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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) promotes TNF-α and IL-1β production in lipopolysaccharide (LPS)-activated THP-1. Expression (a) and secretion (b) of Sema4A were analyzed by qRT-PCR and ELISA in THP-1 after LPS stimulation. Secretion of TNF-α (c) and IL-1β (d) was determined by ELISA after recombinant human semaphorin 4A (rhSema4A), or in combination with LPS treatment. *P <0.05 (statistically significant differences)
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Fig6: Semaphorin 4A (Sema4A) promotes TNF-α and IL-1β production in lipopolysaccharide (LPS)-activated THP-1. Expression (a) and secretion (b) of Sema4A were analyzed by qRT-PCR and ELISA in THP-1 after LPS stimulation. Secretion of TNF-α (c) and IL-1β (d) was determined by ELISA after recombinant human semaphorin 4A (rhSema4A), or in combination with LPS treatment. *P <0.05 (statistically significant differences)

Mentions: As RASFs do not secrete either TNF-α or IL-1β [24], we next determined whether Sema4A overexpression could affect the production of these cytokines by macrophages, which also play an important role in RA. We thus treated the macrophage cell line THP-1 with LPS and performed qRT-PCR analysis of Sema4A. In these cells Sema4A expression was also strongly upregulated in a dose-dependent manner upon LPS treatment (Fig. 6a). We confirmed that as in RASFs, LPS treatment also induced Sema4A secretion in THP-1 cells (Fig. 6b). We next tested whether Sema4A affected release of TNF-α and IL-1β. rhSema4A treatment for 48 h induced TNF-α and IL-1β more so than cells treated with PBS vehicle alone (Fig. 6c, d). Next, we measured TNF-α and IL-1β production by THP-1 cells treated with Sema4A for 48 h and then activated with LPS (1 μg/ml) for 24 h. Compared with the control and consistent with the observations made in RASFs, we observed that Sema4A significantly promoted the release of these cytokines in response to LPS, compared with the control without LPS stimulation (Fig. 6c, d).Fig. 6


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) promotes TNF-α and IL-1β production in lipopolysaccharide (LPS)-activated THP-1. Expression (a) and secretion (b) of Sema4A were analyzed by qRT-PCR and ELISA in THP-1 after LPS stimulation. Secretion of TNF-α (c) and IL-1β (d) was determined by ELISA after recombinant human semaphorin 4A (rhSema4A), or in combination with LPS treatment. *P <0.05 (statistically significant differences)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Semaphorin 4A (Sema4A) promotes TNF-α and IL-1β production in lipopolysaccharide (LPS)-activated THP-1. Expression (a) and secretion (b) of Sema4A were analyzed by qRT-PCR and ELISA in THP-1 after LPS stimulation. Secretion of TNF-α (c) and IL-1β (d) was determined by ELISA after recombinant human semaphorin 4A (rhSema4A), or in combination with LPS treatment. *P <0.05 (statistically significant differences)
Mentions: As RASFs do not secrete either TNF-α or IL-1β [24], we next determined whether Sema4A overexpression could affect the production of these cytokines by macrophages, which also play an important role in RA. We thus treated the macrophage cell line THP-1 with LPS and performed qRT-PCR analysis of Sema4A. In these cells Sema4A expression was also strongly upregulated in a dose-dependent manner upon LPS treatment (Fig. 6a). We confirmed that as in RASFs, LPS treatment also induced Sema4A secretion in THP-1 cells (Fig. 6b). We next tested whether Sema4A affected release of TNF-α and IL-1β. rhSema4A treatment for 48 h induced TNF-α and IL-1β more so than cells treated with PBS vehicle alone (Fig. 6c, d). Next, we measured TNF-α and IL-1β production by THP-1 cells treated with Sema4A for 48 h and then activated with LPS (1 μg/ml) for 24 h. Compared with the control and consistent with the observations made in RASFs, we observed that Sema4A significantly promoted the release of these cytokines in response to LPS, compared with the control without LPS stimulation (Fig. 6c, d).Fig. 6

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