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Expression of Semaphorin 4A and its potential role in rheumatoid arthritis.

Wang L, Song G, Zheng Y, Tan W, Pan J, Zhao Y, Chang X - Arthritis Res. Ther. (2015)

Bottom Line: Treatment with recombinant human Sema4A (rhSema4A) or small interfering RNA (siRNA) was applied to examine its effect on the biological activity of synovial fibroblasts of RA (RASFs).Increased levels of Sema4A were detected in the synovial tissue and fluid of patients with RA compared with those with OA.Further, increased expression of Sema4A is required to promote inflammation of RA.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Medicinal Biotechnology, Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Shandong Academy of Medicinal Sciences, Jinan, China. wanglin.83@163.com.

ABSTRACT

Introduction: Semaphorin 4A (Sema4A) plays critical roles in many physiological and pathological processes including neuronal development, angiogenesis, immune response regulation, autoimmunity, and infectious diseases. The present study aimed to investigate its expression and biological activity in rheumatoid arthritis (RA).

Methods: RNA and protein were isolated from synovial tissues in RA and osteoarthritis (OA) patients. Treatment with recombinant human Sema4A (rhSema4A) or small interfering RNA (siRNA) was applied to examine its effect on the biological activity of synovial fibroblasts of RA (RASFs). Expression of Sema4A and NF-κB were measured by quantitative RT-PCR (qRT-PCR) and Western blot after lipopolysaccharide (LPS) stimulation. Chromatin immunoprecipitation (ChIP) and siRNA targeting p50 and p60 were applied to detect the regulation of Nuclear factor kappa (NF-κB) on Sema4A. Sema4A, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) secretion were measured by ELISA-based assays.

Results: Increased levels of Sema4A were detected in the synovial tissue and fluid of patients with RA compared with those with OA. Furthermore, synovial fluid level of Sema4A correlated with Disease Activity Score (DAS) in RA. Treatment with rhSema4A promoted invasion of RASFs by upregulating the expression of Matrix metallopeptidase3 (MMP3), MMP9, alpha-smooth muscle actin(α-SMA), and Vimentin, and exacerbated inflammation by promoting the production of IL-6 in RASFs, as well as IL-1β and TNF-α in THP-1 cells. The induction of IL-6 and TNF-α by Sema4A was confirmed at the protein level in fluid samples from patients with RA. Knock-down experiments showed the participation of Plexin B1 towards rhSema4A in the induction of cytokines. In addition, LPS stimulation induced Sema4A expression in RASFs in an NF-κB-dependent manner, and rhSema4A treatment could also activate NF-κB signaling.

Conclusions: These findings suggest an NF-κB-dependent modulation of Sema4A in the immune response. Further, increased expression of Sema4A is required to promote inflammation of RA.

No MeSH data available.


Related in: MedlinePlus

Regulation of semaphorin 4A (Sema4A) expression by NF-κB. a Schematic illustration as the binding of NF-κB to the promoter of Sema4A. b Synovial fibroblasts of rheumatoid arthritis (RASFs) and THP-1 cells were transfected with 50 nM of control, p50 or p65-siRNA, or p50 and p65 combined and incubated for the indicated time. Total extracts were separated by SDS-PAGE and western blot analysis was conducted for measurement of Sema4A protein levels. c Chromatin immunoprecipitation (ChIP) analysis showed that p65 was recruited to the Sema4A promoter after treatment with lipopolysaccharide (LPS) for 90 minutes. On ChIP analysis with control primers against distinct promoter regions there was no recruitment of p65. Input, chromatin input before immunoprecipitation; Anti-p65, immunoprecipitated chromatin with anti-p65 antibody; IgG, immunoprecipitated chromatin with control IgG. Effect of NF-κB inhibitor treatment on the LPS-induced Sema4A was detected by qRT-PCR (d) and western blot (e)
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Fig4: Regulation of semaphorin 4A (Sema4A) expression by NF-κB. a Schematic illustration as the binding of NF-κB to the promoter of Sema4A. b Synovial fibroblasts of rheumatoid arthritis (RASFs) and THP-1 cells were transfected with 50 nM of control, p50 or p65-siRNA, or p50 and p65 combined and incubated for the indicated time. Total extracts were separated by SDS-PAGE and western blot analysis was conducted for measurement of Sema4A protein levels. c Chromatin immunoprecipitation (ChIP) analysis showed that p65 was recruited to the Sema4A promoter after treatment with lipopolysaccharide (LPS) for 90 minutes. On ChIP analysis with control primers against distinct promoter regions there was no recruitment of p65. Input, chromatin input before immunoprecipitation; Anti-p65, immunoprecipitated chromatin with anti-p65 antibody; IgG, immunoprecipitated chromatin with control IgG. Effect of NF-κB inhibitor treatment on the LPS-induced Sema4A was detected by qRT-PCR (d) and western blot (e)

Mentions: Using the position-weight matrices available in GeneCards (Genomics for SEMA4A Gene, Regulatory Elements for SEMA4A Gene) [22], an NF-κB binding site on the Sema4A promoter was identified (Fig. 4a). To further clarify the role of the NF-κB subunits p50 and p65 in the regulation of Sema4A [23], we used siRNA knockdown (both 100 nM) to silence p50 and p65 in RASFs. siRNA constructs effective at silencing p50 or p65 (Fig. 4b), individually and in combination, also inhibited Sema4A expression (Fig. 4b). We also confirmed the above results in THP-1 cells with p50 and p65 knocked down individually and in combination, resulting in suppressed Sema4A expression (Fig. 4b). As NF-κB could regulate Sema4A expression at the transcriptional level, we next investigated whether NF-κB could directly bind to its promoter region. The results of ChIP analysis showed that more p65 was bound to the Sema4A promoter after treatment (Fig. 4c). Importantly, blocking NF-κB signaling attenuated the LPS-induced expression of Sema4A at the transcriptional (Fig. 4d) and translational (Fig. 4e) levels, which demonstrates that the increased recruitment of NF-κB to the Sema4A promoter after LPS stimulation is indeed responsible for its induction in RASFs.Fig. 4


Expression of Semaphorin 4A and its potential role in rheumatoid arthritis.

Wang L, Song G, Zheng Y, Tan W, Pan J, Zhao Y, Chang X - Arthritis Res. Ther. (2015)

Regulation of semaphorin 4A (Sema4A) expression by NF-κB. a Schematic illustration as the binding of NF-κB to the promoter of Sema4A. b Synovial fibroblasts of rheumatoid arthritis (RASFs) and THP-1 cells were transfected with 50 nM of control, p50 or p65-siRNA, or p50 and p65 combined and incubated for the indicated time. Total extracts were separated by SDS-PAGE and western blot analysis was conducted for measurement of Sema4A protein levels. c Chromatin immunoprecipitation (ChIP) analysis showed that p65 was recruited to the Sema4A promoter after treatment with lipopolysaccharide (LPS) for 90 minutes. On ChIP analysis with control primers against distinct promoter regions there was no recruitment of p65. Input, chromatin input before immunoprecipitation; Anti-p65, immunoprecipitated chromatin with anti-p65 antibody; IgG, immunoprecipitated chromatin with control IgG. Effect of NF-κB inhibitor treatment on the LPS-induced Sema4A was detected by qRT-PCR (d) and western blot (e)
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Fig4: Regulation of semaphorin 4A (Sema4A) expression by NF-κB. a Schematic illustration as the binding of NF-κB to the promoter of Sema4A. b Synovial fibroblasts of rheumatoid arthritis (RASFs) and THP-1 cells were transfected with 50 nM of control, p50 or p65-siRNA, or p50 and p65 combined and incubated for the indicated time. Total extracts were separated by SDS-PAGE and western blot analysis was conducted for measurement of Sema4A protein levels. c Chromatin immunoprecipitation (ChIP) analysis showed that p65 was recruited to the Sema4A promoter after treatment with lipopolysaccharide (LPS) for 90 minutes. On ChIP analysis with control primers against distinct promoter regions there was no recruitment of p65. Input, chromatin input before immunoprecipitation; Anti-p65, immunoprecipitated chromatin with anti-p65 antibody; IgG, immunoprecipitated chromatin with control IgG. Effect of NF-κB inhibitor treatment on the LPS-induced Sema4A was detected by qRT-PCR (d) and western blot (e)
Mentions: Using the position-weight matrices available in GeneCards (Genomics for SEMA4A Gene, Regulatory Elements for SEMA4A Gene) [22], an NF-κB binding site on the Sema4A promoter was identified (Fig. 4a). To further clarify the role of the NF-κB subunits p50 and p65 in the regulation of Sema4A [23], we used siRNA knockdown (both 100 nM) to silence p50 and p65 in RASFs. siRNA constructs effective at silencing p50 or p65 (Fig. 4b), individually and in combination, also inhibited Sema4A expression (Fig. 4b). We also confirmed the above results in THP-1 cells with p50 and p65 knocked down individually and in combination, resulting in suppressed Sema4A expression (Fig. 4b). As NF-κB could regulate Sema4A expression at the transcriptional level, we next investigated whether NF-κB could directly bind to its promoter region. The results of ChIP analysis showed that more p65 was bound to the Sema4A promoter after treatment (Fig. 4c). Importantly, blocking NF-κB signaling attenuated the LPS-induced expression of Sema4A at the transcriptional (Fig. 4d) and translational (Fig. 4e) levels, which demonstrates that the increased recruitment of NF-κB to the Sema4A promoter after LPS stimulation is indeed responsible for its induction in RASFs.Fig. 4

Bottom Line: Treatment with recombinant human Sema4A (rhSema4A) or small interfering RNA (siRNA) was applied to examine its effect on the biological activity of synovial fibroblasts of RA (RASFs).Increased levels of Sema4A were detected in the synovial tissue and fluid of patients with RA compared with those with OA.Further, increased expression of Sema4A is required to promote inflammation of RA.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Medicinal Biotechnology, Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Shandong Academy of Medicinal Sciences, Jinan, China. wanglin.83@163.com.

ABSTRACT

Introduction: Semaphorin 4A (Sema4A) plays critical roles in many physiological and pathological processes including neuronal development, angiogenesis, immune response regulation, autoimmunity, and infectious diseases. The present study aimed to investigate its expression and biological activity in rheumatoid arthritis (RA).

Methods: RNA and protein were isolated from synovial tissues in RA and osteoarthritis (OA) patients. Treatment with recombinant human Sema4A (rhSema4A) or small interfering RNA (siRNA) was applied to examine its effect on the biological activity of synovial fibroblasts of RA (RASFs). Expression of Sema4A and NF-κB were measured by quantitative RT-PCR (qRT-PCR) and Western blot after lipopolysaccharide (LPS) stimulation. Chromatin immunoprecipitation (ChIP) and siRNA targeting p50 and p60 were applied to detect the regulation of Nuclear factor kappa (NF-κB) on Sema4A. Sema4A, interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) secretion were measured by ELISA-based assays.

Results: Increased levels of Sema4A were detected in the synovial tissue and fluid of patients with RA compared with those with OA. Furthermore, synovial fluid level of Sema4A correlated with Disease Activity Score (DAS) in RA. Treatment with rhSema4A promoted invasion of RASFs by upregulating the expression of Matrix metallopeptidase3 (MMP3), MMP9, alpha-smooth muscle actin(α-SMA), and Vimentin, and exacerbated inflammation by promoting the production of IL-6 in RASFs, as well as IL-1β and TNF-α in THP-1 cells. The induction of IL-6 and TNF-α by Sema4A was confirmed at the protein level in fluid samples from patients with RA. Knock-down experiments showed the participation of Plexin B1 towards rhSema4A in the induction of cytokines. In addition, LPS stimulation induced Sema4A expression in RASFs in an NF-κB-dependent manner, and rhSema4A treatment could also activate NF-κB signaling.

Conclusions: These findings suggest an NF-κB-dependent modulation of Sema4A in the immune response. Further, increased expression of Sema4A is required to promote inflammation of RA.

No MeSH data available.


Related in: MedlinePlus