Regulated Expression of ADAMTS-12 in Human Trophoblastic Cells: A Role for ADAMTS-12 in Epithelial Cell Invasion? Alexander G. Beristain1,2*, Hua Zhu2, Peter C. K. Leung2 1 Division of Cell Signaling Biology, Ontario Cancer Institute, Toronto, Ontario, Canada, 2 Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada Abstract Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-b1 and interleukin-1b, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the avb3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function. Citation: Beristain AG, Zhu H, Leung PCK (2011) Regulated Expression of ADAMTS-12 in Human Trophoblastic Cells: A Role for ADAMTS-12 in Epithelial Cell Invasion? PLoS ONE 6(4): e18473. doi:10.1371/journal.pone.0018473 Editor: Neil A. Hotchin, University of Birmingham, United Kingdom Received December 1, 2010; Accepted March 7, 2011; Published April 11, 2011 Copyright: 2011 Beristain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a research grant (#MOP-102578) to Dr. Colin D. MacCalman and PCKL from the Canadian Institutes for Health Research (http://www.cihr-irsc.gc.ca/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: aberista@uhnres.utoronto.ca Introduction Human placental development and function are dependent upon mononucleate cytotrophoblasts entering one of two distinct and mutually exclusive differentiation pathways [1,2]. The villous pathway culminates in the formation of the syncytial trophoblast, a multinucleated, terminally differentiated cell that contributes to the majority of placental transport, immunoregulation and endocrine functions throughout pregnancy. Alternatively, cytotro- phoblasts entering the extravillous pathway develop a highly invasive phenotype, which in turn allows these cells to invade deeply into the underlying maternal tissues and vasculature, thereby ensuring a continuous supply of blood to the developing fetus. Extravillous cytotrophoblast (EVT) invasion, unlike carci- noma cell metastasis, is a tightly controlled, developmental process [3,4]. The onset of trophoblastic cell differentiation along the extravillous pathway is dependent upon the proteolytic degrada- tion and/or activation of distinct extracellular matrix (ECM) components, and regulated changes in cell-cell and cell-ECM interactions [5,6]. Consequently, most studies to date have focused upon the roles of matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs), cytokines, integrins, and cadherins in this cellular event. However, there is increasing evidence to suggest that the regulated expression of members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family may represent a significant molecular mechanism for mediation of the terminal differentiation of human cytotrophoblasts and the development of an invasive phenotype. The ADAMTS are a family of secreted proteins that are generally characterized by four structural and functional domains; an amino terminal prodomain, a catalytic domain, a disintegrin- like domain, and an ECM-binding domain (composed of a central thrombospondin type 1 (TSP1) motif, a spacer region and a variable number of TSP-like repeats) at the carboxyl terminal of the mature protein species [7,8]. Thus, all members of this gene family have the potential to act as metalloproteinases and to regulate cell adhesion. Some ADAMTS subtypes have been further subclassified according to the presence of additional C- terminal modules or the identification of common substrates. Furthermore, distinct ADAMTS subtypes have also been shown to play integral roles in the growth and development of tissues and in the onset and progression of degradative diseases including cancer [9–11], arthritis [12], Alzheimer’s disease and a number of inflammatory and thrombotic conditions [13,14]. Here, we have examined the function and regulation of the ADAMTS in the differentiation of human cytotrophoblasts along the extravillous pathway. We first determined that multiple ADAMTS subtypes are present in first trimester human placenta, PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18473