Physiological loading of joints prevents cartilage degradation through CITED2

Abstract

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Figure 1.

Passive motion loading prevents cartilage degradation. Schematics: representations of the short protocol (A), where the rat hind limb is immobilized for 6 h interrupted by 1 h of passive motion or release from immobilization under anesthesia, or the extended protocol (D), where the rat limb is immobilized for 7 d with or without a 1-h/d passive motion protocol. Graphs: qPCR showing fold-change in mRNA levels of MMP-1 (B, E) or CITED2 (C, F), and MMP-1 (enzyme) activity (G) after immobilization (Im), passive motion loading (Pm), sham treatment (Sh), or no treatment (Con). Images: Safranin O staining and immunohistochemical localization of MMP-1, type II collagen denaturation (Col2–3/4M antibody), and CITED2 in articular cartilage from rats undergoing the extended protocol (H). For statistical analysis, 1-way ANOVA and Tukey's post hoc test were performed; n = 5 rats/group. Scale bar = 100 μm. *P < 0.05 vs. control.

Figure 2.

CITED2 is required for load-induced down-regulation of MMP-1. A–E) qPCR showing the effect of IHP loads (MPa as shown, 1 Hz, 1 h) applied to articular cartilage explants (n=5 rats/group) (A, B) or to chondrocytes on CITED2 and MMP-1 mRNA expression (C, D) and CITED2 and MMP-1 protein expression (E) detected by Western blot. F–H) qPCR (F, H) and Western blots (G) showing, respectively, the effect of transfecting chondrocytes with pcDNA3.1-CITED2 or small interfering RNAs for CITED2 (si-CITED2) or scrambled RNA (si-Scramble) on MMP-1 mRNA and protein expression in response to IHP (2.5 MPa, 1 Hz, 1 h) in the absence or presence of IL-1β. Experiments were performed in triplicate. *P < 0.05 vs. control.

Figure 3.

Direct effects of CITED2 and p300 on MMP-1 promoter transactivation. Effect of cotransfecting chondrocytes with CITED2, Ets-1, or p300 in various combinations (shown) with or without siRNA for p300 (si-p300) or IHP (2.5 MPa, 1 Hz, 1 h) on MMP-1 promoter activity in luciferase assays. Experiments were performed in triplicate. *P < 0.05 vs. vector-transfected cells.

Figure 4.

CITED2 competes with Ets-1 for p300 binding. A) Immunoprecipitation with anti-p300 antibody and Western blot (WB) with either anti-CITED2 or anti-Ets-1 antibodies to reveal protein-protein interactions between Ets-1, CITED2, and p300. B, C) qPCR showing the effects of overexpressing p300 or dominant-negative p300 (p300-DN) on the relative expression of MMP-1, CITED2, and p300 in response to IHP (2.5 MPa, 1 Hz, 1 h; B) or cotransfection with WT CITED2 without IHP (C). D, E) Mean IC₅₀ concentrations (nM) for CITED2 on Ets-1-p300 (D) or Ets-1 on CITED2-p300 (E).

Figure 5.

CITED2 regulates MMP-1 through specific motif. A, B) Effect of transfecting chondrocytes with plasmids encoding either WT CITED2, or CITED2 deleted, truncated, or point mutants on MMP-1 and CITED2 mRNA (PCR; A) and/or protein (Western blot) expression in the presence of IL-1β (B). CR, conserved region; Δsrj, CITED2 WT with deletion of 39-aa serine-glycine rich junction; EPEE, mutated LPEL (aa 243–246) motif. C, D) Effect of cotransfecting WT Ets-1 or Ets-1 deleted or truncated mutants with CITED2 (C) on MMP-1 and CITED2 mRNA (PCR) and/or protein (Western blot) expression (D) in the presence of IL-1β.

Figure 6.

MAP kinase p38δ is the upstream mediator of CITED2 in response to moderate loading. A, B) qPCR showing the effect of IHP (MPa as shown, 1 Hz, 1 h) on the expression of CITED2 (A) and MMP-1 (B). C) Western blot showing p38δ and p38α activation, measured as phosphorylation of the p38 substrate, ATF2. D) Luciferase activity in chondrocytes transfected with the CITED2 promoter-reporter construct after application of IHP (2.5 MPa, 1 Hz, 1 h). E) Cotransfection with WT or dominant-negative p38δ or p38α, or with sequential deletion constructs of CITED2 cotransfected with p38δ. Experiments were performed in triplicate. *P < 0.05 vs. control, WT, or previous deletion construct.