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IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation.

Lee SY, Kwok SK, Son HJ, Ryu JG, Kim EK, Oh HJ, Cho ML, Ju JH, Park SH, Kim HY - Arthritis Res. Ther. (2013)

Bottom Line: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA).STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients.Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process.

Methods: Bcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay.

Results: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).

Conclusions: Our data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.

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Bcl-2 and Bax expression in synoviocytes in rheumatoid arthritis (RA). (A) Fibroblast-like synoviocytes (FLSs) from RA patients (n = 3) and osteoarthritis (OA) patients (n = 3) were cultured. The expression of Bcl-2 and Bax mRNA in synoviocytes was evaluated by real-time PCR. *P < 0.05, **P < 0.01 compared with OA. (B) Expression of Bcl-2 and Bax in synoviocytes was evaluated by western blot. The representative figure is shown in the left panel. The optical density (OD) ratio of Bcl-2 and Bax between FLSs in RA and OA is shown in the right panel. *P < 0.05 compared with OA. (C) Expression of Bcl-2 in FLSs in RA and OA was evaluated by confocal microscopy. Bcl-2 expressing FLSs are shown as green. Staining for nuclei by 4',6-diamidino-2-phenylindole (DAPI) is shown as blue. The representative figure is shown. (D) Apoptosis was induced by treating the cells with 1 mM sodium nitroprusside (SNP). The degree of apoptosis was assessed by flow cytometry using propidium iodide (PI) and Annexin V. A (apoptotic cells) were defined as PI-Annexin V+ cells. **P < 0.01 compared with OA.
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Figure 1: Bcl-2 and Bax expression in synoviocytes in rheumatoid arthritis (RA). (A) Fibroblast-like synoviocytes (FLSs) from RA patients (n = 3) and osteoarthritis (OA) patients (n = 3) were cultured. The expression of Bcl-2 and Bax mRNA in synoviocytes was evaluated by real-time PCR. *P < 0.05, **P < 0.01 compared with OA. (B) Expression of Bcl-2 and Bax in synoviocytes was evaluated by western blot. The representative figure is shown in the left panel. The optical density (OD) ratio of Bcl-2 and Bax between FLSs in RA and OA is shown in the right panel. *P < 0.05 compared with OA. (C) Expression of Bcl-2 in FLSs in RA and OA was evaluated by confocal microscopy. Bcl-2 expressing FLSs are shown as green. Staining for nuclei by 4',6-diamidino-2-phenylindole (DAPI) is shown as blue. The representative figure is shown. (D) Apoptosis was induced by treating the cells with 1 mM sodium nitroprusside (SNP). The degree of apoptosis was assessed by flow cytometry using propidium iodide (PI) and Annexin V. A (apoptotic cells) were defined as PI-Annexin V+ cells. **P < 0.01 compared with OA.

Mentions: Rheumatoid synoviocytes, the principal components of pannus, proliferate abnormally in a fashion similar to that of a tumor, resist apoptosis and also exhibit other features in common with metastatic cancer cells [2]. Therefore, we first examined whether there was an imbalance between pro-apoptotic and anti-apoptotic genes in FLSs from RA patients compared with FLSs from OA patients. As shown in Figure 1A, the expression of anti-apoptotic Bcl-2 mRNA was higher in FLSs from RA patients while the expression of pro-apoptotic Bax mRNA was lower in FLSs from RA patients. Western blot analysis also showed similar results (Figure 1B). Using confocal microscopy, we identified the expression of Bcl-2 at the single FLS level. As shown in Figure 1C, Bcl-2 was highly expressed in RA FLSs. These findings verified that an imbalance between pro-apoptotic and anti-apoptotic genes exists in FLSs from RA patients.


IL-17-mediated Bcl-2 expression regulates survival of fibroblast-like synoviocytes in rheumatoid arthritis through STAT3 activation.

Lee SY, Kwok SK, Son HJ, Ryu JG, Kim EK, Oh HJ, Cho ML, Ju JH, Park SH, Kim HY - Arthritis Res. Ther. (2013)

Bcl-2 and Bax expression in synoviocytes in rheumatoid arthritis (RA). (A) Fibroblast-like synoviocytes (FLSs) from RA patients (n = 3) and osteoarthritis (OA) patients (n = 3) were cultured. The expression of Bcl-2 and Bax mRNA in synoviocytes was evaluated by real-time PCR. *P < 0.05, **P < 0.01 compared with OA. (B) Expression of Bcl-2 and Bax in synoviocytes was evaluated by western blot. The representative figure is shown in the left panel. The optical density (OD) ratio of Bcl-2 and Bax between FLSs in RA and OA is shown in the right panel. *P < 0.05 compared with OA. (C) Expression of Bcl-2 in FLSs in RA and OA was evaluated by confocal microscopy. Bcl-2 expressing FLSs are shown as green. Staining for nuclei by 4',6-diamidino-2-phenylindole (DAPI) is shown as blue. The representative figure is shown. (D) Apoptosis was induced by treating the cells with 1 mM sodium nitroprusside (SNP). The degree of apoptosis was assessed by flow cytometry using propidium iodide (PI) and Annexin V. A (apoptotic cells) were defined as PI-Annexin V+ cells. **P < 0.01 compared with OA.
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Figure 1: Bcl-2 and Bax expression in synoviocytes in rheumatoid arthritis (RA). (A) Fibroblast-like synoviocytes (FLSs) from RA patients (n = 3) and osteoarthritis (OA) patients (n = 3) were cultured. The expression of Bcl-2 and Bax mRNA in synoviocytes was evaluated by real-time PCR. *P < 0.05, **P < 0.01 compared with OA. (B) Expression of Bcl-2 and Bax in synoviocytes was evaluated by western blot. The representative figure is shown in the left panel. The optical density (OD) ratio of Bcl-2 and Bax between FLSs in RA and OA is shown in the right panel. *P < 0.05 compared with OA. (C) Expression of Bcl-2 in FLSs in RA and OA was evaluated by confocal microscopy. Bcl-2 expressing FLSs are shown as green. Staining for nuclei by 4',6-diamidino-2-phenylindole (DAPI) is shown as blue. The representative figure is shown. (D) Apoptosis was induced by treating the cells with 1 mM sodium nitroprusside (SNP). The degree of apoptosis was assessed by flow cytometry using propidium iodide (PI) and Annexin V. A (apoptotic cells) were defined as PI-Annexin V+ cells. **P < 0.01 compared with OA.
Mentions: Rheumatoid synoviocytes, the principal components of pannus, proliferate abnormally in a fashion similar to that of a tumor, resist apoptosis and also exhibit other features in common with metastatic cancer cells [2]. Therefore, we first examined whether there was an imbalance between pro-apoptotic and anti-apoptotic genes in FLSs from RA patients compared with FLSs from OA patients. As shown in Figure 1A, the expression of anti-apoptotic Bcl-2 mRNA was higher in FLSs from RA patients while the expression of pro-apoptotic Bax mRNA was lower in FLSs from RA patients. Western blot analysis also showed similar results (Figure 1B). Using confocal microscopy, we identified the expression of Bcl-2 at the single FLS level. As shown in Figure 1C, Bcl-2 was highly expressed in RA FLSs. These findings verified that an imbalance between pro-apoptotic and anti-apoptotic genes exists in FLSs from RA patients.

Bottom Line: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA).STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients.Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades adjacent cartilage and bone in rheumatoid arthritis (RA). The study was undertaken to determine the effect of interleukin-17 (IL-17) on the survival and/or proliferation of FLSs from RA patients and to investigate whether signal tranducer and activator of transcription 3 (STAT3) is implicated in this process.

Methods: Bcl-2 and Bax expression in FLSs was determined using the real-time PCR and western blot analysis. The expression of Bcl-2 and phosphoSTAT3 in synovial tissues was investigated by confocal microscope. Apoptosis of FLSs was detected by Annexin V/propidium iodide staining and/or phase contrast microscopy. The proliferation of FLSs was determined by CCK-8 ELISA assay.

Results: The pro-apoptotic Bax is decreased and anti-apoptotic Bcl-2 is increased in FLSs from RA patients compared with those from patients with osteoarthritis (OA). IL-17 upregulated the expression of Bcl-2 in FLSs from RA patients, but not in FLSs from OA patients. STAT3 was found to mediate IL-17-induced Bcl-2 upregulation in FLSs from RA patients. Additionally, IL-17 promoted the survival and proliferation of FLSs from RA patients. Most importantly, treatment with STAT3 inhibitor reversed the protective effect of IL-17 on FLSs apoptosis induced by sodium nitroprusside (SNP).

Conclusions: Our data demonstrate that STAT3 is critical in IL-17-induced survival of FLS from RA patients. Therefore, therapeutic strategies that target the IL-17/STAT3 pathway might be strong candidates for RA treatment modalities.

Show MeSH
Related in: MedlinePlus