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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

Semaphorin 4A (Sema4A) regulates the invasive phenotype of synovial fibroblasts of rheumatoid arthritis (RASFs). The invasive ability of RASFs as investigated by transwell apparatus after recombinant human semaphorin 4A (rhSema4A) (a) or silencing Sema4A (b) treatment, which were depicted in × 100 magnification. Western blot was applied to detect the expression of matrix metalloproteinase (MMP)3 and MMP9 (c), as well as alpha smooth actin (α-SMA) and vimentin (d) in RASFs after rhSema4A treatment at different concentrations. GAPDH glyceraldehyde-3-phosphate dehydrogenase
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Fig2: Semaphorin 4A (Sema4A) regulates the invasive phenotype of synovial fibroblasts of rheumatoid arthritis (RASFs). The invasive ability of RASFs as investigated by transwell apparatus after recombinant human semaphorin 4A (rhSema4A) (a) or silencing Sema4A (b) treatment, which were depicted in × 100 magnification. Western blot was applied to detect the expression of matrix metalloproteinase (MMP)3 and MMP9 (c), as well as alpha smooth actin (α-SMA) and vimentin (d) in RASFs after rhSema4A treatment at different concentrations. GAPDH glyceraldehyde-3-phosphate dehydrogenase

Mentions: The effect of Sema4A on the biological activity of human RASFs was examined. RASFs were treated with various concentrations of rhSema4A in serum-containing medium for 24 h and the invasive viability was determined using the transwell chamber. As shown in Fig. 2a, the in vitro invasive ability increased when RASFs in the upper compartment were stimulated with rhSema4A at different concentrations for 24 h. In contrast, silencing Sema4A (100 nM) with siRNA showed that the invasion was blocked in comparison with the control groups (Fig. 2b). Matrix metalloproteinases (MMPs) degrade the extracellular matrix, promoting cell invasion and their upregulation has also been linked to the pathological process of RA [18]. To further support the pro-invasion ability of Sema4A, we then examined the effect of Sema4A on MMP expression. As shown in Fig. 2c, rhSema4A treatment induced MMP-3 and MMP-9 expression, which are important for cell invasion [18]. In addition, RASFs, undergoing myofibroblast or epithelial-mesenchymal transition (EMT)-like transition, would be more likely to invade [19, 20]. Accordingly, our data showed that rhSema4A treatment could also promote the above transition as demonstrated by the induction of the myofibroblast maker, α-smooth muscle actin (SMA) and the EMT marker, vimentin (Fig. 2d) [19, 20].Fig. 2


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)

Semaphorin 4A (Sema4A) regulates the invasive phenotype of synovial fibroblasts of rheumatoid arthritis (RASFs). The invasive ability of RASFs as investigated by transwell apparatus after recombinant human semaphorin 4A (rhSema4A) (a) or silencing Sema4A (b) treatment, which were depicted in × 100 magnification. Western blot was applied to detect the expression of matrix metalloproteinase (MMP)3 and MMP9 (c), as well as alpha smooth actin (α-SMA) and vimentin (d) in RASFs after rhSema4A treatment at different concentrations. GAPDH glyceraldehyde-3-phosphate dehydrogenase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549119&req=5

Fig2: Semaphorin 4A (Sema4A) regulates the invasive phenotype of synovial fibroblasts of rheumatoid arthritis (RASFs). The invasive ability of RASFs as investigated by transwell apparatus after recombinant human semaphorin 4A (rhSema4A) (a) or silencing Sema4A (b) treatment, which were depicted in × 100 magnification. Western blot was applied to detect the expression of matrix metalloproteinase (MMP)3 and MMP9 (c), as well as alpha smooth actin (α-SMA) and vimentin (d) in RASFs after rhSema4A treatment at different concentrations. GAPDH glyceraldehyde-3-phosphate dehydrogenase
Mentions: The effect of Sema4A on the biological activity of human RASFs was examined. RASFs were treated with various concentrations of rhSema4A in serum-containing medium for 24 h and the invasive viability was determined using the transwell chamber. As shown in Fig. 2a, the in vitro invasive ability increased when RASFs in the upper compartment were stimulated with rhSema4A at different concentrations for 24 h. In contrast, silencing Sema4A (100 nM) with siRNA showed that the invasion was blocked in comparison with the control groups (Fig. 2b). Matrix metalloproteinases (MMPs) degrade the extracellular matrix, promoting cell invasion and their upregulation has also been linked to the pathological process of RA [18]. To further support the pro-invasion ability of Sema4A, we then examined the effect of Sema4A on MMP expression. As shown in Fig. 2c, rhSema4A treatment induced MMP-3 and MMP-9 expression, which are important for cell invasion [18]. In addition, RASFs, undergoing myofibroblast or epithelial-mesenchymal transition (EMT)-like transition, would be more likely to invade [19, 20]. Accordingly, our data showed that rhSema4A treatment could also promote the above transition as demonstrated by the induction of the myofibroblast maker, α-smooth muscle actin (SMA) and the EMT marker, vimentin (Fig. 2d) [19, 20].Fig. 2

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