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Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

Zheng X, Li AS, Zheng H, Zhao D, Guan D, Zou H - PLoS ONE (2015)

Bottom Line: The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK.Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low.Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, ShengJing Hospital of China Medical University, Shenyang, Liaoning, China.

ABSTRACT
In response to interleukin 6 (IL-6) stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT)3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB pathways.

No MeSH data available.


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Phosphorylation levels of STAT3 and ERK are different between CD45+ and CD45-myeloma cells.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.
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pone.0119780.g003: Phosphorylation levels of STAT3 and ERK are different between CD45+ and CD45-myeloma cells.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.

Mentions: We have demonstrated that CD45 RO, RB, but not RA move to lipid rafts and facilitate IL-6-induced signaling [17]. This study was then aimed at understanding the mechanism behind IL-6-induced proliferation regulated by different CD45 isoforms. First, we carefully evaluate whether the phosphorylation level of IL-6-mediated signaling molecules differed between CD45+ and CD45- myeloma cells. As shown in Fig. 3A, IL-6 stimulation induced specific phosphorylation of STAT1, STAT3 and ERK. Surprisingly, the tyrosine phosphorylation level of STAT3, but not STAT1, was greatly enhanced in CD45+ U266 cells, which are CD45RO+, CD45RB+, CD45RA-, in comparison with the cells without expression of CD45. In contrast, ERK phosphorylation was significantly reduced in CD45+ U266 cells. Because IL-6 elicits distinct responses and activates various signaling pathways in a cell-specific manner [8,37], we evaluate our results in other cell types. Similar to CD45+ U266, ILKM2 myeloma cells were also highly expressed by CD45RO, RB but not for CD45RA expression (Fig. 3B). Western blotting data clearly showed that IL-6-induced STAT3 phosphorylation level was markedly higher in CD45+ ILKM2 cells, while ERK phosphorylation level was significantly lower. To rule out the cell line based phenomena, primary patient samples were also included and similar results were obtained by comparing CD45+ CD138+ with CD45- CD138+ myeloma cells (Fig. 3C). This data suggests that CD45 (CD45RO, CD45RB) may upregulate IL-6-induced STAT3 tyrosine phosphorylation while downregulating ERK tyrosine phosphorylation.


Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

Zheng X, Li AS, Zheng H, Zhao D, Guan D, Zou H - PLoS ONE (2015)

Phosphorylation levels of STAT3 and ERK are different between CD45+ and CD45-myeloma cells.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363322&req=5

pone.0119780.g003: Phosphorylation levels of STAT3 and ERK are different between CD45+ and CD45-myeloma cells.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.
Mentions: We have demonstrated that CD45 RO, RB, but not RA move to lipid rafts and facilitate IL-6-induced signaling [17]. This study was then aimed at understanding the mechanism behind IL-6-induced proliferation regulated by different CD45 isoforms. First, we carefully evaluate whether the phosphorylation level of IL-6-mediated signaling molecules differed between CD45+ and CD45- myeloma cells. As shown in Fig. 3A, IL-6 stimulation induced specific phosphorylation of STAT1, STAT3 and ERK. Surprisingly, the tyrosine phosphorylation level of STAT3, but not STAT1, was greatly enhanced in CD45+ U266 cells, which are CD45RO+, CD45RB+, CD45RA-, in comparison with the cells without expression of CD45. In contrast, ERK phosphorylation was significantly reduced in CD45+ U266 cells. Because IL-6 elicits distinct responses and activates various signaling pathways in a cell-specific manner [8,37], we evaluate our results in other cell types. Similar to CD45+ U266, ILKM2 myeloma cells were also highly expressed by CD45RO, RB but not for CD45RA expression (Fig. 3B). Western blotting data clearly showed that IL-6-induced STAT3 phosphorylation level was markedly higher in CD45+ ILKM2 cells, while ERK phosphorylation level was significantly lower. To rule out the cell line based phenomena, primary patient samples were also included and similar results were obtained by comparing CD45+ CD138+ with CD45- CD138+ myeloma cells (Fig. 3C). This data suggests that CD45 (CD45RO, CD45RB) may upregulate IL-6-induced STAT3 tyrosine phosphorylation while downregulating ERK tyrosine phosphorylation.

Bottom Line: The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK.Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low.Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, ShengJing Hospital of China Medical University, Shenyang, Liaoning, China.

ABSTRACT
In response to interleukin 6 (IL-6) stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT)3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB pathways.

No MeSH data available.


Related in: MedlinePlus