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

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.

Figure 1

Lipopolysaccharide induces YB-1 secretion in primary monocytic cells. (A) In acute kidney-transplant rejection, infiltrating monocytes are double immunopositive for YB-1 and ED1. The inset (left panel) shows immunohistochemical analyses carried out with healthy kidney. (B) LPS stimulation of primary monocytes leads to a concentration-dependent release of YB-1 into the supernatant. Detection was carried out with TCA-precipitated conditioned medium, (C) and quantified to 25% of the total cellular YB-1 content at maximum. (D) Macrophage migration inhibitory factor concentration in the supernatant is increased in a similar LPS concentration range. (E) LPS stimulation of monocytes with subsequent vesicle preparation (see supplementary Fig 2 online) enriches YB-1 protein. Vesicles successfully protect YB-1 against protease activity (trypsin, second panel). Detergent pretreatment (Triton X-100, third panel) disrupts vesicles and trypsin degrades YB-1. Spill-over of cytoplasmic protein is excluded by glyceraldehyde 3-phosphate dehydrogenase detection (fourth panel) and proper vesicle preparation is verified by the presence of RAB7 (fifth panel). YB-1 was also detected in TCA-precipitated medium after removal of vesicular structures by centrifugation (sixth panel). Control for total cellular protein and intracellular RAB7 content is given in the lowest panel. CON, control; LPS, lipopolysaccharide; MIF, macrophage migration inhibitory factor; TCA, trichloroacetic acid; w/o, without; YB, Y-box protein.

Figure 2

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.

Figure 3

Extracellular YB-1 stimulates cell migration. (A) Rat mesangial cells grown to confluency and arrested by use of mitomycin C are assessed for cell migration under different conditions for 48 h. Increased migration in the scratched region is observed for cells stimulated with rYB-1 and FCS (10%, positive control), whereas denatured YB-1 has no effect. (B) Quantification was carried out at four regions of the migration front in three independent experiments. (C) Similarly, human mesangial cells (hMCs) show increased migration on stimulation with synthetic oligopeptides corresponding to a YB-1 cold-shock domain motif compared with unstimulated cells, whereas the control peptide does not have any influence. (D) These findings correlate to cytoskeletal changes in mesangial cells induced by YB-1 peptides, with a marked upregulation of actin stress fibres within 1 h, as detected by phalloidin staining. A. dest, Aqua dest; rMCs, rat mesangial cells; YB, Y-box protein.