Activation of the P38/CREB/MMP13 axis is associated with osteoarthritis

Abstract

Purposes: Osteoarthritis (OA) is a common joint disease characterized by the degradation of articular cartilage and joint inflammation. Interleukin-1ß induces P38/cAMP response element binding protein (CREB) pathway activation, resulting in increased expression of matrix metallopeptidase-13 (MMP13) in chondrocytes. However, the role of the P38/CREB/MMP13 axis is unclear in the progression of OA. In this study, we aimed to answer the following questions: (1) how does the P38/CREB/MMP13 axis in cartilage from patients with OA compare with control specimens? (2) Can the P38 agonist anisomycin (ANS) induce mouse OA?

Materials and methods: Surgical specimens of human cartilage were divided into OA and control groups. Surgical specimens of mouse cartilage were divided into control and ANS-induced groups. Safranin O staining of the cartilage tissues was performed to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed using these tissues to investigate messenger RNA expressions of type II collagen, aggrecan, MMP13, and ADAM metallopeptidase with thrombospondin type 1 motif 5. Phosphorylated (p)-P38, p-CREB, and MMP13 were evaluated by Western blot analysis. Anisomycin was used to activate P38, and p-P38, p-CREB, and MMP13 were evaluated by immunofluorescence and Western blot analysis.

Results: Safranin O staining showed that the extracellular matrix degraded in humans with OA and ANS-induced mouse cartilage samples. The expressions of p-P38, p-CREB, and MMP13 were all upregulated in osteoarthritic cartilage or anisomycin-induced chondrocytes, suggesting that the P38/CREB/MMP13 axis may play a role in the progression of OA.

Conclusions: The P38/CREB/MMP13 axis is active in osteoarthritic chondrocytes and may cause the degeneration of cartilage. Effective new therapy directed against this pathway could be developed.

Keywords: CREB; anisomycin; degenerative joint disease; mice.

Figure 1

P38/CREB/MMP13 is active in human osteoarthritic chondrocytes. (A) X-rays of the hip from patients with OA and the control. (B) Safranin O and fast green staining of the cartilage tissues from patients with OA and the control. Relative messenger RNA expression of (C) ADAM metallopeptidase with thrombospondin type 1 motif 5, (D) MMP13, (E) Col2, and (F) aggrecan in human osteoarthritic and control cartilage samples. (G) Western blot analysis of p-P38, p-CREB, and MMP13 in human chondrocytes from osteoarthritic and the control cartilage. The band density analysis of (H) p-P38, (I) p-CREB, and (J) MMP13 were analyzed by Image J software and normalized to ß-actin. n=10, data represent the mean±standard error of the mean. *P<0.05. Abbreviations: CREB, cAMP response element binding protein; MMP13, matrix metallopeptidase-13; OA, osteoarthritis; p, phosphorylated.

Figure 2

P38/CREB/MMP13 is active in ANS-induced human and mouse chondrocytes. (A) Immunofluorescence analysis of p-P38, p-CREB, and MMP13 expression in chondrocytes treated with or without ANS (10 µM) for 24 hrs. (B) Western blot analysis of p-P38, p-CREB, and MMP13 in chondrocytes treated with or without ANS (10 µM) for 24 hrs. The band density analysis of p-P38, p-CREB, and MMP13 from (C) human chondrocytes and (D) mouse chondrocytes were analyzed by Image J software and normalized to ß-actin. n=10, data are expressed as the mean±standard error of the mean. *P<0.05. Abbreviations: CREB, cAMP response element binding protein; MMP13, matrix metallopeptidase-13; p, phosphorylated; ANS, anisomycin.

Figure 3

ANS-induced mouse osteoarthritis. (A) X-ray of the knee from the control ANS-induced mice. (B) Safranin O and fast green staining of cartilage tissues from mice in the CON and ANS groups. (C) Immunohistochemistry analysis of MMP13 expression in the CON group and ANS-induced mouse cartilage. (D) Osteoarthritis Research Society International scores were determined according to the safranin O and fast green staining results. (E) The percentage of MMP13-positive cells was calculated according to immunohistochemistry. (F) Relative messenger RNA expression of MMP13 in the CON group and ANS-induced mouse cartilage samples. (G) Western blot analysis of p-P38, p-CREB, and MMP13 in the CON group and ANS-induced mouse cartilage. The band density analysis of (H) p-P38, (I) p-CREB, and (J) MMP13 was performed using Image J software and normalized to β-actin. n=15, Data are expressed as the mean±standard error of the mean. *P<0.05. Abbreviations: CREB, cAMP response element binding protein; MMP13, matrix metallopeptidase-13; p, phosphorylated; ANS, anisomycin.