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SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways.

Santoro A, Conde J, Scotece M, Abella V, Lois A, Lopez V, Pino J, Gomez R, Gomez-Reino JJ, Gualillo O - PLoS ONE (2015)

Bottom Line: We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression.This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

View Article: PubMed Central - PubMed

Affiliation: SERGAS (Servizo Galego de Saude) and IDIS (Instituto de Investigación Sanitaria de Santiago), the NEIRID Lab (Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases), Research Laboratory 9, Santiago University Clinical Hospital, Santiago de Compostela, Spain; University of Naples Federico II, Dept. of Pharmacy, 80138, Naples, Italy.

ABSTRACT

Objectives: Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored.

Methods: SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis.

Results: Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.

Conclusions: Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

No MeSH data available.


Related in: MedlinePlus

MMP-13 mRNA and protein levels were determined by real-time PCR and western blot analysis respectively.A. Human MMP-13 mRNA expression in T/C-28a2 chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. B. Representative western blot of human MMP-13 protein expression in lysates obtained from T/C-28a2 chondrogenic cells treated with with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Low panel. Densitometric analysis of at least three independent experiments. C. Human MMP-13 mRNA expression in human primary chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. Data are means ± S.E.M. of at least 3 independent experiments. **P<0.01 and ***P<0.001 vs untreated control cells; ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
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pone.0135979.g002: MMP-13 mRNA and protein levels were determined by real-time PCR and western blot analysis respectively.A. Human MMP-13 mRNA expression in T/C-28a2 chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. B. Representative western blot of human MMP-13 protein expression in lysates obtained from T/C-28a2 chondrogenic cells treated with with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Low panel. Densitometric analysis of at least three independent experiments. C. Human MMP-13 mRNA expression in human primary chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. Data are means ± S.E.M. of at least 3 independent experiments. **P<0.01 and ***P<0.001 vs untreated control cells; ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.

Mentions: Once determined the modulation of SERPINE2 expression by IL-1α, we sought to analyze whether this protein has a regulatory effect on the expression of one of the most relevant collagenases, MMP-13. As shown in Fig 2A, IL-1α showed a significant up-regulation of MMP-13 mRNA expression after 24h challenge in T/C-28a2 chondrocytes. Interestingly, recombinant SERPINE2 was able to revert, efficiently, the induction of MMP-13 elicited by IL-1α. These results were also confirmed in terms of protein expression (Fig 2B).


SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways.

Santoro A, Conde J, Scotece M, Abella V, Lois A, Lopez V, Pino J, Gomez R, Gomez-Reino JJ, Gualillo O - PLoS ONE (2015)

MMP-13 mRNA and protein levels were determined by real-time PCR and western blot analysis respectively.A. Human MMP-13 mRNA expression in T/C-28a2 chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. B. Representative western blot of human MMP-13 protein expression in lysates obtained from T/C-28a2 chondrogenic cells treated with with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Low panel. Densitometric analysis of at least three independent experiments. C. Human MMP-13 mRNA expression in human primary chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. Data are means ± S.E.M. of at least 3 independent experiments. **P<0.01 and ***P<0.001 vs untreated control cells; ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
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Related In: Results  -  Collection

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pone.0135979.g002: MMP-13 mRNA and protein levels were determined by real-time PCR and western blot analysis respectively.A. Human MMP-13 mRNA expression in T/C-28a2 chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. B. Representative western blot of human MMP-13 protein expression in lysates obtained from T/C-28a2 chondrogenic cells treated with with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. β-actin was used to ensure equal sample loading. Low panel. Densitometric analysis of at least three independent experiments. C. Human MMP-13 mRNA expression in human primary chondrocytes incubated with SERPINE2 (0.4 ng/mL) in presence or not of IL-1α (0.5 ng/mL) for 24 h. Data are means ± S.E.M. of at least 3 independent experiments. **P<0.01 and ***P<0.001 vs untreated control cells; ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
Mentions: Once determined the modulation of SERPINE2 expression by IL-1α, we sought to analyze whether this protein has a regulatory effect on the expression of one of the most relevant collagenases, MMP-13. As shown in Fig 2A, IL-1α showed a significant up-regulation of MMP-13 mRNA expression after 24h challenge in T/C-28a2 chondrocytes. Interestingly, recombinant SERPINE2 was able to revert, efficiently, the induction of MMP-13 elicited by IL-1α. These results were also confirmed in terms of protein expression (Fig 2B).

Bottom Line: We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression.This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

View Article: PubMed Central - PubMed

Affiliation: SERGAS (Servizo Galego de Saude) and IDIS (Instituto de Investigación Sanitaria de Santiago), the NEIRID Lab (Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases), Research Laboratory 9, Santiago University Clinical Hospital, Santiago de Compostela, Spain; University of Naples Federico II, Dept. of Pharmacy, 80138, Naples, Italy.

ABSTRACT

Objectives: Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored.

Methods: SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis.

Results: Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.

Conclusions: Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

No MeSH data available.


Related in: MedlinePlus