Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin

Abstract

Objective: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP.

Methods: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed.

Results: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner.

Conclusion: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Fig. 1. Western blotting analysis of human OA cartilage samples and ADAMTS-7 (TS-7)- and ADAMTS-12 (TS-12)-mediated COMP digestion

Samples were resolved on 8% SDS-PAGE gels, under reducing conditions, and COMP was detected using an anti-COMP antiserum. Intact COMP monomer, 110-kDa fragment and additional fragment in OA cartilage are indicated with arrow 1, 2, 3, respectively.

Fig. 2. ADAMTS-12 and ADAMTS-7 blocking antibodies inhibit TNF-α- or IL-1β-induced COMP degradation

(A) Upregulation of ADAMTS-7 and ADAMTS-12 by TNF-α and IL-1β. The units are arbitrary and the leftmost bar in each group indicates a relative level of 1. * p < .05 vs. untreated controls. (B) & (C) Antibody blocking assays. OA cartilage explants were cultured in the presence of 5 ng/ml of TNF-α (B) or 5 ng/ml of IL-1β (C) with blocking antibodies, as indicated, for 7 days. The media were separated on non-reduced SDS-PAGE gels and COMP was detected using an anti-COMP antibody. Intact COMP and its degradative fragment are indicated with arrow and arrowhead respectively.

Fig. 3. Reduced expression of ADAMTS7 or/and ADAMTS-12 by siRNA silencing inhibits the degradation of COMP in human chondrocytes

(A) siRNAs against ADAMTS-7 and ADAMTS-12 efficiently suppress the expression of their target molecules, assayed by immumofluorescence cell staining. Immortalized human chondrocytes, C-28/I2, transfected with either pSUPER plasmid (CTR), pSUPER-ADAMTS-7 siRNA (siTS7) or pSUPER-ADAMTS-12 siRNA (siTS12) were stained with either anti-ADAMTS-7 (left panel) or anti-ADAMTS-12 (right panel). The nuclei were stained with DAPI and the overlapping of these two signals is shown as “merge”. (B) & (C) Knockdown of either ADAMTS-7 or/and ADAMTS-12 dramatically inhibits COMP degradation. The C-28/12 cells were transfected with either ADAMTS-7 siRNA (siTS7), ADAMTS-12 siRNA (siTS7), both (siTS7 +siTS12) or pSUPER control (CTR), were cultured in the presence of 5 ng/ml of TNF- α (B) or 5 ng/ml of Il-1β (C) for 7 days. The media were separated on reduced SDS-PAGE gels and COMP was detected using an anti-COMP antibody. Intact COMP and its degradative fragment are indicated with arrow and arrowhead, respectively.

Fig. 4. Cleavage of α ₂M by ADAMTS-7 and ADAMTS-12

In vitro digestion assay of α ₂M by lower (A) or higher (B) amount of ADAMTS-7. 0.14 uM a₂M was incubated in the absence or presence of various amount of ADAMTS-7, as indicated, for 2 h at 37 °C. The products were then separated by 8% non-reduced SDS-PAGE and visualized by Coomassie blue staining. (C) Digestion of α ₂M by ADAMTS-12. 0.10 μM α ₂M was incubated in the absence or presence of various amount of ADAMTS-12, as indicated, for 2 h at 37 °C. The products were then separated by 6% non-reduced SDS-PAGE and visualized by Coomassie blue staining. Arrow 1, 2, 3 indicate the intact α 2M, ∼180 kDa and 105 kDa resulted fragments, respectively.

Fig. 5. α₂M inhibits ADAMTS-7-mediated COMP degradation in a dose-dependent manner, assayed by Coomassie blue staining (A) and Western Blotting (B)

0.33 μM ADAMTS-7 was first incubated with increasing concentrations of α ₂M, as indicated, for 2 h at 37 °C, then 0.17 μM COMP was added and allowed to incubate for additional 2 h at 37 °C. The resulting products were analyzed by 8% non-reduced SDS-PAGE and visualized by either Coomassie blue staining (A) or Western blotting with anti-COMP antibody (B). Arrows 1, 2, 3 and 4 in (A) indicate the intact COMP, the 180-kDa fragment of α 2M, the 110-kDa fragment of COMP and the 105-kDa fragment of α2M, respectively. In panel (B), arrow and arrowhead indicate intact COMP and its degradative fragment, respectively. Lanes 1 and 2 indicate that anti-COMP antibody does not cross-recognize either intact a2M nor its 180 kD degradative fragment. Lanes 4-8 indicate that intact COMP was protected from ADAMTS-7 cleavage by α ₂M in a dose-dependent manner.

Fig. 6. α₂M inhibits ADAMTS-12-mediated COMP degradation in a does-dependent manner, assayed by Coomassie blue staining (A) and Western Blotting (B)

0.20 uM ADAMTS-12 was first incubated with increasing concentrations of α ₂M, as indicated, for 2 h at 37 °C, then 0.10 uM COMP was added and allowed to incubate for an additional 2 h at 37 °C. The resultant products were analyzed by 8% non-reduced SDS-PAGE and visualized by either Coomassie blue staining (A) or Western blotting with anti-COMP antibody (B). Arrows 1, 2, 3 and 4 in panel A indicate the intact COMP, the 180-kDa fragment of α 2M, the 110-kDa fragment of COMP and the 105-kDa fragment of α2M, respectively. In panel B, the intact COMP and its 110-kDa degradative fragment are indicated. Lanes 1 and 2 indicate that anti-COMP antibody does not cross-react with either intact α2M or its 180 kD degradative fragment. Lanes 4-9 indicate that intact COMP was protected from ADAMTS-7 cleavage by α2M in a dose-dependent manner.