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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.


Related in: MedlinePlus

IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts.(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.
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pone.0119780.g001: IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts.(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.

Mentions: First, we examined whether IL-6 signaling molecules are distributed in lipid rafts of myeloma cells. To prepare lipid rafts, CD45+ U266 cell lysates were subjected to a sucrose gradient ultracentrifuge and the gradient samples were fractionated into 12 fractions. As shown in Fig. 1A, both STAT3 and STAT1 were detected in both the raft and soluble fractions regardless of the presence of IL-6. IL-6 treatment induced the phosphorylation of both STAT3 and STAT1 and movement of P-STAT3 and P-STAT1 into the rafts fractions. Surprisingly, SHP2, SOS1 and ERK were not significantly distributed in the lipid raft fraction. Although IL-6 could induce tyrosine phosphorylation of ERK (P-ERK), P-ERK did not exist in the raft fraction. Raft marker CTB, which binds GM1 gangliosides, was detected only in the raft fraction (around fraction 5–8), while CD71, a nonraft marker [17,29], was found in virtually every soluble fraction (fractions 10–12). To determine the importance of P-STAT3 and P-STAT1 translocation to lipid rafts in intracellular signaling events, the activation of these molecules was monitored in CD45+ U266 cells depleted of cholesterol using MCD. IL-6 treatment resulted in time-dependent STAT1 and STAT3 phosphorylation. Such a response was dramatically abrogated in MCD treatment cells compared to untreated control cells (Fig. 1B). On the other hand, MCD could not effectively block IL-6-medicated activation of ERK. It seems that MCD abrogates the early stage of the activation of ERK but not the late stage. Overall, this data suggests that IL-6-mediated activation of STAT3 and STAT1, but not ERK, is specifically required for the integrity of lipid rafts.


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)

IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119780.g001: IL-6-induced STAT3 and STAT1 phosphorylation is required for the integrity of lipid rafts.(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.
Mentions: First, we examined whether IL-6 signaling molecules are distributed in lipid rafts of myeloma cells. To prepare lipid rafts, CD45+ U266 cell lysates were subjected to a sucrose gradient ultracentrifuge and the gradient samples were fractionated into 12 fractions. As shown in Fig. 1A, both STAT3 and STAT1 were detected in both the raft and soluble fractions regardless of the presence of IL-6. IL-6 treatment induced the phosphorylation of both STAT3 and STAT1 and movement of P-STAT3 and P-STAT1 into the rafts fractions. Surprisingly, SHP2, SOS1 and ERK were not significantly distributed in the lipid raft fraction. Although IL-6 could induce tyrosine phosphorylation of ERK (P-ERK), P-ERK did not exist in the raft fraction. Raft marker CTB, which binds GM1 gangliosides, was detected only in the raft fraction (around fraction 5–8), while CD71, a nonraft marker [17,29], was found in virtually every soluble fraction (fractions 10–12). To determine the importance of P-STAT3 and P-STAT1 translocation to lipid rafts in intracellular signaling events, the activation of these molecules was monitored in CD45+ U266 cells depleted of cholesterol using MCD. IL-6 treatment resulted in time-dependent STAT1 and STAT3 phosphorylation. Such a response was dramatically abrogated in MCD treatment cells compared to untreated control cells (Fig. 1B). On the other hand, MCD could not effectively block IL-6-medicated activation of ERK. It seems that MCD abrogates the early stage of the activation of ERK but not the late stage. Overall, this data suggests that IL-6-mediated activation of STAT3 and STAT1, but not ERK, is specifically required for the integrity of lipid rafts.

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