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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

A. Analysis of ERK1/2 phosphorylation by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min. Total ERK was used to ensure equal sample loading. B. IκB-α protein expression determined by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 15 min. β-actin was used to ensure equal sample loading. C, D. p65 and c-Jun expression determined by western blot using nuclear extract obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min.LaminB1 was used to ensure equal sample loading. Low panels. Densitometric analysis of at least three independent experiments. *P<0.05 and ***P<0.001 vs untreated control cells; #P<0.05, ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
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pone.0135979.g004: A. Analysis of ERK1/2 phosphorylation by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min. Total ERK was used to ensure equal sample loading. B. IκB-α protein expression determined by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 15 min. β-actin was used to ensure equal sample loading. C, D. p65 and c-Jun expression determined by western blot using nuclear extract obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min.LaminB1 was used to ensure equal sample loading. Low panels. Densitometric analysis of at least three independent experiments. *P<0.05 and ***P<0.001 vs untreated control cells; #P<0.05, ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.

Mentions: As shown in Fig 4A, IL-1α treatment led to an increase in ERK-1/2 phosphorylation, which was completely inhibited by recombinant SERPINE2 addition.


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)

A. Analysis of ERK1/2 phosphorylation by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min. Total ERK was used to ensure equal sample loading. B. IκB-α protein expression determined by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 15 min. β-actin was used to ensure equal sample loading. C, D. p65 and c-Jun expression determined by western blot using nuclear extract obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min.LaminB1 was used to ensure equal sample loading. Low panels. Densitometric analysis of at least three independent experiments. *P<0.05 and ***P<0.001 vs untreated control cells; #P<0.05, ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
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pone.0135979.g004: A. Analysis of ERK1/2 phosphorylation by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min. Total ERK was used to ensure equal sample loading. B. IκB-α protein expression determined by western blot using lysates obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 15 min. β-actin was used to ensure equal sample loading. C, D. p65 and c-Jun expression determined by western blot using nuclear extract obtained from T/C-28a2 chondrogenic cells pretreated with SERPINE2 (0.4 ng/mL) for 12 h and then challenged with IL-1α (0.5 ng/mL) for 30 min.LaminB1 was used to ensure equal sample loading. Low panels. Densitometric analysis of at least three independent experiments. *P<0.05 and ***P<0.001 vs untreated control cells; #P<0.05, ##P<0.01 and ### P<0.001 vs IL-1α-stimulated chondrocytes.
Mentions: As shown in Fig 4A, IL-1α treatment led to an increase in ERK-1/2 phosphorylation, which was completely inhibited by recombinant SERPINE2 addition.

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