Interaction of HIF1α and β-catenin inhibits matrix metalloproteinase 13 expression and prevents cartilage damage in mice

Abstract

Low oxygen tension (hypoxia) regulates chondrocyte differentiation and metabolism. Hypoxia-inducible factor 1α (HIF1α) is a crucial hypoxic factor for chondrocyte growth and survival during development. The major metalloproteinase matrix metalloproteinase 13 (MMP13) is also associated with chondrocyte hypertrophy in adult articular cartilage, the lack of which protects from cartilage degradation and osteoarthritis (OA) in mice. MMP13 is up-regulated by the Wnt/β-catenin signaling, a pathway involved in chondrocyte catabolism and OA. We studied the role of HIF1α in regulating Wnt signaling in cartilage and OA. We used mice with conditional knockout of Hif1α (∆Hif1α(chon)) with joint instability. Specific loss of HIF1α exacerbated MMP13 expression and cartilage destruction. Analysis of Wnt signaling in hypoxic chondrocytes showed that HIF1α lowered transcription factor 4 (TCF4)-β-catenin transcriptional activity and inhibited MMP13 expression. Indeed, HIF1α interacting with β-catenin displaced TCF4 from MMP13 regulatory sequences. Finally, ΔHif1α(chon) mice with OA that were injected intraarticularly with PKF118-310, an inhibitor of TCF4-β-catenin interaction, showed less cartilage degradation and reduced MMP13 expression in cartilage. Therefore, HIF1α-β-catenin interaction is a negative regulator of Wnt signaling and MMP13 transcription, thus reducing catabolism in OA. Our study contributes to the understanding of the role of HIF1α in OA and highlights the HIF1α-β-catenin interaction, thus providing new insights into the impact of hypoxia in articular cartilage.

Keywords: Wnt signaling; chondrocyte; hypoxia-inducible factor 1α; matrix metalloprotease 13; osteoarthritis.

Fig. 1.

Hypoxia and hypoxia-inducible factor 1α (HIF1α) are reduced in cartilage of mice with OA. (A) Immunohistofluorescence staining of Hypoxyprobe adducts in healthy and OA wild-type mouse cartilage. Hypoxyprobe adducts were revealed in hypoxic cells (P_O2 < 10 mmHg) by a fluorescein-conjugated antibody (HP-FITC-MAb). Graph shows proportion of hypoxic positive cells in cartilage of the tibial plateau and internal femoral condyle. Data are mean ± SEM. *P < 0.05 compared with control (n = 7 animals per group). (Scale bar, 100 μm.) (B, Upper) HIF1α immunostaining and OA score (Safranin-O staining) in control mice at 0, 4, and 6 wk post-OA induction. Graphs show percentage of HIF1α(+) cells and OA score in articular cartilage of the tibial plateau and internal femoral condyle. (Scale bar, 100 μm.) *P < 0.05 compared with control (n = 7 animals per group) (C) Safranin-O staining and (D) TUNEL assay of Hif1α^fl/fl and ΔHif1α^chon mouse joints with OA or sham operation (ct) at week 6. (Scale bar, 100 μm.) OA score in OA and sham-operated knees of Hif1α^fl/fl and ΔHif1α^chon mice (week 6). *P < 0.05 compared with control. ^#P < 0.05 (n = 8–11 animals per group). (E) Immunostaining for MMP13 in Hif1α^fl/fl and ΔHif1α^chon mouse joints (week 6) and quantification. (Scale bar, 100 μm.) *P < 0.05 compared with control. ^#P < 0.05 (n = 8–11 animals per group).

Fig. S1.

β-Galactosidase immunostaining in Col2-Cre^ERT; R26R-LacZ and R26R-LacZ mice with injection of tamoxifen (n = 3).

Fig. S2.

(A) EPAS1 expression in Hif1α^fl/fl and ΔHif1α^chon mice at week 6 (immunohistochemistry) in control knees. (B) Immunocytofluorescence staining of EPAS1 in normoxic and hypoxic chondrocytes (n = 4). Quantification of EPAS1 translocation in chondrocytes: intensity of Epas1 signal into the nucleus of chondrocytes cultured in hypoxia and normoxia after Hif1α siRNA silencing (pixels) (n = 198–277). (C) PCR analysis of relative gene expression in primary chondrocytes with 21% and 1% O₂ for Wnt targets (Axin and Wisp1) with EPAS1 siRNA silencing (n = 6). (D) Coimmunoprecipitation of β-catenin in nuclear protein extracts. Western blot (WB) analysis of protein levels of EPAS1 and β-catenin.

Fig. 2.

HIF1α inhibits the transcription of Wnt targets in Wnt3a-induced chondrocytes. (A) Proteoglycan release in chondrocyte culture media (n = 9). qPCR analysis of relative gene expression in primary chondrocytes with 21% and 1% O₂ for: (B) anabolic marker (collagen 2A, COL2A) and catabolic marker (Mmp13) (n = 7); and (C) direct transcriptional targets of Wnt3a (Axin and Wisp1) (n = 14). (D) Expression of anabolic and catabolic genes (COL2A and Mmp13) with HIF1α siRNA silencing (n = 6). (E) Direct transcriptional targets of Wnt3a (Axin and Wisp1) with HIF1α siRNA silencing (n = 6). (F) Catabolic marker Mmp13 in HIF1α-lacking chondrocytes (Cre-lox recombination in vitro) (n = 5). (G) Direct transcriptional targets of Wnt3a (Axin and Wisp1) in HIF1α-lacking chondrocytes (Cre-lox recombination in vitro) (n = 5). Data are mean ± SEM. *P < 0.05 compared with control, ^#P < 0.05. (H) Western blot analysis of HIF1α expression in von Hippel–Lindau tumor suppressor protein (VHL)-lacking chondrocytes (with 21% O₂), and quantification (n = 3). qPCR analysis of relative gene expression in VHL-lacking chondrocytes (Cre-lox recombination in vitro) for: (I) anabolic marker COL2A; catabolic marker MMP13; and direct transcriptional targets of Wnt (Axin and Wisp1) (with 21% O₂). (J) Western blot analysis of stabilized HIF1α expression using a tag antibody (HA) in primary chondrocytes and qPCR analysis of relative gene expression of Mmp13. Data are mean ± SEM (n = 5 experiments); *P < 0.05 compared with control; ^#P < 0.05.

Fig. 3.

Wnt3a promotes β-catenin translocation into the nucleus in hypoxia and normoxia. (A) Immunocytofluorescence staining of β-catenin in normoxic and hypoxic chondrocytes. Bar 100 μm (n = 4). Quantification of β-catenin translocation in chondrocytes: intensity of β-catenin signal into the nucleus of chondrocytes cultured in hypoxia and normoxia after Wnt3a stimulation (pixels) (n = 198–277). (B) Western blot analysis of β-catenin protein level in normoxic and hypoxic chondrocytes and quantification. Data are mean ± SEM. *P < 0.05 compared with control (Ct).

Fig. 4.

HIF1α binds β-catenin and inhibits TCF4 binding to the MMP13 regulatory region. (A) Immunocytofluorescence staining of β-catenin and HIF1α in hypoxic chondrocytes (63×). (n = 3). (B) Coimmunoprecipitation of β-catenin in nuclear protein extracts. Western blot (WB) analysis of protein levels of HIF1α, TCF4, and β-catenin and quantification of HIF1α–β-catenin and TCF4–β-catenin complexes. (C) ChIP analysis of TCF4 binding to the Mmp13 regulatory region. Sequence contains Wnt responsive elements. RNA Pol, RNA polymerase (positive control); IgG, mouse IgG (negative control). qPCR analysis of TCF4 binding to Mmp13 regulatory regions (n = 3). *P < 0.05 compared with control; ^#P < 0.05. (D) Luciferase reporter assay in C3H10 cells. Data are ratio of firefly luciferase to control (Renilla) luciferase activity (n = 3). (E) qPCR analysis of relative gene expression (with HIF1α siRNA silencing) with 21% and 1% O₂ for Mmp13 (n = 8) and direct transcriptional targets of Wnt (Axin and Wisp1) (n = 8). Data are mean ± SEM. *P < 0.05 compared with control; ^#P < 0.05.

Fig. 5.

Loss of HIF1α increases TCF4–β-catenin complexes and cartilage lesions in OA mice with control or PKF118-310. (A) Safranin-O staining and OA score of Hif1α^fl/fl and ΔHif1α^chon mouse knees after OA induction or control (Ct) at week 6 after treatment or not with PKF118-310. (Scale bar, 100 μm.) (B) MMP13 expression in Hif1α^fl/fl and ΔHif1α^chon mice at week 6 and quantification. (Scale bar, 100 μm.) (C) TUNEL assay, quantification in Hif1α^fl/fl and ΔHif1α^chon mouse joints with OA or control at week 6. Data are mean ± SEM. *P < 0.05, ^#P < 0.05 compared with control Hif-1α^fl/fl (n = 8–11 animals per group).