Limits...
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) activates mitogen-activated protein (MAP) kinases through αvβ3-integrin. (A and B) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with rhMFG-E8 (500 ng/ml) for different periods of time. Following incubation, the cell lysates were harvested and subjected to western blot analysis using monoclonal antibodies for phospho and total p38 and ERK. Results are normalized with total p38 and ERK as loading control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments.*P<0.05 vs. 0 min. (C) 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 30 min and then subjected to western blot analysis using antibodies for p-p38, and ERK, and total p38 and ERK. Representative blots obtained from 3 independent experiments are shown. *P<0.05 vs. 0 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494603&req=5

f3-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) activates mitogen-activated protein (MAP) kinases through αvβ3-integrin. (A and B) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with rhMFG-E8 (500 ng/ml) for different periods of time. Following incubation, the cell lysates were harvested and subjected to western blot analysis using monoclonal antibodies for phospho and total p38 and ERK. Results are normalized with total p38 and ERK as loading control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments.*P<0.05 vs. 0 min. (C) 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 30 min and then subjected to western blot analysis using antibodies for p-p38, and ERK, and total p38 and ERK. Representative blots obtained from 3 independent experiments are shown. *P<0.05 vs. 0 min.

Mentions: To determine whether rhMFG-E8 alters MAP kinase phosphorylation, the dHL-60 cells were treated with rhMFG-E8 for different periods of time and we then measured the p38 and ERK phosphorylation levels by western blot analysis. As shown in Fig. 3A and B, the dHL-60 cells stimulated with rhMFG-E8 showed a significant upregulation in p38 and ERK phosphorylation in a time-dependent manner with the highest induction in their phosphorylation observed at the 20- and 30-min time points; after these time points, their phosphorylation decreased to basal levels. Since αvβ3-integrin recognizes MFG-E8, we wished to determine whether this integrin is involved in the MFG-E8-mediated signal transduction of p38 and ERK phosphorylation in the dHL-60 cells. For this purpose, we first treated the dHL-60 cells with the neutralizing antibody for αv-integrin or IgG isotype antibody followed by stimulation with rhMFG-E8, which evidently revealed that the promoting effects of rhMFG-E8 on p38 and ERK phosphorylation were notably diminished (Fig. 3C), indicating the role of αvβ3-integrin in transducing MFG-E8-mediated downstream signaling and 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) activates mitogen-activated protein (MAP) kinases through αvβ3-integrin. (A and B) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with rhMFG-E8 (500 ng/ml) for different periods of time. Following incubation, the cell lysates were harvested and subjected to western blot analysis using monoclonal antibodies for phospho and total p38 and ERK. Results are normalized with total p38 and ERK as loading control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments.*P<0.05 vs. 0 min. (C) 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 30 min and then subjected to western blot analysis using antibodies for p-p38, and ERK, and total p38 and ERK. Representative blots obtained from 3 independent experiments are shown. *P<0.05 vs. 0 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijmm-36-01-0018: Recombinant human milk fat globule-epidermal growth factor-factor 8 (rhMFG-E8) activates mitogen-activated protein (MAP) kinases through αvβ3-integrin. (A and B) Differentiated HL-60 cells (1.5×106/ml) were placed into 1.5 ml microfuge tubes with Opti-MEM and then stimulated with rhMFG-E8 (500 ng/ml) for different periods of time. Following incubation, the cell lysates were harvested and subjected to western blot analysis using monoclonal antibodies for phospho and total p38 and ERK. Results are normalized with total p38 and ERK as loading control and are expressed as the fold induction in comparison to the 0 min time point. Data are expressed as the means ± SE (n=3 samples/group), obtained from 3 independent experiments.*P<0.05 vs. 0 min. (C) 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 30 min and then subjected to western blot analysis using antibodies for p-p38, and ERK, and total p38 and ERK. Representative blots obtained from 3 independent experiments are shown. *P<0.05 vs. 0 min.
Mentions: To determine whether rhMFG-E8 alters MAP kinase phosphorylation, the dHL-60 cells were treated with rhMFG-E8 for different periods of time and we then measured the p38 and ERK phosphorylation levels by western blot analysis. As shown in Fig. 3A and B, the dHL-60 cells stimulated with rhMFG-E8 showed a significant upregulation in p38 and ERK phosphorylation in a time-dependent manner with the highest induction in their phosphorylation observed at the 20- and 30-min time points; after these time points, their phosphorylation decreased to basal levels. Since αvβ3-integrin recognizes MFG-E8, we wished to determine whether this integrin is involved in the MFG-E8-mediated signal transduction of p38 and ERK phosphorylation in the dHL-60 cells. For this purpose, we first treated the dHL-60 cells with the neutralizing antibody for αv-integrin or IgG isotype antibody followed by stimulation with rhMFG-E8, which evidently revealed that the promoting effects of rhMFG-E8 on p38 and ERK phosphorylation were notably diminished (Fig. 3C), indicating the role of αvβ3-integrin in transducing MFG-E8-mediated downstream signaling and 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