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Y-box protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen.

Frye BC, Halfter S, Djudjaj S, Muehlenberg P, Weber S, Raffetseder U, En-Nia A, Knott H, Baron JM, Dooley S, Bernhagen J, Mertens PR - EMBO Rep. (2009)

Bottom Line: Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications.The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration.Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology & Immunology, University Hospital RWTH Aachen, Aachen, Germany.

ABSTRACT
Y-box protein (YB)-1 of the cold-shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB-1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB-1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB-1 was secreted through a non-classical mode resembling that of the macrophage migration inhibitory factor. YB-1 release requires ATP-binding cassette transporters, and microvesicles protect YB-1 from protease degradation. Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications. The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

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YB-1 is secreted through a non-classical, vesicle-mediated pathway by LPS-stimulated cells. (A) Confocal laser-scanning microscopy visualized formation of YB-1–GFP-enriched vesicles in 2 h after LPS stimulation of rMCs, however, this was not found for GFP-stimulation alone. Time-lapse live-cell microscopy was carried out for over 120 min with images taken every 2 min (supplementary videos 1 and 2 online). (B) Biochemical characterization of the YB-1 export mechanism was carried out with LPS-stimulated MM6 monocytic cells. Preincubation with brefeldin A as an inhibitor of the classical export pathway enhances YB-1 secretion. Preincubation with inhibitors of ATP-binding cassette transporters (C) probenicid and (D) glyburide reduces the release of YB-1. (E) The ionophore ionomycin superinduces LPS-dependent YB-1 secretion. (F) Disruption of the electrochemical gradient by reserpine successfully blocks the LPS effect on YB-1 secretion. (G) Schematic drawing of the YB-1 domains and distribution of 16 lysines (Ks) in the protein. (H) Expression plasmids for Flag–YB-1 and double-mutated Flag–YB-1 Lys301/304Ala proteins were introduced into rMCs and equal expression levels were determined by Flag antibody. After LPS stimulation a time-dependent release of Flag–YB-1, but not of mutated Flag–YB-1 (Lys301/304Ala), is detected with the supernatant (upper panels). Controls for cellular protein and precipitation efficiency are provided in the lower panels. CSD, cold shock domain; GFP, green fluorescent protein; LPS, lipopolysaccharide; mm6, MonoMac-6; rMCs, rat mesangial cells; YB, Y-box protein.
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f2: YB-1 is secreted through a non-classical, vesicle-mediated pathway by LPS-stimulated cells. (A) Confocal laser-scanning microscopy visualized formation of YB-1–GFP-enriched vesicles in 2 h after LPS stimulation of rMCs, however, this was not found for GFP-stimulation alone. Time-lapse live-cell microscopy was carried out for over 120 min with images taken every 2 min (supplementary videos 1 and 2 online). (B) Biochemical characterization of the YB-1 export mechanism was carried out with LPS-stimulated MM6 monocytic cells. Preincubation with brefeldin A as an inhibitor of the classical export pathway enhances YB-1 secretion. Preincubation with inhibitors of ATP-binding cassette transporters (C) probenicid and (D) glyburide reduces the release of YB-1. (E) The ionophore ionomycin superinduces LPS-dependent YB-1 secretion. (F) Disruption of the electrochemical gradient by reserpine successfully blocks the LPS effect on YB-1 secretion. (G) Schematic drawing of the YB-1 domains and distribution of 16 lysines (Ks) in the protein. (H) Expression plasmids for Flag–YB-1 and double-mutated Flag–YB-1 Lys301/304Ala proteins were introduced into rMCs and equal expression levels were determined by Flag antibody. After LPS stimulation a time-dependent release of Flag–YB-1, but not of mutated Flag–YB-1 (Lys301/304Ala), is detected with the supernatant (upper panels). Controls for cellular protein and precipitation efficiency are provided in the lower panels. CSD, cold shock domain; GFP, green fluorescent protein; LPS, lipopolysaccharide; mm6, MonoMac-6; rMCs, rat mesangial cells; YB, Y-box protein.

Mentions: Primary human monocytes were exposed to increasing lipopolysaccharide (LPS) concentrations for 4 h and the supernatants were trichloroacetic acid (TCA)-precipitated. Immunoblotting showed dose-dependent secretion of YB-1, which peaked at a concentration of 5 ng/ml LPS (Fig 1B), whereas no non-specific cytosolic protein release, for example that of glyceraldehyde 3-phosphate dehydrogenase, was observed. A Trypan-blue exclusion assay confirmed that protein release was not due to a lack of cell integrity (viability >95%). By contrast, intracellular YB-1 protein levels essentially did not change after LPS exposure (Fig 2B, lower panel). Calculation of protein loading and band intensities indicated that approximately 25% of total YB-1 protein was secreted by primary monocytes on stimulation, using 5 ng/ml LPS (Fig 1C). Quantification of macrophage migration inhibitory factor (MIF; Kleemann et al, 2000) in the same supernatant by ELISA indicated a similar dose response to LPS (Fig 1D). LPS-induced secretion of both YB-1 and MIF was also observed in MonoMac-6 (MM6) cells (supplementary Fig 1B–D online), underpinning the above findings. MM6 cells are well-characterized LPS-responsive monocytic cells. In this system, YB-1 secretion was also apparent in a narrow LPS concentration range between 1 and 7.5 ng/ml, which paralleled MIF secretion (supplementary Fig 1B,C online). Kinetic studies carried out at the peak LPS concentration of 5 ng/ml showed low-level baseline secretion of YB-1 up to 2 h, followed by an apparent marked LPS effect after 4 h (supplementary Fig 1D online).


Y-box protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen.

Frye BC, Halfter S, Djudjaj S, Muehlenberg P, Weber S, Raffetseder U, En-Nia A, Knott H, Baron JM, Dooley S, Bernhagen J, Mertens PR - EMBO Rep. (2009)

YB-1 is secreted through a non-classical, vesicle-mediated pathway by LPS-stimulated cells. (A) Confocal laser-scanning microscopy visualized formation of YB-1–GFP-enriched vesicles in 2 h after LPS stimulation of rMCs, however, this was not found for GFP-stimulation alone. Time-lapse live-cell microscopy was carried out for over 120 min with images taken every 2 min (supplementary videos 1 and 2 online). (B) Biochemical characterization of the YB-1 export mechanism was carried out with LPS-stimulated MM6 monocytic cells. Preincubation with brefeldin A as an inhibitor of the classical export pathway enhances YB-1 secretion. Preincubation with inhibitors of ATP-binding cassette transporters (C) probenicid and (D) glyburide reduces the release of YB-1. (E) The ionophore ionomycin superinduces LPS-dependent YB-1 secretion. (F) Disruption of the electrochemical gradient by reserpine successfully blocks the LPS effect on YB-1 secretion. (G) Schematic drawing of the YB-1 domains and distribution of 16 lysines (Ks) in the protein. (H) Expression plasmids for Flag–YB-1 and double-mutated Flag–YB-1 Lys301/304Ala proteins were introduced into rMCs and equal expression levels were determined by Flag antibody. After LPS stimulation a time-dependent release of Flag–YB-1, but not of mutated Flag–YB-1 (Lys301/304Ala), is detected with the supernatant (upper panels). Controls for cellular protein and precipitation efficiency are provided in the lower panels. CSD, cold shock domain; GFP, green fluorescent protein; LPS, lipopolysaccharide; mm6, MonoMac-6; rMCs, rat mesangial cells; YB, Y-box protein.
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Related In: Results  -  Collection

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f2: YB-1 is secreted through a non-classical, vesicle-mediated pathway by LPS-stimulated cells. (A) Confocal laser-scanning microscopy visualized formation of YB-1–GFP-enriched vesicles in 2 h after LPS stimulation of rMCs, however, this was not found for GFP-stimulation alone. Time-lapse live-cell microscopy was carried out for over 120 min with images taken every 2 min (supplementary videos 1 and 2 online). (B) Biochemical characterization of the YB-1 export mechanism was carried out with LPS-stimulated MM6 monocytic cells. Preincubation with brefeldin A as an inhibitor of the classical export pathway enhances YB-1 secretion. Preincubation with inhibitors of ATP-binding cassette transporters (C) probenicid and (D) glyburide reduces the release of YB-1. (E) The ionophore ionomycin superinduces LPS-dependent YB-1 secretion. (F) Disruption of the electrochemical gradient by reserpine successfully blocks the LPS effect on YB-1 secretion. (G) Schematic drawing of the YB-1 domains and distribution of 16 lysines (Ks) in the protein. (H) Expression plasmids for Flag–YB-1 and double-mutated Flag–YB-1 Lys301/304Ala proteins were introduced into rMCs and equal expression levels were determined by Flag antibody. After LPS stimulation a time-dependent release of Flag–YB-1, but not of mutated Flag–YB-1 (Lys301/304Ala), is detected with the supernatant (upper panels). Controls for cellular protein and precipitation efficiency are provided in the lower panels. CSD, cold shock domain; GFP, green fluorescent protein; LPS, lipopolysaccharide; mm6, MonoMac-6; rMCs, rat mesangial cells; YB, Y-box protein.
Mentions: Primary human monocytes were exposed to increasing lipopolysaccharide (LPS) concentrations for 4 h and the supernatants were trichloroacetic acid (TCA)-precipitated. Immunoblotting showed dose-dependent secretion of YB-1, which peaked at a concentration of 5 ng/ml LPS (Fig 1B), whereas no non-specific cytosolic protein release, for example that of glyceraldehyde 3-phosphate dehydrogenase, was observed. A Trypan-blue exclusion assay confirmed that protein release was not due to a lack of cell integrity (viability >95%). By contrast, intracellular YB-1 protein levels essentially did not change after LPS exposure (Fig 2B, lower panel). Calculation of protein loading and band intensities indicated that approximately 25% of total YB-1 protein was secreted by primary monocytes on stimulation, using 5 ng/ml LPS (Fig 1C). Quantification of macrophage migration inhibitory factor (MIF; Kleemann et al, 2000) in the same supernatant by ELISA indicated a similar dose response to LPS (Fig 1D). LPS-induced secretion of both YB-1 and MIF was also observed in MonoMac-6 (MM6) cells (supplementary Fig 1B–D online), underpinning the above findings. MM6 cells are well-characterized LPS-responsive monocytic cells. In this system, YB-1 secretion was also apparent in a narrow LPS concentration range between 1 and 7.5 ng/ml, which paralleled MIF secretion (supplementary Fig 1B,C online). Kinetic studies carried out at the peak LPS concentration of 5 ng/ml showed low-level baseline secretion of YB-1 up to 2 h, followed by an apparent marked LPS effect after 4 h (supplementary Fig 1D online).

Bottom Line: Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications.The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration.Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology & Immunology, University Hospital RWTH Aachen, Aachen, Germany.

ABSTRACT
Y-box protein (YB)-1 of the cold-shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB-1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB-1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB-1 was secreted through a non-classical mode resembling that of the macrophage migration inhibitory factor. YB-1 release requires ATP-binding cassette transporters, and microvesicles protect YB-1 from protease degradation. Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications. The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

Show MeSH
Related in: MedlinePlus