We have previously reported that CD45 functions as a positive regulator for myeloma cell proliferation in the downstream of interleukin-6 (IL-6) by regulating activation of Lyn (

). It remains to be clarified how CD45 regulates IL-6-induced proliferation in myeloma cells. In this study, we aim to explore the regulation and isoform-specific difference on the mechanism of CD45 translocation induced by IL-6 in human myeloma cells. We used myeloma cell lines, U266 and ILKM2, which express CD45RO and CD45RB but not CD45RA in this study. We showed that methyl-beta-cyclodextrin, a lipid rafts inhibitor, significantly inhibited IL-6 induced proliferation, diminished phosphorylation of Lyn and signal transducer and activator of transcription 3 (STAT3), and abolished nuclear localization of STAT3, suggesting the significant role of lipid rafts on IL-6 signaling. Using sucrose density gradient fractionation method, we observed the dramatic change of CD45 distribution by IL-6 stimulation. Without IL-6 stimulation, CD45RO and RB molecules were excluded from lipid rafts. But, IL-6 stimulation results in significant translocation of both isoforms to lipid rafts. On the other hand, IL-6R alpha, gp130, Lyn and a raft integral protein, flotillin-2, were always localized inside lipid rafts. Furthermore, it was also confirmed that IL-6 induced CD45RO and CD45RB translocation to lipid rafts by confocal laser scanning microscopy, while IL-6R alpha always remained inside rafts. These observations have also been confirmed by the exogenous expression of CD45RO, RB tagged with green fluorescent protein in CD45 negative U266 cells. As negative control, Epstein-Barr Virus transformed B cells, C066 or KUS, which express CD45RA and CD45RB, but not CD45RO, were used. BCR cross-linking using anti-IgM stimulation induced IgM and Ig beta translocation to lipid rafts, but both CD45RB and RA showed no marked translocation. And we couldn’t observe marked translocation of CD45RB nor CD45RA to rafts after IL-6 stimulation. As the level of IL-6R alpha was very low in these cells, we introduced IL-6R alpha gene into KUS or C066 cells. Then, translocation of CD45RB to lipid rafts was observed while CD45RA did not in response to IL-6 stimulation. In addition, we confirmed IL-6 induced complex formation of CD45 with IL-6R alpha, Lyn on lipid rafts. We also checked the phosphorylation state of two tyrosine residues (Tyr396 and Tyr507) of Lyn. In CD45 positive cells, IL-6 induced dephosphorylation of the negative regulatory tyrosine, Tyr507, after that, Tyr396, the positive regulatory site of kinase activity, was phosphorylated. While, in CD45 negative cells, Tyr507 dephosphorylation and significant phosphorylation of Tyr396 were not observed. And after IL-6 stimulation, we also observed PAG dephosphorylation and Csk release from lipid rafts, which might finally lead to the dephosphorylation of Tyr507 of Lyn. Considering these results, we conclude that CD45RO and RB translocation to lipid rafts is significant for myeloma cells to proliferate in response to IL-6.

Author notes

Corresponding author

Sign in via your Institution