cells [34]. In addition, CD98/b1 integrin interactions which have no effect on amino acid transport have been shown to be important in modulating CD98-dependent transformation [18], survival [23,27,35], proliferation [23,27,29,30,35], adhesion [18,26], migration [18,26] and tubule formation [26]. PI3-kinase [18,26], Rho A [23] and focal adhesion kinase [18,26] signaling have been implicated in mediating CD98/b1 integrin-dependent cell spreading, migration, transformation and survival. The relative roles of CD98-dependent amino acid transport and other as yet undefined signaling pathways on cell proliferation are currently unclear. Based on the in vivo and in in vitro data showing a requirement for CD98 for cell survival and proliferation, we investigated the mechanisms whereby CD98 regulates these cellular processes in polarized renal epithelial cells. We demonstrate that CD98hc expression is required for these cells to survive, as deleting or downregulating CD98hc expression results in cell death. Utilizing overexpression studies of mutants of CD98hc and Lat-1 we show that CD98 increases cell proliferation by two distinct mechanisms that signal through MAPKs. One requires the CD98hc cytoplasmic tail and is independent of changes in amino acid transport. The second is due to increased CD98-dependent amino acid transport and does not require the CD98hc cytoplasmic tail. Thus CD98 modulates cell proliferation by two distinct mechanisms, namely the induction of signaling via its cytoplasmic tail and its ability to increase amino acid transport into cells. Results CD98 is Required for Inner Medullary Collecting Duct (IMCD) Cell Survival Consistent with previous reports demonstrating that CD98 expression increases cell proliferation [23,27,29,30,35], we ob- served that IMCD cells (which are derived from the kidney collecting ducts) overexpressing CD98 proliferated more rapidly than wild type IMCD cells under normal culture conditions. To define the molecular mechanisms of this increased cell prolifera- tion, we initially attempted to downregulate CD98hc in IMCD cells using 3 different hairpin siRNAs expressed by lentiviruses. Although we successfully downregulated CD98hc expression using all 3 lentiviruses, the cells died within a week (data not shown). We next attempted to generate IMCD cells from CD98hc floxed mice and delete the gene in vitro using cre recombinant adenovirus. While we were able to obtain CD98hcfl/fl cells, we were unable to obtain CD98hc-null cells, as they also died within a week (data not shown). To demonstrate the requirement for CD98hc for IMCD cell survival, we transfected CD98hcfl/fl cells with a human CD98hc cDNA and then deleted the floxed gene in vitro with adeno cre. In figure 1, we show that CD98fl/fl cells treated with cre show increased caspase-3 activation and this correlates with the degree of downregulated mouse CD98hc protein. In contrast, we did not detect any caspase-3 activation in CD98fl/fl cells that expressed human CD98hc following cre treatment. All the cre- infected CD98hcfl/fl IMCD cells died eventually, while the reconstituted cells survived and continued proliferating. Thus, deleting CD98hc results in significant IMCD cell apoptosis suggesting these cells require CD98hc for survival. The Cytoplasmic Tail of CD98 is Required to Induce Serum Dependent Cell Proliferation Due to our failure to establish CD98hc-null cell populations we utilized previously generated IMCD cells that overexpress full length (CD98) and mutants of human CD98hc [26] to define how CD98 Regulates Proliferation by Activating MAPKs Figure 1. CD98 is required for IMCD cell survival. CD98fl/fl IMCD cells or CD98fl/fl IMCD cells reconstituted with human CD98 (CD98fl/ flhCD98) were infected with adeno-cre virus after which they were harvested at different time points and immunoblotted for cleaved caspase-3, mouse CD98hc, human CD98hc or actin. doi:10.1371/journal.pone.0040026.g001 CD98 regulates cell proliferation. The mutants (Figure 2A) either lacked the first 77 amino acids of the cytoplasmic tail (CD98-77), the full putative cytoplasmic tail (CD98-82) or the cytoplasmic tail as well as the N-terminal five amino acids (WALLL) of the transmembrane domain (CD98-87), which we have shown to be critical for CD98 to interact with the b1 integrin and regulate integrin dependent cell adhesion, migration and tubulogenesis [26]. Equal expression of these mutants in the IMCD cells was verified by flow cytometry with an antibody directed against the extracellular domain of human CD98hc, which demonstrates that the CD98hc is expressed on the cell surface [26]. We further showed that all of these mutants are expressed in membranes by performing western blot analysis with an antibody directed against human CD98hc on cytosolic and membrane fractions derived from the IMCD cells (Figure 2B). We defined the role of CD98 on IMCD cell proliferation in cells grown in complete medium containing low levels (1%) of serum (Figure 2C). CD98 cells showed a 2 fold increase in cell proliferation relative to IMCD cells and consistent with this result, there was increased cyclin D1 expression in CD98 cells 4 hours after the addition of complete medium (Figure 2D). By contrast, there was only approximately a 35% increase in proliferation in the CD98-77, CD98-82 or CD98-87 deletion mutants (Figure 2C). These results suggest that increased CD98hc expression enhances cell proliferation in complete medium by a mechanism that requires the 77 amino acid cytoplasmic tail and another that does not. CD98 Regulates Serum-dependent Cell Proliferation by Increasing Erk and p38 MAPK Signaling Complete medium, which includes fetal calf serum, increases cell proliferation by altering many different signaling pathways. Because the data presented above suggests that the CD98hc cytoplasmic tail regulates cell proliferation and CD98hc has been demonstrated to activate specific integrin-dependent signaling pathways [17–19], we initially defined whether there were differences in serum-dependent (complete medium with 10% serum) activation of PI3K, Erk and p38 MAPK signaling between IMCD cells and CD98 cells. In contrast to the changes seen in integrin-dependent signaling where the PI3K pathway was PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e40026