Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum - PubMed (original) (raw)

Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum

Vadim Budagian et al. Mol Cell Biol. 2005 Nov.

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Abstract

Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

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Figures

FIG. 1.

FIG. 1.

Membrane-bound Axl and sAxl are present in L929 and L929R fibroblasts. (A) RT-PCR analysis of Axl expression in L929 and L929R cells. Amplification of the β-actin message was used to normalize the amount of cDNA. (B) L929 and L929R cells were lysed, and expression of Axl was analyzed by Western blotting with specific Abs. Lysates from Axl−/− fibroblasts were used as a negative control. Expression of β-actin was detected on the same membrane after stripping and served as a loading control. (C) Expression of Axl on the cell membrane of L929 and L929R fibroblasts was assessed by flow cytometry analysis. (D) sAxl was detected in cell-conditioned medium (CM), and membrane-bound Axl was detected in lysates (L) from L929 and L929R cells by Western blotting after immunoprecipitation with specific anti-Axl Abs. (E) Glycosylation pattern of sAxl. sAxl was immunoprecipitated from cell-conditioned medium and treated with N-glycosidase or left untreated, as described in Materials and Methods. After treatment, protein lysates were analyzed by Western blotting with anti-Axl Abs.

FIG. 2.

FIG. 2.

Regulation of Axl shedding. L929 (A) and L929R (B) cells were treated with 200 ng/ml of PMA for different times, and the concentration of sAxl in cell-conditioned medium was evaluated by a specific ELISA. Untreated cells were used as controls. *, P < 0.05 versus control samples. L929 (C) and L929R (D) cells were left untreated or treated with PMA for 2 h. Expression of membrane-bound Axl was evaluated by flow cytometry. (E and F) Cells were treated with PMA (200 ng/ml), Gas6 (50 ng/ml), IL-15 (50 ng/ml), TNF-α (10 ng/ml), and LPS (10 ng/ml) for 2 h, and the concentration of sAxl in the culture medium was quantified by ELISA. Untreated (medium) cells were used as controls. **, P < 0.01 versus control samples.

FIG. 3.

FIG. 3.

Effects of metalloproteinase inhibitors on Axl cleavage. L929 or L929R cells were incubated with 2.5 μM GM6001 or with its inactive analog as a control (A) or with 10 μM hydroxamate inhibitor GW280264X (B) for 1 h prior to treatment with 200 ng/ml of PMA for another 2 h. Untreated cells were used as controls. Cell-conditioned medium (CM) or cell lysates (L) were harvested and analyzed by ELISA for the presence of sAxl or membrane-bound Axl, respectively. (C) Cells were treated with the above inhibitors, followed by PMA stimulation for another 2 h. Expression of membrane-bound Axl was evaluated by flow cytometry analysis. * and **, P < 0.01 versus control samples; neg., negative.

FIG. 4.

FIG. 4.

Analysis of sAxl release in ADAM10- or TACE-deficient MEFs. Expression of Axl in different MEFs was assessed by RT-PCR (A) and Western blotting (B). Amplification of the β-actin message was used to normalize the amount of cDNA to be used. Detection of β-actin protein was used to prove equal loading of cell lysates. (C) MEFs were treated with 200 ng/ml of PMA for 4 h, and the sAxl concentration in the culture medium was detected by ELISA. Untreated cells were used as a control. **, P < 0.05 versus ADAM10+/+ cells; ***, P < 0.01 versus untreated samples. (D) Expression of membrane-bound Axl on ADAM10+/+ and ADAM10−/− MEFs was evaluated by flow cytometry. (E and F) L929 and L929R cells were transfected with ADAM10 or scrambled (control) siRNA oligonucleotides, and expression of ADAM10 was assessed by Western blotting; detection of β-actin is shown as a loading control (E). (F) Forty-eight hours after transfection, cells were treated with PMA for 2 h or left untreated. Next, the concentration of sAxl in cell-conditioned medium was evaluated by ELISA. *, P < 0.05; **, P < 0.01 (versus cells transfected with scrambled control oligonucleotides). The data represent at least three separate experiments with comparable results.

FIG. 5.

FIG. 5.

The membrane-proximal region of Axl is important for sAxl generation. (A) Axl deletion mutants were generated as described in Materials and Methods. COS-7 cells were transiently transfected with full-length Axl (Axl WT) or with Axl deletion mutants (AxlΔ1, AxlΔ2, and AxlΔ3) lacking different 14-aa sequence in the membrane-proximal region of Axl. FNIII, fibronectin type III; TM, transmembrane. (B) Efficiency of transfection was proved by Western blotting with anti-Axl Abs, and detection of β-actin on the same blots is shown as a loading control. (C) After 48 h, the culture medium was replaced with fresh culture medium and the cells were incubated in the presence or absence of PMA for 1 h. Conditioned medium was collected, and the concentration of sAxl was determined by ELISA. *, P < 0.05; **, P < 0.01 (versus Axl WT-transfected cells). The results are representative of three independent experiments.

FIG. 6.

FIG. 6.

Detection of sAxl, Mer, and Tyro3 in culture medium from DCs and tumor cell lines. (A) DCs were generated as described in Materials and Methods, and expression of Axl, Mer, and Tyro3 on the cell membrane of WT and Axl−/− DCs was evaluated by flow cytometry analysis. (B) DCs were pretreated with GW280264X for 30 min prior to stimulation with PMA for another 4 h or left untreated. Unstimulated and Axl−/− DCs served as control cells. The concentration of sAxl or cell-associated Axl in cell-conditioned medium (CM) or cell lysates (L), respectively, and the concentrations of Mer and Tyro3 in cell-conditioned medium were analyzed by ELISA. (C) TS/A and J558 cells were incubated in the presence or absence of PMA for 4 h. Culture medium was collected, and the concentration of sAxl was evaluated by ELISA. *, P < 0.05 versus untreated cells; **, P < 0.05 versus control samples.

FIG. 7.

FIG. 7.

sAxl is present in mouse serum and associates with Gas6. (A) Sera from C57BL/6, CH3, BALB/c, Axl+/+, Axl+/−, and Axl−/− mice were tested for the presence of sAxl by ELISA. Each bar represents the mean of at least five animals per strain (*, P < 0.05; **, P < 0.01). (B) The concentration of Gas6 in mouse sera was determined by a two-site ELISA. The concentration of sAxl or Gas6 was determined by a specific ELISA for Axl or Gas6, respectively, and is shown for comparison. (C) Axl was precipitated from mouse serum with specific Abs. Immunocomplexes were subjected to SDS-PAGE and transferred onto nitrocellulose membrane, and Gas6 was detected by probing the membrane with specific Abs. Detection of Axl on the same blots served as a loading control. IgG, immunoglobulin G.

FIG. 8.

FIG. 8.

Immobilized Axl-Fc chimeric protein promotes cell migration and induces the phosphorylation of Axl and PI3K. (A) L929R cells were plated onto Axl-Fc- or IL-3R-Fc (control)-coated six-well plates and allowed to grow in the presence of 10% FCS. After 18 h, a wound was created by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) L929R cells were serum starved for 4 h and incubated in Axl-Fc-coated wells for 15 min. Incubation in IL-3R-Fc (control)-coated wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. The picture is representative of three independent experiments, all of which yielded highly comparable results. IP, immunoprecipitation; WB, Western blotting.

FIG. 9.

FIG. 9.

Immobilized Axl-Fc chimeric protein induces cell migration and phosphorylation of Axl and PI3K in WT but not Axl−/− MEFs. (A) MEFs were plated onto Axl-Fc- or IL-3R-Fc (control)-coated six-well plates. A confluent cell monolayer was wounded by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) MEFs were serum starved for 4 h and incubated in Axl-Fc-coated wells for 15 min. Incubation in IL-3R-Fc-coated (control) wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. IP, immunoprecipitation; WB, Western blotting.

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