CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs

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 β1 and β3 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

IMCD cell proliferation increases in response to enhanced amino acid transport.

<p>(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 ± 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.</p

The cytoplasmic tail of CD98 is required for serum-dependent cell proliferation. (A)

<p>Schematic representation of the CD98 constructs used for CD cell transfection. CD, cytoplasmic domain; TM, transmembrane domain; EC, extracellular domain. (B) Full-length CD98hc and its truncation mutants are expressed in membrane fractions of IMCD cells. IMCD cells expressing CD98hc or the different truncation mutants were fractionated into cytoplasmic (Cyt) Triton X-100–soluble and – insoluble membrane fractions (MF). These fractions were subjected to SDS-PAGE and immunoblotted with an antibody directed against human CD98hc. (C) CD98hc induces cell proliferation in serum. IMCD 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 ± s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p<0.05) between CD98 and deletion mutants. (**) denotes a statistically significant difference between CD98 and IMCD. (D) Time course of Cyclin D1 expression. Sub-confluent cells were serum deprived for 24 hours and then treated with complete medium. Lysates were isolated at the time points indicated and analyzed by SDS-PAGE.</p

CD98hc/Lat-1-dependent amino acids transport increases cell proliferation mediated by Erk- and p38 MAPK.

<p>(A) CD98hc/Lat-1-dependent amino acids transport increases cell proliferation. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for 2 hours. ³H-Thymidine was then added in serum free media with a full complement of amino acids for an 8 hour pulse. ³H-Thymidine incorporation was determined as described in Experimental Procedures. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (*)indicates statistically significant differences (p<0.05) compared to IMCD cells. (**) denotes a statistically significant difference between CD98 and CD98/Lat-1. (B, C) treatment of CD98/Lat-1 cells with amino acids increases cyclin D1 expression and MAPK activation. Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0). Cells were then treated with serum free media containing a full complement of amino acids and lysates were isolated at the time points indicated. Lysates were analyzed by SDS-PAGE and immunoblots were performed with the antibodies against cyclin D1 (B) and Erk, Akt and p38 MAPK. β-Actin is a loading control (C). (D) MAPK increases CD98/Lat-1 dependent amino acid induced proliferation. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for an additional 2 hours and pretreated with inhibitors prior to addition of ³H-Thymidine in serum free media with a full complement of amino acids for an 8 hour pulse. ³H-Thymidine incorporation was determined as described in Experimental Procedures. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p<0.05) compared to untreated cells. U+SB denotes concomitant treatment with the MEK inhibitor U0126 (U) the p38 MAPK inhibitor SB203580 (SB). (**) denotes statistically significant differences between U+SB versus either inhibitor alone.</p

mTOR pathway is activated by amino acids but does not affect amino acid-dependent proliferation.

<p>(A) Amino acid treatment increases mTor signaling in CD98/Lat-1 cells. Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0). Cells were then treated with serum free media containing a full complement of amino acids. Lysates were isolated at the time points indicated and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as antibodies recognizing the multiple phosphorylated forms of 4EBP1. (B) mTor pathways do not regulate amino acid-dependent proliferation of CD98/Lat-1 cells. Cells were plated onto 96-well plates, serum deprived for 24 hours then deprived of amino acids for an additional 2 hours and pretreated with Rapamycin (100 nM) and PP242 (2.5 µM) prior to addition of ³H-Thymidine in serum free media with a full complement of amino acids for an 8 hour pulse. ³H-Thymidine incorporation was determined as described in Experimental Procedures then. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (C and D) Inhibitors of mTor, MAPK and PI3 kinase were effective in inhibiting amino acid-dependent signaling. (C) Cells were serum deprived for 24 hours then subjected to amino acid deprivation for an additional two hours (time 0) and pretreated with Rapamycin and PP242 . Cells were treated with serum free media containing a full complement of amino acids in the presence of inhibitors. Lysates were isolated after 2hours of amino acid treatment and subjected to SDS-PAGE. Immunoblots were probed with phos-p70S6K (Thr389) as well as an antibody recognizing the hyperphosphorylated forms of 4EBP1. (D) Cells were treated as described in C in the presence of LY294002, SB 203580 and U0126, all at 10 µM. Lysates were isolated after two hours of amino acid treatment in the presence of inhibitors and subjected to SDS-PAGE.</p

Schematic diagram demonstrating how CD98 regulates cell proliferation.

<p>Overexpression of CD98 results in increased cell proliferation which is dependent on MAPK signaling mediated by the cytoplasmic (CT) domain and increases amino acid transport which is dependent on the extracellular (EC) domain. CD98 overexpression increases mTOR signaling but this has no effect on cell proliferation.</p

CD98 is required for IMCD cell survival.

<p>CD98^fl/fl IMCD cells or CD98^fl/fl IMCD cells reconstituted with human CD98 (CD98^fl/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.</p

CD98hc and Lat-1 increase cell proliferation.

<p>(A, B) Overexpressed Lat-1 interacts with CD98hc and CD98hc deletion mutants. <b>(A)</b> Western blot analysis of clones of IMCD cells transfected with Lat-1. Total Akt was used as a loading control. (B) Cells overexpressing CD98 or CD98 deletion mutants as well as Flag-tagged human Lat-1 were lysed and immunoprecipitated with a human specific CD98 antibody. Non-reduced (NR) and reduced (R) immunoprecipitates were separated by SDS-PAGE and immunoblotted with either a human specific CD98 antibody (upper panel) or Flag antibody (lower panel). (C, D) Co-expression of CD98hc and Lat-1 increases amino acid transport and cell proliferation. (C) ³H-Isoleucine uptake was performed as described in Experimental Procedures. IMCD/Lat-1cells are IMCD cells transfected with Lat-1 only and CD98/Lat-1 cells were the Lat-1 cells that were further transfected with CD98 and selected by flow cytometry. (*) denotes statistically significant differences (p<0.05) compared to IMCD cells whereas (**) indicates significant differences compared to CD98 cells. (D) Transfected 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 ± s.e.m. of a representative experiment performed in eight replicates. (*) indicates statistically significant differences (p<0.05) compared to IMCD cells. (**) denotes a statistically significant difference between CD98 and CD98/Lat-1. (E) Cells expressing CD98 deletion mutants and Lat-1 proliferated less than CD98/Lat-1 cells. IMCD cells overexpressing Lat-1 were transfected with either CD98 or CD98 deletion mutant constructs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040026#pone-0040026-g001" target="_blank">Fig.1</a>. Thymidine incorporation was determined as described above. Values are the mean ± s.e.m. of a representative experiment performed in eight replicates. (*)indicates statistically significant differences (p<0.05) between double transfectants and cells transfected with CD98 or deletion mutants only.</p

CD98 regulates serum dependent cell proliferation by increasing Erk and p38 MAPK signaling.

<p>(A, C) CD98hc expression increases MAPK, Akt and mTor signaling in response to serum. Subconfluent cells were serum deprived for 24 hours then treated with complete medium. Lysates were prepared at the time points indicated and analyzed by western blot with the antibodies indicated. (B, D) MAPK regulates CD98hc-dependent IMCD cell proliferation. Cells seeded onto 96-well plates were serum deprived for 24 hours, pretreated with inhibitors prior to addition of ³H-Thymidine in complete medium as described in Experimental Procedures. U+SB denotes concomitant treatment with the MEK inhibitor U0126 (U) and the p38 MAPK inhibitor SB203580 (SB). (*) indicates statistically significant differences (p<0.05) between treated and untreated cells. (**) denotes statistically significant differences between U+SB versus either inhibitor alone.</p