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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 αvβ3-integrin. (A) Differentiated HL-60 (dHL-60) cells (1.5×106) were placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to flow cytometry using PE-labeled anti-CXCR2 antibody. The mean fluorescence intensities (MFI) of the isotype- and anti-αv-integrin-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (B) A total of 1.5×106 dHL-60 cells was placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were then pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to 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 and corresponding densitometric bar diagram obtained from 2 independent experiments are shown.
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f4-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 αvβ3-integrin. (A) Differentiated HL-60 (dHL-60) cells (1.5×106) were placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to flow cytometry using PE-labeled anti-CXCR2 antibody. The mean fluorescence intensities (MFI) of the isotype- and anti-αv-integrin-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (B) A total of 1.5×106 dHL-60 cells was placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were then pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to 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 and corresponding densitometric bar diagram obtained from 2 independent experiments are shown.

Mentions: To determine the involvement of αvβ3-integrin in the rhMFG-E8-mediated alteration in CXCR2 and GRK2 expression, the dHL-60 cells were pre-treated with anti-αv-integrin antibody to block the MFG-E8 receptor for the transmission of downstream signaling. As shown in Fig. 4A, pre-treatment of the cells with anti-αv-integrin antibody neutralized the rhMFG-E8-induced downregulation in the surface expression of CXCR2, while a significant downregulation in CXCR2 surface expression was observed in the cells treated with the IgG isotype control. Similarly, the expression of GRK2 was induced in the rhMFG-E8-stimulated dHL-60 cells pre-treated with the IgG isotype control; conversely the rhMFG-E8-induced upregulation in GRK2 expression was diminished in the cells pre-treated with anti-αv-integrin (Fig. 4B). Taken together, these data clearly indicate that the MFG-E8-mediated down-regulation of CXCR2 and the upregulation of GRK2 expression are transmitted through the αvβ3-integrin pathway.


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 αvβ3-integrin. (A) Differentiated HL-60 (dHL-60) cells (1.5×106) were placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to flow cytometry using PE-labeled anti-CXCR2 antibody. The mean fluorescence intensities (MFI) of the isotype- and anti-αv-integrin-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (B) A total of 1.5×106 dHL-60 cells was placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were then pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to 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 and corresponding densitometric bar diagram obtained from 2 independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-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 αvβ3-integrin. (A) Differentiated HL-60 (dHL-60) cells (1.5×106) were placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to flow cytometry using PE-labeled anti-CXCR2 antibody. The mean fluorescence intensities (MFI) of the isotype- and anti-αv-integrin-treated samples are shown. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments. *P<0.05 vs. PBS treatment. (B) A total of 1.5×106 dHL-60 cells was placed into 1.5 ml microfuge tubes containing 1 ml of Opti-MEM. The cells were then pre-treated with 1 µg/ml of each of the IgG isotype control or anti-αv-integrin neutralizing antibody for 1 h at 37°C. The cells were then stimulated with rhMFG-E8 (500 ng/ml) or PBS for 2 h and then subjected to 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 and corresponding densitometric bar diagram obtained from 2 independent experiments are shown.
Mentions: To determine the involvement of αvβ3-integrin in the rhMFG-E8-mediated alteration in CXCR2 and GRK2 expression, the dHL-60 cells were pre-treated with anti-αv-integrin antibody to block the MFG-E8 receptor for the transmission of downstream signaling. As shown in Fig. 4A, pre-treatment of the cells with anti-αv-integrin antibody neutralized the rhMFG-E8-induced downregulation in the surface expression of CXCR2, while a significant downregulation in CXCR2 surface expression was observed in the cells treated with the IgG isotype control. Similarly, the expression of GRK2 was induced in the rhMFG-E8-stimulated dHL-60 cells pre-treated with the IgG isotype control; conversely the rhMFG-E8-induced upregulation in GRK2 expression was diminished in the cells pre-treated with anti-αv-integrin (Fig. 4B). Taken together, these data clearly indicate that the MFG-E8-mediated down-regulation of CXCR2 and the upregulation of GRK2 expression are transmitted through the αvβ3-integrin pathway.

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