Limits...
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 S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts.(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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4363322&req=5

pone.0119780.g002: IL-6-induced S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts.(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.

Mentions: It is well known that in response to stimulation by IL-6, tyrosine phosphorylated STATs undergo dimerization and are translocated from cytoplasm to nucleus [39,40]. To evaluate STAT’s nuclear trafficking in a live cell, stable transfectants of STAT1-EGFP and STAT3-EGFP in CD45+ U266 cells were stimulated with IL-6. Then, specific tyrosine 701 for STAT1 and 705 for STAT3 phosphorylation were examined. Both EGFP-tagged and untagged STAT1 and STAT3 exhibited robust tyrosine phosphorylation with IL-6 stimulation (Fig. 2A), suggesting the potential biological function of STAT3-EGFP and STAT1-EGFP. To further confirm whether IL-6-induced activation of STATs is dependent upon lipid rafts, we carried out real-time microscopic imaging of the translocation of STATs to the nucleus. As shown in Fig. 2B, stably expressed STAT3-EGFP in CD45+ U266 cells was localized in the cytoplasm and nucleus even after IL-6 removal for 12 hours. STAT3-EGFP was found to be prominently nuclear soon after the IL-6 stimulation. The peak nuclear fluorescence intensity was about 10 minutes. However, STAT1-EGFP was clearly mainly localized in the cytoplasm prior to treatment with IL-6, which was different from STAT3-EGFP. The fluorescence intensity of the nucleus only rapidly increased between 10 and 30 minutes and peaked at 30 minutes. Furthermore, when STAT3 and STAT1-EGFP expressed cells were treated with MCD, a cholesterol sequestrating agent, the IL-6-induced STAT1 and STAT3 nuclear translocation was blocked. Our previous report already found that MCD could significantly inhibit IL-6-induced proliferation in CD45+ U266 cells [17]. Therefore, current data suggests that the integrity of lipid rafts is also required for nuclear localization of STATs and may potentiate subsequent proliferation in multiple myeloma cells.


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 S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119780.g002: IL-6-induced S.TAT3 and STAT1 nuclear translocation is required for the integrity of lipid rafts.(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.
Mentions: It is well known that in response to stimulation by IL-6, tyrosine phosphorylated STATs undergo dimerization and are translocated from cytoplasm to nucleus [39,40]. To evaluate STAT’s nuclear trafficking in a live cell, stable transfectants of STAT1-EGFP and STAT3-EGFP in CD45+ U266 cells were stimulated with IL-6. Then, specific tyrosine 701 for STAT1 and 705 for STAT3 phosphorylation were examined. Both EGFP-tagged and untagged STAT1 and STAT3 exhibited robust tyrosine phosphorylation with IL-6 stimulation (Fig. 2A), suggesting the potential biological function of STAT3-EGFP and STAT1-EGFP. To further confirm whether IL-6-induced activation of STATs is dependent upon lipid rafts, we carried out real-time microscopic imaging of the translocation of STATs to the nucleus. As shown in Fig. 2B, stably expressed STAT3-EGFP in CD45+ U266 cells was localized in the cytoplasm and nucleus even after IL-6 removal for 12 hours. STAT3-EGFP was found to be prominently nuclear soon after the IL-6 stimulation. The peak nuclear fluorescence intensity was about 10 minutes. However, STAT1-EGFP was clearly mainly localized in the cytoplasm prior to treatment with IL-6, which was different from STAT3-EGFP. The fluorescence intensity of the nucleus only rapidly increased between 10 and 30 minutes and peaked at 30 minutes. Furthermore, when STAT3 and STAT1-EGFP expressed cells were treated with MCD, a cholesterol sequestrating agent, the IL-6-induced STAT1 and STAT3 nuclear translocation was blocked. Our previous report already found that MCD could significantly inhibit IL-6-induced proliferation in CD45+ U266 cells [17]. Therefore, current data suggests that the integrity of lipid rafts is also required for nuclear localization of STATs and may potentiate subsequent proliferation in multiple myeloma cells.

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