Dissection of Wnt5a-Ror2 signaling leading to matrix metalloproteinase (MMP-13) expression

Abstract

It has been shown that constitutively active Wnt5a-Ror2 signaling in osteosarcoma cell lines plays crucial roles in induced expression of matrix metalloproteinase-13 (MMP-13), required for their invasiveness; however, it remains largely unclear about the molecular basis of MMP-13 gene induction by Wnt5a-Ror2 signaling. Here we show by reporter assay that the activator protein 1 (AP1) (binding site in the promoter region of MMP-13 gene is primarily responsible for its transcriptional activation by Wnt5a-Ror2 signaling in osteosarcoma cell lines SaOS-2 and U2OS. Chromatin immunoprecipitation assays revealed that c-Jun and ATF2 are crucial transcription factors recruited to the AP1-binding site in the MMP-13 gene promoter during Wnt5a-Ror2 signaling in SaOS-2 cells. Using siRNA-mediated suppression or specific inhibitors, we also show that Dishevelled2 (Dvl2) and c-Jun N-terminal kinase are required for MMP-13 gene induction presumably via phosphorylation of c-Jun and ATF2 during Wnt5a-Ror2 signaling in SaOS-2 cells. Interestingly, Dvl2 and Rac1, but not Dvl3, are required for MMP-13 expression in SaOS-2 cells, whereas Dvl3, but not Dvl2 and Rac1, is required for its expression in U2OS cells, indicating the presence of distinct intracellular signaling machineries leading to expression of the same gene, in this case MMP-13 gene in different osteosarcoma cell lines. Moreover, we provide evidence suggesting that Wnt5a-Ror2 signaling might also be required for expression of MMP-13 gene during the development of the cartilaginous tissue.

FIGURE 1.

Constitutively active Wnt5a-Ror2 signaling is critical for transcriptional activation of MMP-13 gene promoter via AP1-binding site in SaOS-2 cells. A, SaOS-2 cells transfected with Ctrl #1, Ror2 #1, or Ror2 #2 siRNA were cultured for 4 days, and WCLs were analyzed by immunoblotting with the indicated antibodies. B, SaOS-2 cells were transfected with Ctrl #1, Ror2 #1, or Ror2 #2 siRNA and cultured for 4 days. Subsequently, SaOS-2 cells were transfected with the indicated MMP-13 gene promoter-luciferase (Luc) vectors along with the renilla luciferase reporter construct and were cultured for 24 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. C, SaOS-2 cells were transfected with either Ctrl #1 or Ror2 #1 siRNA and cultured for 4 days. Then cells were transfected with the indicated MMP-13 gene promoter-luc vectors, whose promoters contain mutations within the OSE2- and AP1-binding sites, respectively, along with the renilla luciferase reporter construct and were cultured for 24 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. D, SaOS-2 cells were transfected with Ctrl #2, Wnt5a #1, or Wnt5a #2 siRNA and cultured for 4 days. Total RNAs from the respective cells were then isolated and subjected to qRT-PCR to monitor mRNA expression of either Wnt5a or GAPDH. Relative amounts of Wnt5a transcripts were determined after normalization by those of GAPDH transcripts. *, p < 0.01, t test. E, SaOS-2 cells were transfected with Ctrl #2, Wnt5a #1, or Wnt5a #2 siRNA and cultured for 4 days. Subsequently, SaOS-2 cells were transfected with the indicated MMP-13 gene promoter-luciferase (Luc) vectors along with the renilla luciferase reporter construct and were cultured for 24 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. F, SaOS-2 cells were transfected with the respective MMP-13 gene promoter-luc vectors for 12 h. The cells were further serum-starved for 12 h and then treated with either Wnt5a (400 ng/ml) or vehicle for 12 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test.

FIGURE 2.

Constitutively active Wnt5a-Ror2 signaling is critical for transcriptional activation of MMP-13 gene promoter via AP1-binding site in U2OS cells. A, U2OS cells were transfected with Ctrl #1, Ror2 #1, or Ror2 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. *, p < 0.01, t test. B, U2OS cells were transfected with Ctrl #1, Ror2 #1, or Ror2 #2 siRNA and cultured for 4 days. Subsequently, the cells were transfected with the indicated MMP-13 gene promoter-luc vectors along with the renilla luciferase reporter construct and cultured for 24 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. C, U2OS cells were transfected with either Ctrl #1 or Ror2 #1 siRNA and cultured for 4 days. Then cells were transfected with the indicated MMP-13 gene promoter-luc vectors, whose promoters contain mutations within the OSE2- and AP1-binding sites, respectively, along with the renilla luciferase reporter construct and were cultured for 24 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. D, U2OS cells were transfected with the indicated MMP-13 promoter-luc vectors along with the renilla luciferase reporter construct and cultured for 12 h. The cells were further serum-starved for 12 h and then stimulated with either Wnt5a (400 ng/ml) or vehicle for 12 h. Reporter assays were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test.

FIGURE 3.

AP1 components c-Jun and ATF2 are recruited to MMP-13 gene promoter in a Wnt5a-dependent manner. A, the diagram represents the positions of the respective primers in the MMP-13 gene promoter used for ChIP analysis. The proximal region, but not the distal one, flanks the AP1-binding site in the MMP-13 gene promoter. B, SaOS-2 cells were transfected with Ctrl #2, Wnt5a #1, or Wnt5a #2 siRNA and cultured for 4 days. ChIP analysis was performed using the indicated antibodies as described under “Experimental Procedures.” C, SaOS-2 cells were transfected with either Ctrl #2 or Wnt5a #1 siRNA and cultured for 4 days. WCLs were analyzed by immunoblotting with the indicated antibodies. Note that the asterisk (*) and double asterisks (**) indicate hypophosphorylated and hyperphosphorylated forms of Dvl2, respectively. D, SaOS-2 cells were serum-starved for 12 h and then stimulated with either Wnt5a (400 ng/ml) or vehicle for 1 h. WCLs were analyzed by immunoblotting with the indicated antibodies. E, SaOS-2 cells were serum-starved for 12 h and then stimulated with either Wnt5a (400 ng/ml) or vehicle for 1 h. ChIP analysis was performed using the indicated antibodies. F, SaOS-2 cells were serum-starved for 12 h and then stimulated with either Wnt5a (400 ng/ml) or vehicle for 1 h. WCLs were analyzed by immunoblotting with the indicated antibodies.

FIGURE 4.

JNK is critical for the expression of MMP-13 gene by Wnt5a-Ror2 signaling in SaOS-2 cells. A, SaOS-2 cells were transfected with Ctrl #2, Wnt5a #1, or Wnt5a #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. *, p < 0.01, t test. B, SaOS-2 cells were transfected with Ctrl #1, Ror2 #1, or Ror2 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. *, p < 0.01, t test. C, SaOS-2 cells were treated with vehicle or SP600125 (an inhibitor of JNK, 20 μm) for 10 h. Total RNAs from the respective cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. *, p < 0.01, t test.

FIGURE 5.

Dvl2, but not Dvl3, is required for the expression of MMP-13 gene via phosphorylation of both c-Jun and ATF2 in SaOS-2 cells. A, SaOS-2 cells were transfected with Ctrl #1, Dvl2 #1, Dvl2 #2, Dvl3 #1, or Dvl3 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. B, SaOS-2 cells were transfected with Ctrl #1, Dvl2 #1, or Dvl2 #2 siRNA. After 4 days in culture, WCLs were analyzed by immunoblotting with the indicated antibodies. The histograms indicate the relative extents of phosphorylated forms of c-Jun or ATF2 (phosphorylated c-Jun or ATF2 levels normalized by total c-Jun or ATF2 levels, respectively). Quantifications were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. *, p < 0.01, t test. C, SaOS-2 cells were transfected with Ctrl #1, Dvl3 #1, or Dvl3 #2 siRNA. After 4 days in culture, WCLs were analyzed by immunoblotting with the indicated antibodies. The histograms indicate the relative extents of phosphorylated forms of c-Jun or ATF2 (phosphorylated c-Jun or ATF2 levels normalized by total c-Jun or ATF2 levels, respectively). Quantifications were performed as described under “Experimental Procedures.” The data are representative of three independent experiments. The bars represent the mean ± SD of triplicate experiments. D, SaOS-2 cells were transfected with either Ctrl #2 or Dvl2 #3 siRNA for 4 days in culture, and then Dvl2 #3 siRNA-treated cells were further transfected with either siRNA-resistant Dvl2 WT or KM (K446M) mutant expression vector and cultured for 24 h. Total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. E, SaOS-2 cells were transfected with Ctrl #2, Rac1 #1, or Rac1 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. Note that the asterisk (*) and double asterisks (**) indicate hypophosphorylated and hyperphosphorylated forms of Dvl2, respectively.

FIGURE 6.

Dvl3, but not Dvl2, is critical for transcriptional activation of MMP-13 gene promoter by Wnt5a-Ror2 signaling in U2OS cells. A, U2OS cells were transfected with Ctrl #1, Dvl2 #1, Dvl2 #2, Dvl3 #1, or Dvl3 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies. *, p < 0.01, t test. B, U2OS cells were transfected with Ctrl #2, Rac1 #1, or Rac1 #2 siRNA. After 4 days in culture, total RNAs from the respective transfected cells were isolated and subjected to qRT-PCR to monitor mRNA expression of either MMP-13 or GAPDH. WCLs were also prepared and subjected to immunoblotting with the indicated antibodies.

FIGURE 7.

Expression of MMP-13 by Wnt5a-Ror2 signaling in the cartilaginous tissues of mouse embryos. The paraffin specimens were prepared from the cartilaginous tissues from both Ror2^+/+ and Ror2^−/− male mouse embryos at E15.5, fixed, and stained with the indicated antibodies as described under “Experimental Procedures.” Nuclei were visualized with Mayer's hematoxylin. Magnified images of boxed regions are shown (magnified).