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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

Effects of lipopolysaccharide (LPS) on the expression and secretion of semaphorin 4A (Sema4A). qRT-PCR was applied to detect the expression of Sema4A after stimulation by LPS at various concentrations (a) or periods (b) in synovial fibroblasts of rheumatoid arthritis RASFs, respectively. c Sema4A protein was assayed by western blot after LPS treatment in RASFs at indicated time points. d Secretion of Sema4A in the supernatant of RASFs was detected by ELISA after LPS stimulation. *P <0.05 and **P <0.01 (statistically significant differences) GAPDH glyceraldehyde-3-phosphate dehydrogenase
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Fig3: Effects of lipopolysaccharide (LPS) on the expression and secretion of semaphorin 4A (Sema4A). qRT-PCR was applied to detect the expression of Sema4A after stimulation by LPS at various concentrations (a) or periods (b) in synovial fibroblasts of rheumatoid arthritis RASFs, respectively. c Sema4A protein was assayed by western blot after LPS treatment in RASFs at indicated time points. d Secretion of Sema4A in the supernatant of RASFs was detected by ELISA after LPS stimulation. *P <0.05 and **P <0.01 (statistically significant differences) GAPDH glyceraldehyde-3-phosphate dehydrogenase

Mentions: To analyze the influence of Sema4A in immune response, RASFs were treated with the toll-like receptor (TLR) ligand [21] LPS for 24 h. On qRT-PCR there was a modest induction of Sema4A mRNA expression after stimulation of RASFs with LPS at different concentrations (Fig. 3a). Time-course analysis showed that Sema4A expression increased after 3 h upon stimulation with LPS (1 μg/mL, Fig. 3b). Western blot analysis of LPS-stimulated RASFs (1 μg/mL) confirmed the induction of Sema4A protein after 12 h (Fig. 3c). Further, the secreted level of Sema4A in the supernatant of RASFs increased steadily after LPS stimulation (Fig. 3d). These findings prompted us to study the function of Sema4A during inflammation in detail.Fig. 3


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)

Effects of lipopolysaccharide (LPS) on the expression and secretion of semaphorin 4A (Sema4A). qRT-PCR was applied to detect the expression of Sema4A after stimulation by LPS at various concentrations (a) or periods (b) in synovial fibroblasts of rheumatoid arthritis RASFs, respectively. c Sema4A protein was assayed by western blot after LPS treatment in RASFs at indicated time points. d Secretion of Sema4A in the supernatant of RASFs was detected by ELISA after LPS stimulation. *P <0.05 and **P <0.01 (statistically significant differences) 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

Fig3: Effects of lipopolysaccharide (LPS) on the expression and secretion of semaphorin 4A (Sema4A). qRT-PCR was applied to detect the expression of Sema4A after stimulation by LPS at various concentrations (a) or periods (b) in synovial fibroblasts of rheumatoid arthritis RASFs, respectively. c Sema4A protein was assayed by western blot after LPS treatment in RASFs at indicated time points. d Secretion of Sema4A in the supernatant of RASFs was detected by ELISA after LPS stimulation. *P <0.05 and **P <0.01 (statistically significant differences) GAPDH glyceraldehyde-3-phosphate dehydrogenase
Mentions: To analyze the influence of Sema4A in immune response, RASFs were treated with the toll-like receptor (TLR) ligand [21] LPS for 24 h. On qRT-PCR there was a modest induction of Sema4A mRNA expression after stimulation of RASFs with LPS at different concentrations (Fig. 3a). Time-course analysis showed that Sema4A expression increased after 3 h upon stimulation with LPS (1 μg/mL, Fig. 3b). Western blot analysis of LPS-stimulated RASFs (1 μg/mL) confirmed the induction of Sema4A protein after 12 h (Fig. 3c). Further, the secreted level of Sema4A in the supernatant of RASFs increased steadily after LPS stimulation (Fig. 3d). These findings prompted us to study the function of Sema4A during inflammation in detail.Fig. 3

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