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Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion.

Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Teasdale RD, Vanhaesebroeck B, Meunier FA, Taguchi T, Stow JL - J. Cell Biol. (2010)

Bottom Line: Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated.Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2.These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

ABSTRACT
Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.

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Impaired trafficking and secretion of TNF in macrophages from genetically inactivated p110δ mice. (A and B) Supernatants from LPS-stimulated WT and p110δD910A/D910A BMM cultures were collected every 2 h over a 10-h time course for ELISA measurements of secreted TNF (A) and IL-6 (B). Results indicate mean ± SEM from three littermates of each genotype. ND, not detected. (C) Flow cytometric analysis of intracellular and surface TNF staining in WT and p110δD910A/D910A BMMs treated with LPS and TAPI for 2 h. Mean fluorescence intensity in each TNF channel expressed as mean ratio ± SEM relative to LPS-stimulated WT from three independent experiments. ***, P < 0.001. Representative overlay histograms are shown for LPS-stimulated WT and p110δD910A/D910A BMMs. (D and E) Confocal analysis of TNF staining in activated WT and p110δD910A/D910A BMMs in the presence (D) or absence (E) of TAPI for 2 h. (D) Surface (red) and intracellular (green) TNF staining. Magnified images in D show BMMs costained with phalloidin (blue) to depict surface actin. (E) Intracellular TNF (green) in BMMs costained with the Golgi marker GM130 (red). Differential interference contrast images are included to define cells. Bars, 10 µm.
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fig3: Impaired trafficking and secretion of TNF in macrophages from genetically inactivated p110δ mice. (A and B) Supernatants from LPS-stimulated WT and p110δD910A/D910A BMM cultures were collected every 2 h over a 10-h time course for ELISA measurements of secreted TNF (A) and IL-6 (B). Results indicate mean ± SEM from three littermates of each genotype. ND, not detected. (C) Flow cytometric analysis of intracellular and surface TNF staining in WT and p110δD910A/D910A BMMs treated with LPS and TAPI for 2 h. Mean fluorescence intensity in each TNF channel expressed as mean ratio ± SEM relative to LPS-stimulated WT from three independent experiments. ***, P < 0.001. Representative overlay histograms are shown for LPS-stimulated WT and p110δD910A/D910A BMMs. (D and E) Confocal analysis of TNF staining in activated WT and p110δD910A/D910A BMMs in the presence (D) or absence (E) of TAPI for 2 h. (D) Surface (red) and intracellular (green) TNF staining. Magnified images in D show BMMs costained with phalloidin (blue) to depict surface actin. (E) Intracellular TNF (green) in BMMs costained with the Golgi marker GM130 (red). Differential interference contrast images are included to define cells. Bars, 10 µm.

Mentions: Therefore, we isolated bone marrow–derived macrophages (BMMs) from p110δD910A/D910A mice and WT littermates to study cytokine trafficking and secretion. Unstimulated WT and p110δD910A/D910A BMMs showed negligible TNF production or secretion (Fig. 3, A and C), as expected. In response to LPS stimulation, TNF secretion was detected in supernatants of both WT and p110δD910A/D910A BMMs by 2 h, and its secretion was maintained at high levels from 4–10 h only in WT cultures (Fig. 3 A). In contrast, TNF secretion from p110δD910A/D910A BMMs was severely reduced by 70–80% throughout the time course. This closely recapitulated the effects of IC87114 on LPS-stimulated secretion of TNF from RAW264.7 macrophages (Fig. 1 B). We also tested the ability of p110δD910A/D910A BMMs to effectively secrete cytokines other than TNF. Interleukin-6 (IL-6) is another proinflammatory cytokine whose biosynthetic trafficking pathway partially overlaps with that of TNF in RAW264.7 cells (Manderson et al., 2007). Upon LPS activation, both WT and p110δD910A/D910A BMMs produce IL-6, which was secreted alongside TNF (Fig. 3 B). Activated p110δD910A/D910A BMMs secreted IL-6 at levels similar to WT cells, which is consistent with only a modest impact of p110δ inactivation on secretion of this cytokine. Therefore, loss of p110δ activity selectively inhibits the trafficking and secretion of TNF but not IL-6 in BMMs. Although IL-6 secretion is not regulated by p110δ, it could nevertheless be reduced in RAW264.7 cells treated with LY294002 or wortmannin (Fig. S1, C and D, respectively), suggesting that other PI3K isoforms might be involved.


Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion.

Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Teasdale RD, Vanhaesebroeck B, Meunier FA, Taguchi T, Stow JL - J. Cell Biol. (2010)

Impaired trafficking and secretion of TNF in macrophages from genetically inactivated p110δ mice. (A and B) Supernatants from LPS-stimulated WT and p110δD910A/D910A BMM cultures were collected every 2 h over a 10-h time course for ELISA measurements of secreted TNF (A) and IL-6 (B). Results indicate mean ± SEM from three littermates of each genotype. ND, not detected. (C) Flow cytometric analysis of intracellular and surface TNF staining in WT and p110δD910A/D910A BMMs treated with LPS and TAPI for 2 h. Mean fluorescence intensity in each TNF channel expressed as mean ratio ± SEM relative to LPS-stimulated WT from three independent experiments. ***, P < 0.001. Representative overlay histograms are shown for LPS-stimulated WT and p110δD910A/D910A BMMs. (D and E) Confocal analysis of TNF staining in activated WT and p110δD910A/D910A BMMs in the presence (D) or absence (E) of TAPI for 2 h. (D) Surface (red) and intracellular (green) TNF staining. Magnified images in D show BMMs costained with phalloidin (blue) to depict surface actin. (E) Intracellular TNF (green) in BMMs costained with the Golgi marker GM130 (red). Differential interference contrast images are included to define cells. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3101599&req=5

fig3: Impaired trafficking and secretion of TNF in macrophages from genetically inactivated p110δ mice. (A and B) Supernatants from LPS-stimulated WT and p110δD910A/D910A BMM cultures were collected every 2 h over a 10-h time course for ELISA measurements of secreted TNF (A) and IL-6 (B). Results indicate mean ± SEM from three littermates of each genotype. ND, not detected. (C) Flow cytometric analysis of intracellular and surface TNF staining in WT and p110δD910A/D910A BMMs treated with LPS and TAPI for 2 h. Mean fluorescence intensity in each TNF channel expressed as mean ratio ± SEM relative to LPS-stimulated WT from three independent experiments. ***, P < 0.001. Representative overlay histograms are shown for LPS-stimulated WT and p110δD910A/D910A BMMs. (D and E) Confocal analysis of TNF staining in activated WT and p110δD910A/D910A BMMs in the presence (D) or absence (E) of TAPI for 2 h. (D) Surface (red) and intracellular (green) TNF staining. Magnified images in D show BMMs costained with phalloidin (blue) to depict surface actin. (E) Intracellular TNF (green) in BMMs costained with the Golgi marker GM130 (red). Differential interference contrast images are included to define cells. Bars, 10 µm.
Mentions: Therefore, we isolated bone marrow–derived macrophages (BMMs) from p110δD910A/D910A mice and WT littermates to study cytokine trafficking and secretion. Unstimulated WT and p110δD910A/D910A BMMs showed negligible TNF production or secretion (Fig. 3, A and C), as expected. In response to LPS stimulation, TNF secretion was detected in supernatants of both WT and p110δD910A/D910A BMMs by 2 h, and its secretion was maintained at high levels from 4–10 h only in WT cultures (Fig. 3 A). In contrast, TNF secretion from p110δD910A/D910A BMMs was severely reduced by 70–80% throughout the time course. This closely recapitulated the effects of IC87114 on LPS-stimulated secretion of TNF from RAW264.7 macrophages (Fig. 1 B). We also tested the ability of p110δD910A/D910A BMMs to effectively secrete cytokines other than TNF. Interleukin-6 (IL-6) is another proinflammatory cytokine whose biosynthetic trafficking pathway partially overlaps with that of TNF in RAW264.7 cells (Manderson et al., 2007). Upon LPS activation, both WT and p110δD910A/D910A BMMs produce IL-6, which was secreted alongside TNF (Fig. 3 B). Activated p110δD910A/D910A BMMs secreted IL-6 at levels similar to WT cells, which is consistent with only a modest impact of p110δ inactivation on secretion of this cytokine. Therefore, loss of p110δ activity selectively inhibits the trafficking and secretion of TNF but not IL-6 in BMMs. Although IL-6 secretion is not regulated by p110δ, it could nevertheless be reduced in RAW264.7 cells treated with LY294002 or wortmannin (Fig. S1, C and D, respectively), suggesting that other PI3K isoforms might be involved.

Bottom Line: Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated.Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2.These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

ABSTRACT
Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.

Show MeSH
Related in: MedlinePlus