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The role of protein kinase C alpha translocation in radiation-induced bystander effect.

Fang Z, Xu A, Wu L, Hei TK, Hong M - Sci Rep (2016)

Bottom Line: However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear.Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells.Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China.

ABSTRACT
Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

No MeSH data available.


Related in: MedlinePlus

PKC activation affected TNFR1 distribution within AL cells.(a) TNFR1 expression in bystander cells. Membrane fraction of the bystander cells was isolated, subjected to western blotting and probed with TNFR1 antibody. Integrin was used as loading control. (b) Accumulation of TNFR1 in membrane fraction after PKC activation. Cells were treated with 1 μM PKC activator PMA and analyzed as described before. (c) PKC inhibitors suppressed TNFR1 accumulation along cell membrane. Cells were treated with 1 μM PMA for 30 min without or with PKC inhibitors chelerythrine chloride (10 μM) or Gö6976 (5 nM). For all western blottings, three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). (d,e) PMA accelerated internalization and recycling of TNFR1. Cells were labeled with biotin, then treated without or with 1 μM PMA for 30 min at 37 °C for protein internalization and recycling as described in the “Methods” section. Biotin-labeled proteins were pull-downed by streptavidin beads, separated by SDS-PAGE, followed by western blotting. (f ) Relative level of internalized and recycled TNFR1 after PKC activation. Data are pooled from three independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between PMA treatment and the untreated control (p = 0.004 for internalization and p = 0.0005 for recycling).
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f3: PKC activation affected TNFR1 distribution within AL cells.(a) TNFR1 expression in bystander cells. Membrane fraction of the bystander cells was isolated, subjected to western blotting and probed with TNFR1 antibody. Integrin was used as loading control. (b) Accumulation of TNFR1 in membrane fraction after PKC activation. Cells were treated with 1 μM PKC activator PMA and analyzed as described before. (c) PKC inhibitors suppressed TNFR1 accumulation along cell membrane. Cells were treated with 1 μM PMA for 30 min without or with PKC inhibitors chelerythrine chloride (10 μM) or Gö6976 (5 nM). For all western blottings, three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). (d,e) PMA accelerated internalization and recycling of TNFR1. Cells were labeled with biotin, then treated without or with 1 μM PMA for 30 min at 37 °C for protein internalization and recycling as described in the “Methods” section. Biotin-labeled proteins were pull-downed by streptavidin beads, separated by SDS-PAGE, followed by western blotting. (f ) Relative level of internalized and recycled TNFR1 after PKC activation. Data are pooled from three independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between PMA treatment and the untreated control (p = 0.004 for internalization and p = 0.0005 for recycling).

Mentions: Cellular response to TNFα is mediated through interaction with two TNF receptors TNFR1 and TNFR2. TNFR1 is expressed in various tissues and cytotoxicity elicited by TNF mostly acts through TNFR1; while TNFR2 is typically found in cells of the immune system, and mainly responds to the membrane-bound form of TNFα2728. We therefore examined the expression of TNFR1 in bystander AL cells and found an increased level of TNFR1 in the membrane fraction (Fig. 3a, left panel). We further performed biotinylation labeling and western blotting to confirm the location of accumulated TNFR1 and found out that the receptor was accumulated on plasma membrane. When cells were pretreated with chelerythrine chloride before they were subjected to irradiation, suppression of such an increment was observed (Fig. 3a, right panel), suggesting a link between PKC translocation and the accumulation of TNFR1 along the cell membrane. We next wanted to see whether the accumulation of TNFR1 in AL cell surface was due to PKC activation. When AL cells were treated with PMA, an activator of PKC, PKC level was elevated in the membrane fraction (Supplementary Fig. S3). The level of TNFR1 was increased as well (Fig. 3b). When cells were co-treated with PMA and PKC inhibitors chelerythrine chloride or Gö6976, the elevation of TNFR1 on cell membrane was attenuated (Fig. 3c), implicating that activation of PKC may increase TNFR1 level on plasma membrane.


The role of protein kinase C alpha translocation in radiation-induced bystander effect.

Fang Z, Xu A, Wu L, Hei TK, Hong M - Sci Rep (2016)

PKC activation affected TNFR1 distribution within AL cells.(a) TNFR1 expression in bystander cells. Membrane fraction of the bystander cells was isolated, subjected to western blotting and probed with TNFR1 antibody. Integrin was used as loading control. (b) Accumulation of TNFR1 in membrane fraction after PKC activation. Cells were treated with 1 μM PKC activator PMA and analyzed as described before. (c) PKC inhibitors suppressed TNFR1 accumulation along cell membrane. Cells were treated with 1 μM PMA for 30 min without or with PKC inhibitors chelerythrine chloride (10 μM) or Gö6976 (5 nM). For all western blottings, three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). (d,e) PMA accelerated internalization and recycling of TNFR1. Cells were labeled with biotin, then treated without or with 1 μM PMA for 30 min at 37 °C for protein internalization and recycling as described in the “Methods” section. Biotin-labeled proteins were pull-downed by streptavidin beads, separated by SDS-PAGE, followed by western blotting. (f ) Relative level of internalized and recycled TNFR1 after PKC activation. Data are pooled from three independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between PMA treatment and the untreated control (p = 0.004 for internalization and p = 0.0005 for recycling).
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Related In: Results  -  Collection

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f3: PKC activation affected TNFR1 distribution within AL cells.(a) TNFR1 expression in bystander cells. Membrane fraction of the bystander cells was isolated, subjected to western blotting and probed with TNFR1 antibody. Integrin was used as loading control. (b) Accumulation of TNFR1 in membrane fraction after PKC activation. Cells were treated with 1 μM PKC activator PMA and analyzed as described before. (c) PKC inhibitors suppressed TNFR1 accumulation along cell membrane. Cells were treated with 1 μM PMA for 30 min without or with PKC inhibitors chelerythrine chloride (10 μM) or Gö6976 (5 nM). For all western blottings, three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). (d,e) PMA accelerated internalization and recycling of TNFR1. Cells were labeled with biotin, then treated without or with 1 μM PMA for 30 min at 37 °C for protein internalization and recycling as described in the “Methods” section. Biotin-labeled proteins were pull-downed by streptavidin beads, separated by SDS-PAGE, followed by western blotting. (f ) Relative level of internalized and recycled TNFR1 after PKC activation. Data are pooled from three independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between PMA treatment and the untreated control (p = 0.004 for internalization and p = 0.0005 for recycling).
Mentions: Cellular response to TNFα is mediated through interaction with two TNF receptors TNFR1 and TNFR2. TNFR1 is expressed in various tissues and cytotoxicity elicited by TNF mostly acts through TNFR1; while TNFR2 is typically found in cells of the immune system, and mainly responds to the membrane-bound form of TNFα2728. We therefore examined the expression of TNFR1 in bystander AL cells and found an increased level of TNFR1 in the membrane fraction (Fig. 3a, left panel). We further performed biotinylation labeling and western blotting to confirm the location of accumulated TNFR1 and found out that the receptor was accumulated on plasma membrane. When cells were pretreated with chelerythrine chloride before they were subjected to irradiation, suppression of such an increment was observed (Fig. 3a, right panel), suggesting a link between PKC translocation and the accumulation of TNFR1 along the cell membrane. We next wanted to see whether the accumulation of TNFR1 in AL cell surface was due to PKC activation. When AL cells were treated with PMA, an activator of PKC, PKC level was elevated in the membrane fraction (Supplementary Fig. S3). The level of TNFR1 was increased as well (Fig. 3b). When cells were co-treated with PMA and PKC inhibitors chelerythrine chloride or Gö6976, the elevation of TNFR1 on cell membrane was attenuated (Fig. 3c), implicating that activation of PKC may increase TNFR1 level on plasma membrane.

Bottom Line: However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear.Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells.Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China.

ABSTRACT
Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

No MeSH data available.


Related in: MedlinePlus