Growth Differentiation Factor-15 (GDF-15) Suppresses In Vitro Angiogenesis Through a Novel Interaction With Connective Tissue Growth Factor (CCN2) Ramon J. Whitson, Marshall Scott Lucia, and James R. Lambert * Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045 ABSTRACT Growth differentiation factor-15 (GDF-15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF-15 and CCN2 using yeast two-hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF-15 and His-tagged CCN2 produced in PC-3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF-15 blocks CCN2-mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF-15 inhibits CCN2-mediated angiogenesis, activation of a V b 3 integrins and focal adhesion kinase (FAK) was examined. CCN2-mediated FAK activation was inhibited by GDF-15 and was accompanied by a decrease in a V b 3 integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF-15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF-15 may provide insight into the functional role of GDF-15 in disease states. J. Cell. Biochem. 114: 1424–1433, 2013. ß 2012 Wiley Periodicals, Inc. KEY WORDS: GROWTH DIFFERENTIATION FACTOR-15 (GDF-15); MACROPHAGE INHIBITORY CYTOKINE-1 (MIC-1); PROSTATE DERIVED FACTOR (PDF); TRANSFORMING GROWTH FACTOR BETA (TGFb); CONNECTIVE TISSUE GROWTH FACTOR (CTGF); CYR61, CTGF, AND NOV PROTEIN FAMILY 2 (CCN2); ANGIOGENESIS; FOCAL ADHESION KINASE (FAK) G rowth differentiation factor-15 (GDF-15) is a divergent member of the transforming growth factor beta (TGFb)/bone morphogenetic protein (BMP) superfamily sharing only 15–29% sequence similarity with other members of this family [Bootcov et al., 1997]. GDF-15 is secreted as a mature dimer and is grouped with other members of the TGFb family due to the conserved cysteine knot motif found within the mature peptide. GDF-15 has been identified in a variety of experimental systems and is also known as macrophage inhibitory cytokine-1 (MIC-1), prostate derived factor (PDF), non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1), placental bone morphogenetic protein (PLAB), and placental TGFb (PTGFb) [Bootcov et al., 1997; Hromas et al., 1997; Paralkar et al., 1998; Bottner et al., 1999; Li et al., 2000; Baek et al., 2001]. GDF-15 is primarily secreted from epithelial cells and is aberrantly expressed in tumors [Thomas et al., 2001; Karan et al., 2003; Bauskin et al., 2005; Lind et al., 2009]. Similar to TGFb1, GDF- 15 is reported to have both anti- and pro-tumorigenic properties [Baek et al., 2001; Lambert et al., 2006; Martinez et al., 2006; Cekanova et al., 2009; Senapati et al., 2010; Wang et al., 2011]. GDF- 15 suppresses cell proliferation in breast, colon, and prostate cancer cells [Li et al., 2000; Baek et al., 2001; Lambert et al., 2006] and inhibits tumor growth in prostate xenografts [Lambert et al., 2006]. However, increased serum levels of GDF-15 are associated with metastatic disease in prostate cancer patients [Selander et al., 2007]. Despite the association between GDF-15 and cancer, a signaling pathway activated downstream of GDF-15 in tumors has not been established. As a member of the TGFb family, we hypothesized that Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 114:1424–1433 (2013) 1424 Additional supporting information may be found in the online version of this article. Grant sponsor: Colorado Clinical and Translational Sciences Institute (CCTSI); Grant number: TL1 TR000155. Ramon J. Whitson’s present address is Department of Dermatology, Stanford University, Stanford, CA 94305. *Correspondence to: Dr. James R. Lambert, Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, 12801 E. 17th Avenue, M.S. 8401, Aurora, CO 80045. E-mail: jim.lambert@ucdenver.edu Manuscript Received: 6 December 2012; Manuscript Accepted: 11 December 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 31 December 2012 DOI 10.1002/jcb.24484  ß 2012 Wiley Periodicals, Inc.