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p53 predominantly regulates IL-6 production and suppresses synovial inflammation in fibroblast-like synoviocytes and adjuvant-induced arthritis

View Article: PubMed Central - PubMed

ABSTRACT

Background: Dominant-negative somatic mutations of p53 has been identified in the synovium of patients with rheumatoid arthritis (RA), in which interleukin (IL)-6 has been established as a pivotal inflammatory cytokine. The aim of this study was to clarify the significance of p53 in the longstanding inflammation in RA by modulating IL-6.

Methods: We established adjuvant-induced arthritis (AIA) in Lewis rats and treated them with p53 activator, and then analyzed the histopathology of the synovium and IL-6 expression. Human fibroblast-like synoviocytes (FLS) were cultured and transfected with p53-siRNA or transduced with adenovirus (Ad)-p53, and then assessed with MTT, TUNEL staining, and luciferase assay. IL-1β, tumor necrosis factor (TNF)-α and IL-17 were used to stimulate FLS, and subsequent IL-6 expression as well as relevant signal pathways were explored.

Results: p53 significantly reduced synovitis as well as the IL-6 level in the AIA rats. It controlled cell cycle arrest and proliferation, but not apoptosis. Proinflammatory cytokines inhibited p53 expression in FLS, while p53 significantly suppressed the production of IL-6. Furthermore, IL-6 expression in p53-deficient FLS was profoundly reduced by NF-kappaB, p38, JNK, and ERK inhibitors.

Conclusion: Our findings reveal a novel function of p53 in controlling inflammatory responses and suggest that p53 abnormalities in RA could sustain and accelerate synovial inflammation mainly through IL-6. p53 may be a key modulator of IL-6 in the synovium and plays a pivotal role in suppressing inflammation by interaction with the signal pathways in RA-FLS. Interfering with the p53 pathway could therefore be an effective strategy to treat RA.

No MeSH data available.


Related in: MedlinePlus

p53 did not induce apoptosis in FLS or the synovium (a–d), but did have control over cell growth (e–h). a Trypan blue staining 3 days following transfection with Ad-LacZ and Ad-p53 at different multiplicity of infection (MOI). b MTT assay 3 days following transfection with Ad-LacZ and Ad-p53 at different MOI. c Cell cycle in FLS following transfection with Ad-p53 (MOI = 100) on day (d)3, d5, and d7. d TUNEL staining on slides of collected FLS (left) and synovium 3 days following Ad-p53 treatment (right). e Deletion of p53 in FLS increased cell growth as measured by Alamar blue staining. *,p<0.05 f Deletion of p53 in FLS contribute to the lower expression of p21 thereafter. g Bax expression following knockdown of p53 was analyzed by Western blot. h Effects of p53 expression on bax promotor activity in FLS as measured by luciferase assay. non, blank control. mock, mock transfection. sc, scrambled RNA control
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Fig2: p53 did not induce apoptosis in FLS or the synovium (a–d), but did have control over cell growth (e–h). a Trypan blue staining 3 days following transfection with Ad-LacZ and Ad-p53 at different multiplicity of infection (MOI). b MTT assay 3 days following transfection with Ad-LacZ and Ad-p53 at different MOI. c Cell cycle in FLS following transfection with Ad-p53 (MOI = 100) on day (d)3, d5, and d7. d TUNEL staining on slides of collected FLS (left) and synovium 3 days following Ad-p53 treatment (right). e Deletion of p53 in FLS increased cell growth as measured by Alamar blue staining. *,p<0.05 f Deletion of p53 in FLS contribute to the lower expression of p21 thereafter. g Bax expression following knockdown of p53 was analyzed by Western blot. h Effects of p53 expression on bax promotor activity in FLS as measured by luciferase assay. non, blank control. mock, mock transfection. sc, scrambled RNA control

Mentions: To address the mechanism of p53 on the therapy of inflammatory arthritis, we first delivered p53 to FLS using adenovirus in vitro to identify the possible effects of p53 as a tumor suppressor, including regulation of the cell cycle and induction of apoptosis. As previously noted, ectopic expression of p53 did not induce apoptosis in FLS. There were no significant differences between the Ad-p53 treated group and the Ad-LacZ treated group when counting dead cells with trypan blue staining (Fig. 2a) or when measuring cell viability with the MTT assay (Fig. 2b). Cell cycle analysis by flow cytometry indicated that the p53-overexpressed FLS arrested at phase G1 following transfection with Ad-p53 on days 3, 5, and 7, respectively (Fig. 2c). TUNEL staining demonstrated less DNA fragmentation in Ad-p53 transfected FLS in vitro (Fig. 2d, left panel) and in synovium from the Ad-p53 treatment group in vivo (Fig. 2d, right panel).Fig. 2


p53 predominantly regulates IL-6 production and suppresses synovial inflammation in fibroblast-like synoviocytes and adjuvant-induced arthritis
p53 did not induce apoptosis in FLS or the synovium (a–d), but did have control over cell growth (e–h). a Trypan blue staining 3 days following transfection with Ad-LacZ and Ad-p53 at different multiplicity of infection (MOI). b MTT assay 3 days following transfection with Ad-LacZ and Ad-p53 at different MOI. c Cell cycle in FLS following transfection with Ad-p53 (MOI = 100) on day (d)3, d5, and d7. d TUNEL staining on slides of collected FLS (left) and synovium 3 days following Ad-p53 treatment (right). e Deletion of p53 in FLS increased cell growth as measured by Alamar blue staining. *,p<0.05 f Deletion of p53 in FLS contribute to the lower expression of p21 thereafter. g Bax expression following knockdown of p53 was analyzed by Western blot. h Effects of p53 expression on bax promotor activity in FLS as measured by luciferase assay. non, blank control. mock, mock transfection. sc, scrambled RNA control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5121977&req=5

Fig2: p53 did not induce apoptosis in FLS or the synovium (a–d), but did have control over cell growth (e–h). a Trypan blue staining 3 days following transfection with Ad-LacZ and Ad-p53 at different multiplicity of infection (MOI). b MTT assay 3 days following transfection with Ad-LacZ and Ad-p53 at different MOI. c Cell cycle in FLS following transfection with Ad-p53 (MOI = 100) on day (d)3, d5, and d7. d TUNEL staining on slides of collected FLS (left) and synovium 3 days following Ad-p53 treatment (right). e Deletion of p53 in FLS increased cell growth as measured by Alamar blue staining. *,p<0.05 f Deletion of p53 in FLS contribute to the lower expression of p21 thereafter. g Bax expression following knockdown of p53 was analyzed by Western blot. h Effects of p53 expression on bax promotor activity in FLS as measured by luciferase assay. non, blank control. mock, mock transfection. sc, scrambled RNA control
Mentions: To address the mechanism of p53 on the therapy of inflammatory arthritis, we first delivered p53 to FLS using adenovirus in vitro to identify the possible effects of p53 as a tumor suppressor, including regulation of the cell cycle and induction of apoptosis. As previously noted, ectopic expression of p53 did not induce apoptosis in FLS. There were no significant differences between the Ad-p53 treated group and the Ad-LacZ treated group when counting dead cells with trypan blue staining (Fig. 2a) or when measuring cell viability with the MTT assay (Fig. 2b). Cell cycle analysis by flow cytometry indicated that the p53-overexpressed FLS arrested at phase G1 following transfection with Ad-p53 on days 3, 5, and 7, respectively (Fig. 2c). TUNEL staining demonstrated less DNA fragmentation in Ad-p53 transfected FLS in vitro (Fig. 2d, left panel) and in synovium from the Ad-p53 treatment group in vivo (Fig. 2d, right panel).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Dominant-negative somatic mutations of p53 has been identified in the synovium of patients with rheumatoid arthritis (RA), in which interleukin (IL)-6 has been established as a pivotal inflammatory cytokine. The aim of this study was to clarify the significance of p53 in the longstanding inflammation in RA by modulating IL-6.

Methods: We established adjuvant-induced arthritis (AIA) in Lewis rats and treated them with p53 activator, and then analyzed the histopathology of the synovium and IL-6 expression. Human fibroblast-like synoviocytes (FLS) were cultured and transfected with p53-siRNA or transduced with adenovirus (Ad)-p53, and then assessed with MTT, TUNEL staining, and luciferase assay. IL-1&beta;, tumor necrosis factor (TNF)-&alpha; and IL-17 were used to stimulate FLS, and subsequent IL-6 expression as well as relevant signal pathways were explored.

Results: p53 significantly reduced synovitis as well as the IL-6 level in the AIA rats. It controlled cell cycle arrest and proliferation, but not apoptosis. Proinflammatory cytokines inhibited p53 expression in FLS, while p53 significantly suppressed the production of IL-6. Furthermore, IL-6 expression in p53-deficient FLS was profoundly reduced by NF-kappaB, p38, JNK, and ERK inhibitors.

Conclusion: Our findings reveal a novel function of p53 in controlling inflammatory responses and suggest that p53 abnormalities in RA could sustain and accelerate synovial inflammation mainly through IL-6. p53 may be a key modulator of IL-6 in the synovium and plays a pivotal role in suppressing inflammation by interaction with the signal pathways in RA-FLS. Interfering with the p53 pathway could therefore be an effective strategy to treat RA.

No MeSH data available.


Related in: MedlinePlus