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MFG-E8 inhibits neutrophil migration through αvβ₃-integrin-dependent MAP kinase activation.

Aziz M, Yang WL, Corbo LM, Chaung WW, Matsuo S, Wang P - Int. J. Mol. Med. (2015)

Bottom Line: Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner.There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression.The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.

ABSTRACT
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.

No MeSH data available.


Related in: MedlinePlus

Treatment with anti-αv-integrin antibody and mitogen-activated protein (MAP) kinase inhibitors counteracts recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-mediated downregulation of neutrophil migration. (A) Differentiated HL-60 (dHL-60) cells (3×105) were pre-stimulated with 1 µg/ml of each of the IgG isotype control, anti-αv-integrin neutralizing antibody, the p38 inhibitor, SB203580 (SB), of the ERK inhibitor, PD98059 (PD), at a concentration of 10 µM for 1 h at 37°C in their respective 1.5 ml microfuge tubes. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h, and then plated in 500 µl volume in the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells are plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS. (C) Mechanistic finding. The secretory glycoprotein, MFG-E8 binds to its receptor, αvβ3-integrin, and transduces downstream signaling of MAP kinase (p38 and ERK) activation. The activated MAP kinases then upregulate G protein-coupled receptor kinase 2 (GRK2) which in turn negatively regulates the surface exposure of CXCR2, thereby attenuating neutrophil migration.
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f6-ijmm-36-01-0018: Treatment with anti-αv-integrin antibody and mitogen-activated protein (MAP) kinase inhibitors counteracts recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-mediated downregulation of neutrophil migration. (A) Differentiated HL-60 (dHL-60) cells (3×105) were pre-stimulated with 1 µg/ml of each of the IgG isotype control, anti-αv-integrin neutralizing antibody, the p38 inhibitor, SB203580 (SB), of the ERK inhibitor, PD98059 (PD), at a concentration of 10 µM for 1 h at 37°C in their respective 1.5 ml microfuge tubes. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h, and then plated in 500 µl volume in the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells are plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS. (C) Mechanistic finding. The secretory glycoprotein, MFG-E8 binds to its receptor, αvβ3-integrin, and transduces downstream signaling of MAP kinase (p38 and ERK) activation. The activated MAP kinases then upregulate G protein-coupled receptor kinase 2 (GRK2) which in turn negatively regulates the surface exposure of CXCR2, thereby attenuating neutrophil migration.

Mentions: In order to determine the involvement of αvβ3-integrin and MAP kinases in the rhMFG-E8-mediated inhibition of neutrophil migration, the dHL-60 cells were pre-treated with anti-αv-integrin antibody and the MAP kinase inhibitors, and the effects of rhMFG-E8 on neutrophil migration were antibody evaluated. As shown in Fig. 6A and B, pre-treatment of the cells with anti-αv-integrin antibody and MAP kinase inhibitors markedly reversed the inhibitory effects of rhMFG-E8 on neutrophil migration. Collectively, these findings clearly indicate that MFG-E8 inhibits neutrophil migration by downregulating surface CXCR2 expression through the upregulation of the expression of the intracellular negative regulator, GRK2, and this event is mediated by αvβ3-integrin-mediated MAP kinase activation (Fig. 6C).


MFG-E8 inhibits neutrophil migration through αvβ₃-integrin-dependent MAP kinase activation.

Aziz M, Yang WL, Corbo LM, Chaung WW, Matsuo S, Wang P - Int. J. Mol. Med. (2015)

Treatment with anti-αv-integrin antibody and mitogen-activated protein (MAP) kinase inhibitors counteracts recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-mediated downregulation of neutrophil migration. (A) Differentiated HL-60 (dHL-60) cells (3×105) were pre-stimulated with 1 µg/ml of each of the IgG isotype control, anti-αv-integrin neutralizing antibody, the p38 inhibitor, SB203580 (SB), of the ERK inhibitor, PD98059 (PD), at a concentration of 10 µM for 1 h at 37°C in their respective 1.5 ml microfuge tubes. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h, and then plated in 500 µl volume in the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells are plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS. (C) Mechanistic finding. The secretory glycoprotein, MFG-E8 binds to its receptor, αvβ3-integrin, and transduces downstream signaling of MAP kinase (p38 and ERK) activation. The activated MAP kinases then upregulate G protein-coupled receptor kinase 2 (GRK2) which in turn negatively regulates the surface exposure of CXCR2, thereby attenuating neutrophil migration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494603&req=5

f6-ijmm-36-01-0018: Treatment with anti-αv-integrin antibody and mitogen-activated protein (MAP) kinase inhibitors counteracts recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8)-mediated downregulation of neutrophil migration. (A) Differentiated HL-60 (dHL-60) cells (3×105) were pre-stimulated with 1 µg/ml of each of the IgG isotype control, anti-αv-integrin neutralizing antibody, the p38 inhibitor, SB203580 (SB), of the ERK inhibitor, PD98059 (PD), at a concentration of 10 µM for 1 h at 37°C in their respective 1.5 ml microfuge tubes. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h, and then plated in 500 µl volume in the Boyden chamber inserts. The outer compartment of the inserts contained 500 µl of RPMI medium with 50 ng/ml of recombinant human interleukin-8 (IL-8) as a chemoattractant. After 1.5 h of incubation, the upper surface of the filter was swabbed with cotton-tipped applicators to remove non-migratory cells. Migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with propidium iodide (PI) (1 µg/ml). A total of 6 random microscopic fields per well were counted. Scale bar, 100 µm. (B) The average number of migrated dHL-60 cells are plotted in a bar diagram where the results are expressed as the means ± SE obtained from 6 fields/group of 3 independent experiments. *P<0.05 vs. PBS. (C) Mechanistic finding. The secretory glycoprotein, MFG-E8 binds to its receptor, αvβ3-integrin, and transduces downstream signaling of MAP kinase (p38 and ERK) activation. The activated MAP kinases then upregulate G protein-coupled receptor kinase 2 (GRK2) which in turn negatively regulates the surface exposure of CXCR2, thereby attenuating neutrophil migration.
Mentions: In order to determine the involvement of αvβ3-integrin and MAP kinases in the rhMFG-E8-mediated inhibition of neutrophil migration, the dHL-60 cells were pre-treated with anti-αv-integrin antibody and the MAP kinase inhibitors, and the effects of rhMFG-E8 on neutrophil migration were antibody evaluated. As shown in Fig. 6A and B, pre-treatment of the cells with anti-αv-integrin antibody and MAP kinase inhibitors markedly reversed the inhibitory effects of rhMFG-E8 on neutrophil migration. Collectively, these findings clearly indicate that MFG-E8 inhibits neutrophil migration by downregulating surface CXCR2 expression through the upregulation of the expression of the intracellular negative regulator, GRK2, and this event is mediated by αvβ3-integrin-mediated MAP kinase activation (Fig. 6C).

Bottom Line: Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner.There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression.The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.

ABSTRACT
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.

No MeSH data available.


Related in: MedlinePlus