ERK than SAA1.3 and SAA1.5. Only small differences were observed between the SAA1 proteins tested and SAA2.2 in TLR2-dependent NF-?B luciferase reporter assay. In comparison, SAA1.3 was most effective in stimulating ERK and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from C57BL/10ScN (Tlr4lps-del) mice, we examined the SAA isoforms for their induction of selected pro- and anti-inflammatory cytokines. SAA1.3 was most potent in the induction of TNF? and IL-1rn, whereas SAA1.5 induced robust IL-10 expression. These results show differences of the SAA1 isoforms in their selectivity for the SAA receptors, which may affect their roles in modulating inflammation and immunity.

SUBMITTER: Chen M

PROVIDER: S-EPMC4252704 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Project description:BackgroundCritically ill patients frequently develop muscle atrophy and weakness in the intensive-care-unit setting [intensive care unit-acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute-phase response are major risk factors. We reported earlier that the acute-phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation-induced muscle atrophy.MethodsWe performed cell-based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol-Myers Squibb (BMS)-345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation-induced muscle atrophy and the effects of BMS-345541 treatment.ResultsThe SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll-like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) p65 via TLR2 and TLR4 leading to an increased binding of NF-κB to NF-κB response elements in the promoter region of its target genes resulting in an increased expression of NF-κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF-κB signalling by BMS-345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS-345541 diminished inflammation-induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: -21.2% and BMS-345541: -10.4%; P < 0.05), tibialis anterior (vehicle: -22.7% and BMS-345541: -17.1%; P < 0.05) and soleus (vehicle: -21.1% and BMS-345541: -11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross-sectional area showed that BMS-345541 reduced inflammation-induced atrophy of slow/type I and fast/type II myofibers compared with vehicle-treated septic mice. BMS-345541 reversed the inflammation-induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1.ConclusionsSAA1 activates the TLR2/TLR4//NF-κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF-κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF-κB p65 atrophy pathway could have utility in combatting ICUAW.

Project description:AIM:To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice. METHODS:Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting. RESULTS:Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV. CONCLUSION:SAA-FPR2 pathway composing genes were expressed in normal murine corneas and, upon inflammatory stimuli challenge to the corneas, their expressions were up-regulated, suggesting their roles in pathogenesis of CorNV. The potential usefulness of SAA-FPR2 targets in future management of CorNV-related diseases deserves investigation.

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Serum amyloid A1 isoforms display different efficacy at Toll-like receptor 2 and formyl peptide receptor 2.

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