Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response - PubMed (original) (raw)

Comparative Study

. 2005 May 3;102(18):6356-61.

doi: 10.1073/pnas.0500226102. Epub 2005 Apr 25.

Affiliations

Comparative Study

Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response

Sang Gyu Park et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Although aminoacyl-tRNA synthetases (ARSs) are essential for protein synthesis, they also function as regulators and signaling molecules in diverse biological processes. Here, we screened 11 different human ARSs to identify the enzyme that is secreted as a signaling molecule. Among them, we found that lysyl-tRNA synthetase (KRS) was secreted from intact human cells, and its secretion was induced by TNF-alpha. The secreted KRS bound to macrophages and peripheral blood mononuclear cells to enhance the TNF-alpha production and their migration. The mitogen-activated protein kinases, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, and Galphai were determined to be involved in the signal transduction triggered by KRS. All of these activities demonstrate that human KRS may work as a previously uncharacterized signaling molecule, inducing immune response through the activation of monocyte/macrophages.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

TNF-α-dependent secretion of KRS. (A) Each of 11 different human ARSs was expressed in HEK293 cells by transfection and by their expression in whole-cell lysates (WCL), and secretion into the culture media was determined by Western blotting with an anti-Myc antibody. The expected molecular masses of ARSs, in kDa, are as follows: L, 138; V, 140; A, 107; M, 101; T, 82; D, 57; G, 78; R, 75; K, 68; S, 59; HRS, 57. LRS contains ≈4 kDa extra peptide originating from the vector, KRS runs slightly bigger than the expected size in the gel for unknown reason, and VRS lacking its N-terminal 300 amino acids was used because its full-size version was not expressed well. All of the other ARSs are expressed as full length. (B) HEK293 cells transfected with human KRS were incubated, and the culture medium was harvested at the indicated times. The lack of tubulin in the culture medium indicates the absence of cell lysis resulting from physical damage. (C) The macrophage cell line, RAW264.7, and the colon cancer cell line, HCT116, were treated with TNF-α (10 ng/ml) or TGF-β (2 ng/ml), and the secretion of KRS into the culture medium was determined with its specific antibody. (D) Time course of TNF-α-induced secretion of KRS from HCT116 cells.

Fig. 2.

Fig. 2.

Specific binding of KRS to immune cells. (A) KRS or p18 was biotinylated and incubated with RAW264.7 cells, which were harvested at the indicated times. The cell-bound KRS was reacted with FITC-conjugated streptavidin and visualized by confocal immunofluorescence microscopy. (B) The cell binding specificity of KRS. RAW264.7 cells were pretreated with unlabeled KRS or BSA, and the biotinylated KRS was added subsequently. The biotinylated WRS was also incubated with RAW246.7 cells. (C) RAW264.7 and THP-1 cells were incubated with the indicated concentrations of the His-tagged KRS, and, after they were harvested, the proteins were extracted. The exogenously added (exo) and endogenous (endo) KRS were detected by Western blotting with an anti-KRS antibody. (D) Cell binding by KRS was monitored as above and quantified with a phosphoimage analyzer (Fuji), and the amount of the cell-bound KRS was plotted.

Fig. 3.

Fig. 3.

The effect of KRS on macrophage proliferation. (A) The proliferation of RAW264.7 cells was monitored by the incorporation of tritium-labeled thymidine at different KRS concentrations. Phorbol 12-myristate 13-acetate (PMA) and WRS (7) were used as positive and negative controls, respectively. (B) The effect of KRS on the secretion of TNF-α from RAW264.7 cells. p43 (32) and WRS were used as positive and negative controls, respectively. (C) RAW264.7 and HCT116 cells were treated with 10 nM KRS for the indicated times, and the transcript levels for TNF-α and KRS were determined by RT-PCR as described in Materials and Methods.(D) The two cells were treated with 10 ng/ml TNF-α, and the expression of KRS and p43 was determined by RT-PCR. GAPDH was used as a control.

Fig. 4.

Fig. 4.

The effect of KRS on cell migration. (A) RAW264.7 cells (3 × 105) and KRS (10 nM) were added to the upper and lower wells of Transwell chamber, respectively, and the cells migrated through the membrane were detected by the hematoxylin staining as described in Materials and Methods. (B) Dose-dependent cell migration by KRS. (C) The KRS-dependent induction of MMP-9 was determined by a zymographic assay. RAW264.7 cells, treated as above, were lysed, and the proteins were resolved on SDS/PAGE-containing gelatin. The hydrolysis of gelatin was determined as described in ref. . (D Left) To determine how KRS induces cell migration, RAW264.7 cells were loaded onto the upper well, and KRS or WRS was added to the lower well and allowed them to migrate. (Right) The cells were pretreated with 10 nM KRS or WRS for 30 min and loaded to the upper well and incubated them to migrate without adding KRS or WRS to the lower well. Then, the migrated cells through the membrane were counted as above. (E) The cell migration was also determined in the Transwell chambers in which 10 nM KRS was added to the upper or lower or both wells. As the control, no KRS was added, or 10 nM WRS was added to the lower well.

Fig. 5.

Fig. 5.

Determination of mediators for KRS signaling. RAW264.7 cells were treated for 30 min with different concentrations of KRS (A) or for different times at 10 nM KRS (B). The activation of three major MAPKs, ERK, p38 MAPK, and JNK, was determined by detection of their phosphorylation with their phosphospecific antibodies. The total amount of each kinase was determined by Western blotting with specific antibodies. (C) RAW264.7 cells were pretreated with different kinase inhibitors, 50 μM U0126 (MEK inhibitor), 10 μM SB202190 (p38 MAPK inhibitor), 10 μM SB203580 (p38 MAPK inhibitor), and 10 μM LY294002 (phosphatidylinositol 3-kinase inhibitor), and 10 nM KRS was subsequently added. The specific inhibition of ERK and p38 MAPK was confirmed by using the method above. The effects of kinase inhibitors on the KRS-dependent TNF production (D) and MMP-9 production (E) and cell migration (F) were determined. The effects of cholera toxin (CTX) and pertussis toxin (PTX) on the KRS-induced growth suppression (G) and cell migration (H) are shown as previously described.

Similar articles

Cited by

References

    1. Ko, Y. G., Park, H. & Kim, S. (2002) Proteomics 2, 1304-1310. - PubMed
    1. Han, J. M., Kim, J. Y. & Kim, S. (2003) Biochem. Biophys. Res. Commun. 303, 985-993. - PubMed
    1. Ko, Y. G., Kang, Y. S., Kim, E. K., Park, S. G. & Kim, S. (2000) J. Cell Biol. 149, 567-574. - PMC - PubMed
    1. Ko, Y. G., Kang, Y. S., Park, H., Seol, W., Kim, J., Kim, T., Park, H. S., Choi, E. J. & Kim, S. (2001) J. Biol. Chem. 276, 39103-39106. - PubMed
    1. Kleeman, T. A., Wei, D., Simpson, K. L. & First, E. A. (1997) J. Biol. Chem. 272, 14420-14425. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources