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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

Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) regulates CXCR2 and G protein-coupled receptor kinase 2 (GRK2) expression through the p38 and ERK pathways. (A) Differentiated HL-60 (dHL-60) cells were placed into 1.5 ml microfuge tubes at a density of 1.5×106 cells/ml of Opti-MEM. The cells were then pre-treated with the p38 inhibitor, SB203580, of the ERK inhibitor, PD98059, at a concentration of 10 µM of each for 1 h at 37°C. Following incubation, the cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h followed by the assessment of CXCR2 by flow cytometry. Mean fluorescence intensities (MFI) obtained from 3 independent experiments are plotted into the bar diagram.*P<0.05 vs. PBS treatment. (B) Western blot analysis using anti-GRK2 antibody. The blot was stripped off and re-probed for anti-β-actin antibody, which served as the loading control. Representative blots obtained from 2 independent experiments are shown. Densitometric data are expressed as the means ± SE (n=2 samples/group), obtained from 2 independent experiments.
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f5-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) regulates CXCR2 and G protein-coupled receptor kinase 2 (GRK2) expression through the p38 and ERK pathways. (A) Differentiated HL-60 (dHL-60) cells were placed into 1.5 ml microfuge tubes at a density of 1.5×106 cells/ml of Opti-MEM. The cells were then pre-treated with the p38 inhibitor, SB203580, of the ERK inhibitor, PD98059, at a concentration of 10 µM of each for 1 h at 37°C. Following incubation, the cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h followed by the assessment of CXCR2 by flow cytometry. Mean fluorescence intensities (MFI) obtained from 3 independent experiments are plotted into the bar diagram.*P<0.05 vs. PBS treatment. (B) Western blot analysis using anti-GRK2 antibody. The blot was stripped off and re-probed for anti-β-actin antibody, which served as the loading control. Representative blots obtained from 2 independent experiments are shown. Densitometric data are expressed as the means ± SE (n=2 samples/group), obtained from 2 independent experiments.

Mentions: To determine the role of MAP kinases in the rhMFG-E8-mediated inhibition of CXCR2 expression, the dHL-60 cells were pre-treated with MAP kinase inhibitors and the effects of rhMFG-E8 on CXCR2 expression were then evaluated. As shown in Fig. 5A, pre-treatment of the cells with the specific inhibitors of p38 and ERK diminished the negative regulatory effects of rhMFG-E8 on CXCR2 which led to the downregulation of its surface expression, indicating the involvement of MAP kinases in rhMFG-E8-mediated signaling. Similarly, the rhMFG-E8-induced upregulation of GRK2 was also diminished when the p38 and ERK molecules were blocked by using their specific inhibitors as compared to the DMSO control (Fig. 5B). These data clearly indicate that the rhMFG-E8-mediated downstream signaling which downregulates CXCR2 and upregulates GRK2 is mediated through MAP kinase activation.


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)

Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) regulates CXCR2 and G protein-coupled receptor kinase 2 (GRK2) expression through the p38 and ERK pathways. (A) Differentiated HL-60 (dHL-60) cells were placed into 1.5 ml microfuge tubes at a density of 1.5×106 cells/ml of Opti-MEM. The cells were then pre-treated with the p38 inhibitor, SB203580, of the ERK inhibitor, PD98059, at a concentration of 10 µM of each for 1 h at 37°C. Following incubation, the cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h followed by the assessment of CXCR2 by flow cytometry. Mean fluorescence intensities (MFI) obtained from 3 independent experiments are plotted into the bar diagram.*P<0.05 vs. PBS treatment. (B) Western blot analysis using anti-GRK2 antibody. The blot was stripped off and re-probed for anti-β-actin antibody, which served as the loading control. Representative blots obtained from 2 independent experiments are shown. Densitometric data are expressed as the means ± SE (n=2 samples/group), obtained from 2 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494603&req=5

f5-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) regulates CXCR2 and G protein-coupled receptor kinase 2 (GRK2) expression through the p38 and ERK pathways. (A) Differentiated HL-60 (dHL-60) cells were placed into 1.5 ml microfuge tubes at a density of 1.5×106 cells/ml of Opti-MEM. The cells were then pre-treated with the p38 inhibitor, SB203580, of the ERK inhibitor, PD98059, at a concentration of 10 µM of each for 1 h at 37°C. Following incubation, the cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h followed by the assessment of CXCR2 by flow cytometry. Mean fluorescence intensities (MFI) obtained from 3 independent experiments are plotted into the bar diagram.*P<0.05 vs. PBS treatment. (B) Western blot analysis using anti-GRK2 antibody. The blot was stripped off and re-probed for anti-β-actin antibody, which served as the loading control. Representative blots obtained from 2 independent experiments are shown. Densitometric data are expressed as the means ± SE (n=2 samples/group), obtained from 2 independent experiments.
Mentions: To determine the role of MAP kinases in the rhMFG-E8-mediated inhibition of CXCR2 expression, the dHL-60 cells were pre-treated with MAP kinase inhibitors and the effects of rhMFG-E8 on CXCR2 expression were then evaluated. As shown in Fig. 5A, pre-treatment of the cells with the specific inhibitors of p38 and ERK diminished the negative regulatory effects of rhMFG-E8 on CXCR2 which led to the downregulation of its surface expression, indicating the involvement of MAP kinases in rhMFG-E8-mediated signaling. Similarly, the rhMFG-E8-induced upregulation of GRK2 was also diminished when the p38 and ERK molecules were blocked by using their specific inhibitors as compared to the DMSO control (Fig. 5B). These data clearly indicate that the rhMFG-E8-mediated downstream signaling which downregulates CXCR2 and upregulates GRK2 is mediated through MAP kinase activation.

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