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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

PKC and downstream NF-κB are required for IL-6-induced proliferation in CD45+ myeloma cells.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. ** p < 0.01 vs DMSO control in the absence of IL-6; ## p < 0.01 vs DMSO control in the presence of IL-6 by a one-way ANOVA with HSD test.
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pone.0119780.g007: PKC and downstream NF-κB are required for IL-6-induced proliferation in CD45+ myeloma cells.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. ** p < 0.01 vs DMSO control in the absence of IL-6; ## p < 0.01 vs DMSO control in the presence of IL-6 by a one-way ANOVA with HSD test.

Mentions: We further used reagents that specifically inhibit PKC and IκB to evaluate whether PKC, NF-κB activation were involved in IL-6-induced proliferation. As shown in Fig. 7, treatment of CD45+ U266 cells (Fig. 7A) or CD45+ CD138+ cells (Fig. 7B) with PKC inhibitor abrogated IL-6-induced PKC and IκB phosphorylation, while the NF-κB inhibitor only affected IκB phosphorylation instead of PKC phosphorylation. Furthermore, both inhibitors triggered similar effects on IL-6-induced proliferation confirmed by both the myeloma cell line and primary cells. These results suggest that the activation of PKC and its downstream molecule NF-κB are especially required for IL-6-induced proliferation.


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)

PKC and downstream NF-κB are required for IL-6-induced proliferation in CD45+ myeloma cells.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. ** p < 0.01 vs DMSO control in the absence of IL-6; ## p < 0.01 vs DMSO control in the presence of IL-6 by a one-way ANOVA with HSD test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0119780.g007: PKC and downstream NF-κB are required for IL-6-induced proliferation in CD45+ myeloma cells.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. ** p < 0.01 vs DMSO control in the absence of IL-6; ## p < 0.01 vs DMSO control in the presence of IL-6 by a one-way ANOVA with HSD test.
Mentions: We further used reagents that specifically inhibit PKC and IκB to evaluate whether PKC, NF-κB activation were involved in IL-6-induced proliferation. As shown in Fig. 7, treatment of CD45+ U266 cells (Fig. 7A) or CD45+ CD138+ cells (Fig. 7B) with PKC inhibitor abrogated IL-6-induced PKC and IκB phosphorylation, while the NF-κB inhibitor only affected IκB phosphorylation instead of PKC phosphorylation. Furthermore, both inhibitors triggered similar effects on IL-6-induced proliferation confirmed by both the myeloma cell line and primary cells. These results suggest that the activation of PKC and its downstream molecule NF-κB are especially required for IL-6-induced proliferation.

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