CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs Nada Bulus1, Chloe Feral5, Ambra Pozzi1,2,4, Roy Zent1,2,3,4* 1 Department of Medicine (Division of Nephrology), Vanderbilt University Medical Center, Tennessee, United States of America, 2 Department of Cancer Biology, Vanderbilt University Medical Center, Tennessee, United States of America, 3 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Tennessee, United States of America, 4 Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee, United States of America, 5 Institute for Research on Cancer and Aging, University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, Nice, France Abstract CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with b1 and b3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways. Citation: Bulus N, Feral C, Pozzi A, Zent R (2012) CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs. PLoS ONE 7(6): e40026. doi:10.1371/ journal.pone.0040026 Editor: Jeff M. Sands, Emory University, United States of America Received November 21, 2011; Accepted May 31, 2012; Published June 29, 2012 Copyright: 2012 Bulus 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 work was supported by the Department of Veterans Affairs. 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: roy.zent@vanderbilt.edu Introduction The heterodimeric amino acid transporters consist of a type II transmembrane protein heavy chain and a light chain linked by an extracellular disulfide bridge [1,2]. The heavy chain subunits rBAT and CD98hc (also called 4F2hc) heterodimerize with a number of light chain amino acid transporters [3,4]. The principal function of the heavy chain is to localize the heterodimer to either the apical or basolateral aspect of the cell [1,2]. CD98 is expressed in all cell types with the exception of platelets and its highest levels of expression are in the tubules of the kidney and the gastrointestinal tract [1,2,5], where it plays a critical role in the vectorial transport of amino acids across a polarized epithelium. CD98 is required for normal development in mammals and deletion of CD98hc results in early embryonic lethality in mice [6]. CD98hc heterodimerizes with one of the light chains Lat-1, Lat2, y+Lat-1, y+Lat2 and xCT [7–16] and is required for the surface expression of the heterodimers. These transporters mediate Na+-independent transport of large neutral amino acids (e.g. leucine) and/or Na+-dependent co-transport of positively charged amino acids (e.g. arginine) and neutral amino acids [1,2]. In addition to the light chain, CD98hc associates with b integrins [17–26]. This association is important for altering integrin affinity and integrin dependent signaling resulting in alterations in cell differentiation, proliferation, aggregation, adhesion, migration and malignant transformation [17–21,23–26]. The physiological rele- vance of CD98 function was investigated in mice by either deleting or overexpressing CD98hc [23,24,27–30]. Constitutive deletion of CD98hc resulted in early embryonic lethality [6]. Specifically deleting CD98hc from lymphocytes [27,28] and vascular smooth muscle [29] decreased proliferation of both cell types and altered adaptive humoral immunity or resulted in abnormal vessel repair in the organ specific mice. CD98hc overexpression in the gastrointestinal epithelium induced tumorigenesis by causing barrier dysfunction and stimulating cell proliferation, whereas CD98hc deletion resulted in an attenuated inflammatory response as well as resistance to DSS-induced colitis and colitis-associated tumorigenesis [30]. Together these in vivo data emphasize the important role CD98 plays in regulating cell proliferation in multiple cell types. The mechanism whereby CD98 modulates cell proliferation is not fully understood. Increased amino acid transport increases cell proliferation [31–33], however mechanisms other than this also probably play a role in CD98-dependent proliferation as mutants of CD98hc unable to associate with amino acid transporters can induce malignant transformation of NIH3T3 PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e40026