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J. Biol. Chem., Vol. 283, Issue 7, 4272-4282, February 15, 2008

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Matrix Metalloproteinase 13 (MMP13) and Tissue Inhibitor of Matrix Metalloproteinase 1 (TIMP1), Regulated by the MAPK Pathway, Are Both Necessary for Madin-Darby Canine Kidney Tubulogenesis^*

Nathan E. Hellman¹, June Spector, Jonathan Robinson, Xiaofeng Zuo, Sophie Saunier, Corinne Antignac, John W. Tobias^¶, and Joshua H. Lipschutz^||2

From the Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, INSERM U-574, Hôpital Necker-Enfants Malades, Universite Paris Descartes, 75015 Paris, France, ^¶Genomics Institute, Bioinformatics Core, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the ^||Department of Medicine and Cell and Molecular Biology Graduate Group, University of Pennsylvania and the Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104

A classic model of tubulogenesis utilizes Madin-Darby canine kidney (MDCK) cells. MDCK cells form monoclonal cysts in three-dimensional collagen and tubulate in response to hepatocyte growth factor, which activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. It was shown previously that MAPK activation is necessary and sufficient to induce the first stage of tubulogenesis, the partial epithelial to mesenchymal transition (p-EMT), whereas matrix metalloproteinases (MMPs) are necessary for the second redifferentiation stage. To identify specific MMP genes, their regulators, tissue inhibitors of matrix metalloproteinases (TIMPs), and the molecular pathways by which they are activated, we used two distinct MAPK inhibitors and a technique we have termed subtraction pathway microarray analysis. Of the 19 MMPs and 3 TIMPs present on the Canine Genome 2.0 Array, MMP13 and TIMP1 were up-regulated 198- and 169-fold, respectively, via the MAPK pathway. This was confirmed by two-dimensional and three-dimensional real time PCR, as well as in MDCK cells inducible for the MAPK gene Raf. Knockdown of MMP13 using short hairpin RNA prevented progression past the initial phase of p-EMT. Knockdown of TIMP1 prevented normal cystogenesis, although the initial phase of p-EMT did occasionally occur. The MMP13 knockdown phenotype is likely because of decreased collagenase activity, whereas the TIMP1 knockdown phenotype appears due to increased apoptosis. These data suggest a model, which may also be important for development of other branched organs, whereby the MAPK pathway controls both MDCK p-EMT and redifferentiation, in part by activating MMP13 and TIMP1.

Received for publication, September 25, 2007 , and in revised form, November 12, 2007.

^* This work was supported in part by National Institutes of Health Grants DK069909 and DK070980 and a University of Pennsylvania ITMAT grant (to J. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.

¹ Supported by a Fulbright grant.

² To whom correspondence should be addressed: 431 Hill Pavilion, 380 S. University Ave., Philadelphia, PA 19104-4539. Tel.: 215-573-1848; Fax: 215-898-0189; E-mail: jhlipsch{at}mail.med.upenn.edu.

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