Dissection of Wnt5a-Ror2 Signaling Leading to Matrix Metalloproteinase (MMP-13) Expression

Kaoru Yamagata,Xin Li,Shunkichi Ikegaki,Chitose Oneyama,Masato Okada,Michiru Nishita,Yasuhiro Minami

doi:10.1074/jbc.m111.315127

Kaoru Yamagata, Xin Li + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111.315127

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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.

Highlights

Active Wnt5a-Ror2 signaling in osteosarcoma cells induces matrix metalloproteinases (MMPs)-13 expression
We have previously shown that both Wnt5a and Ror2 are expressed in osteosarcoma SaOS-2 cells at least partly by endogenous expression of Snail in the cells [15] and that as a result Wnt5a-Ror2 signaling is constitutively activated in SaOS-2 cells, leading to the induction of MMP-13, which plays an important role in their invasiveness via activation of a Src-family protein-tyrosine kinase (SFK) [8]
We show by using reporter assays that the proximal promoter region of MMP-13 gene containing both the OSE2 and activator protein 1 (AP1)-binding site is primarily required for expression of MMP-13 gene in SaOS-2 cells (Fig. 1)

Summary

Background

Active Wnt5a-Ror signaling in osteosarcoma cells induces MMP-13 expression. It has been shown that constitutively active Wnt5a-Ror signaling in osteosarcoma cell lines plays crucial roles in induced expression of matrix metalloproteinase-13 (MMP-13), required for their invasiveness; it remains largely unclear about the molecular basis of MMP-13 gene induction by Wnt5a-Ror signaling. We show by reporter assay that the activator protein 1 (AP1) The Wnt/JNK pathway can be mediated by various signaling molecules, including Dishevelled (Dvl), activator protein 1 (AP1), and members of the Rho-family of small GTPases in addition to JNK [12]; it remains elusive how this pathway regulates expression of its target genes.

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION