Model of CD45 isoforms-mediated IL-6 signaling in multiple myeloma cells.

<p>Engagement of IL-6R with IL-6 leads to complex formation of IL-6R, gp130, Lyn as well as CD45RO/RB in raft microdomains. In response to IL-6 stimulation, CD45RO/RB moves into lipid rafts to induce dephosphorylation of the negative regulatory of Tyr507, phosphorylation of Tyr396, and subsequent conformation change and Lyn activation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119780#pone.0119780.ref017" target="_blank">17</a>]. We confirmed our hypothesis that lipid rafts-targeted CD45RO/RB facilitates IL-6-induced STAT3 and Lyn/PKC/NF-κB activation in rafts microdomains, while raft-excluded CD45RA remains outside of lipid rafts after IL-6 stimulation and negatively regulates ERK-activation.</p

PKC and downstream NF-κB are required for IL-6-induced proliferation in CD45⁺ myeloma cells.

<p>CD45⁺ U266 cells (A) or CD45⁺ CD138⁺ primary cells (B) were incubated with or without PKC inhibitor Ro31-8220 (1 μM), or NF-κB inhibitor BAY11-7082 (5 μM) for 1 hour and stimulated with IL-6 (10 ng/ml). PKC phosphorylation level was measured after 10 minutes of stimulation with IL-6, and the IκB phosphorylation level was analyzed after 60 minutes stimulation. Western blotting was performed by using specific antibodies. The representative blots of three independent experiments are shown. BrdU incorporation was used to detect the DNA synthesis by IL-6 after 72 hours. DMSO is used as a control. Data are shown as mean ± SD of triplicate cultures and are from one experiment representative of three performed. ** <i>p</i> < 0.01 vs DMSO control in the absence of IL-6; ## <i>p</i> < 0.01 vs DMSO control in the presence of IL-6 by a one-way ANOVA with HSD test.</p

Phosphorylation levels of STAT3 and ERK are different between CD45⁺ and CD45^-myeloma cells.

<p>CD45^- U266 cells and CD45⁺ U266 cells (A), CD45^- NOP2 cells and CD45⁺ ILKM2 cells (B), and CD45^- CD138⁺ and CD45⁺ CD138⁺ primary cells (C) were stained for CD45RO, RB and RA antibodies and analyzed by flow cytometry. The percentage expression relative to its isotype control is shown in the histogram. Cells were also stimulated with IL-6 for the indicated time. Western blotting was performed by using specific antibodies and the representative blots of three independent experiments are shown.</p

CD45 expression enhances the nuclear localization of STAT3.

<p>(A) CD45^- U266 and CD45⁺ U266 cells transfected with STAT3-EGFP were analyzed by confocal microscopy for EGFP fusion protein. After stimulation with 10 ng/ml of IL-6, the distributions of STAT3-EGFP fusion protein in nuclear, cytoplasm, or cytoplasm plus nuclear were quantified by counting 100 cells at different time points. The mean percentages from three independent experiments were calculated. (B) Cytoplasm and nuclear extracts were isolated from CD45^- U266 cells and CD45⁺ U266 cells, respectively, and analyzed by western blotting with antibodies against phosphorylated STAT3 or STAT3. Cytoplasmic marker β-action and nuclear marker SP1 were used to indicate the purity of our extraction procedure. Data shown are representative of three experiments.</p

IL-6-induced S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts.

<p>(A) CD45⁺ U266 cells were transfected with untagged (mock) or STAT1-EGFP, STAT3-EGFP expression plasmids, and either treated or untreated with IL-6 at different time points. Immunoblotting was performed as above. (B) Subcellular distribution of STATs-EGFP fusion proteins. Nuclear translocation of both STAT3-EGFP and STAT1-EGFP was evaluated by live cell imaging. Cells were pre-incubated with or without 10 mM MCD for 30 minutes and then stimulated with 10 ng/ml of IL-6 and images were generated at different time points. P-REP was used as a mock vector, which expresses EGFP. Data shown are representative of three experiments.</p

IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts.

<p>(A) CD45⁺ U266 cells were grown in IL-6 free medium for 12 hours (IL-6 starvation). Cells were incubated with or without 10 ng/ml of IL-6 for 5 minutes. The cell lysates were subjected to sucrose density gradient centrifugation, and endogenous proteins indicated beside figures from each sucrose fraction were analyzed by immunoblotting. CD71 was detected as a nonraft marker. Lipid raft fractions were confirmed using CTX dot plots for each fraction. (B) CD45⁺ U266 cells were treated or untreated with MCD (10 mM) for 30 minutes at 37°C. Cells were then stimulated with IL-6 at different time points. Whole-cell lysates were subjected to SDS/PAGE and separate plots with antibodies are shown. The representative blots of three independent experiments are shown.</p

Effects of Exogenously expressed CD45RO, RB, or RA-EGFP on phosphorylation of STAT3, MAPK, PKC and IκB.

<p>CD45^- U266 cells were transfected with untagged (mock) or CD45RO-EGFP, CD45RB-EGFP or CD45RA-EGFP expression plasmids, and either treated or untreated with IL-6 (10 ng/ml) at different time points. The representative blots are from three independent experiments and separate blotting using antibodies to P-STAT3, STAT3 (A), P-ERK, ERK (B), P-PKC, PKC (C) and P-IκB, IκB (D) are shown. The densities of protein bands were determined by densitometry and the data represent a change from the control mock density. * <i>p</i> < 0.05 vs mock control in the presence of IL-6; ** <i>p</i> < 0.01 vs mock control in the presence of IL-6 by a one-way ANOVA with HSD test.</p