CD98 Regulates Proliferation by Activating MAPKs Figure 4. IMCD cell proliferation increases in response to enhanced amino acid transport. (A) CD98 expression increases isoleucine transport. 3H-Isoleucine uptake was performed as described in Experimental Procedures. Cless cells are IMCD transfected with a CD98 mutant unable to interact with Lats. (*) denotes statistically significant differences (p,0.05) between IMCD and CD98 cells. (B) Transfected IMCD cells were plated onto 96-well plates, serum deprived for 24 hours then treated for 16 hours with 3H-Thymidine in media containing 1% serum. 3H-Thymidine incorporation was determined as described in Experimental Procedures. Values are the mean 6 s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p,0.05) between IMCD and Cless cells. (**) denotes statistically significant differences between CD98 and Cless cells. doi:10.1371/journal.pone.0040026.g004 incorporation (Figure 6A). This increase in proliferation was not seen in the Cless cells; but proliferation was more than 3 fold and double in the CD98/Lat-1 cells when compared to IMCD and CD98 cells respectively. Consistent with the markedly increased proliferation of the CD98/Lat-1 cells there was increased expression of cyclin D1 when compared to IMCD cells (Figure 6B). Thus CD98 significantly increases cell proliferation in the setting of exposure only to amino acids, confirming this as a mechanism of regulating cell proliferation. We next investigated which pathways regulated CD98 depen- dent cell proliferation in response to amino acids. We initially defined the roles of Erk, Akt and p38 MAPK signaling by serum and amino acid starving IMCD and CD98/Lat-1 cells which were then exposed only to amino acids. Similar to the results seen when the cells were exposed to complete medium with serum, increased Erk and p38 MAPK but not Akt signaling was seen in the CD98/ Lat-1 compared to the control IMCD cells (Figure 6C). We tested the role of these pathways in increasing the proliferation of the CD98/Lat-1 cells by exposing them to specific inhibitors of these pathways. As shown in Figure 6D, inhibiting MEK and p38 MAPK significantly decreases cell proliferation in both cell populations, while the PI3 kinase inhibitors have no effect. Furthermore, there was an additive effect of the MEK and p38 MAPK inhibitors, suggesting that these pathways acted synergis- tically to support CD98/Lat-1 cell proliferation. Increased Amino Acid Transport by CD98 Induces Activation of the mTor Pathway; however this Pathway is not Required for Enhanced Cell Proliferation Because of the effects of amino acids on the mTor pathway, we defined the effects of medium containing only amino acids on this pathway in CD98/Lat-1 cells. As shown in figure 7A there were markedly increased levels of activation of p70S6K in the CD98/ Lat-1 cells. Consistent with this observation there was strikingly elevated phosphorylation of the slower migrating forms of 4EBP1.Taken together, these results confirm that the mTor pathway is dramatically activated when the CD98/Lat-1 cells are exposed to amino acids. We next defined the role of this highly activated pathway in inducing the increased proliferation of the CD98/Lat-1 cells in serum free medium by growing them in the presence of Rapamycin and PP242 (Figure 7B). Addition of either of these inhibitors did not alter CD98/Lat-1 cell proliferation, despite the fact that they inhibited phosphorylation of the downstream targets of mTor, p70S6K and 4EBP1 (Figure 7C). The PI3 kinase inhibitor LY294002, which is also an inhibitor of mTor did not alter cell proliferation although it significantly decreased amino acid activation of the mTor pathway. By contrast, the MEK and p38 MAPK inhibitors which inhibit cell proliferation do not alter activation of the mTor pathway (Figure 7D). Thus although overexpression of CD98 leads to increased amino acid transport and activation of the mTor pathway, it does not play a role in cell proliferation. Discussion A key function of the scaffolding protein, CD98hc, is to regulate cell proliferation. In this manuscript we demonstrate that CD98hc is required for the survival of polarized renal collecting duct cells and that it promotes cell proliferation by two distinct mechanisms. The CD98hc cytoplasmic tail promotes IMCD cell proliferation in response to complete medium with serum by activating Erk and p38 MAPK signaling. In addition, the extracellular domain of CD98hc promotes cell proliferation by increasing amino acid transport which activates the same signaling pathways. Although increased amino transport stimulates mTor signaling, this does not alter cell proliferation (Figure 8). These data clearly show that in polarized renal epithelial cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common signaling pathways. We demonstrate that knocking down CD98 by siRNA or genetically deleting it from CD98flox/flox collecting duct cells resulted in their death, which confirmed that CD98 is required for cell survival. These results are consistent with the early embryonic (E3.5 to 9) lethality of CD98 mice [6]. The severe phenotype in IMCD cells lacking CD98 is likely not only due to its ability to interact with b1 integrin as b1-null collecting duct cells survive and proliferate [37]. We cannot exclude the possibility that CD98/b3 PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e40026