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Cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr Peptide generates a potent inhibitor of trans-endothelial migration of monocytes.

Yousif AM, Minopoli M, Bifulco K, Ingangi V, Di Carluccio G, Merlino F, Motti ML, Grieco P, Carriero MV - PLoS ONE (2015)

Bottom Line: The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility.FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis.PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY].

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University Federico II, Naples, Italy.

ABSTRACT
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus

The peptide [SRSRY] binds to FPR1 on THP-1 cell surface.A. THP-1 cells (1.5 x106 cells/sample) were pre-incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 60 minutes at 4°C, then exposed to 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for additional 60 minutes at 4°C. Fluorimetric measurement of cell-associated fluorescence was assed using 485 nm excitation and 535 nm emission filters. Data are expressed as a percentage of the fluorescence associated to THP-1 cells exposed to FITC-fMLF alone (None = 100%) and represent a mean ± SD from triplicates. *Statistical significance (t-test) against None with p<0.001. B. THP-1 cells incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 30 minutes at 37°C and exposed to 10 nM FITC-fMLF for additional 30 minutes at 37°C. Scale bar: 5 μm. Original magnifications: 630x.
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pone.0126172.g003: The peptide [SRSRY] binds to FPR1 on THP-1 cell surface.A. THP-1 cells (1.5 x106 cells/sample) were pre-incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 60 minutes at 4°C, then exposed to 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for additional 60 minutes at 4°C. Fluorimetric measurement of cell-associated fluorescence was assed using 485 nm excitation and 535 nm emission filters. Data are expressed as a percentage of the fluorescence associated to THP-1 cells exposed to FITC-fMLF alone (None = 100%) and represent a mean ± SD from triplicates. *Statistical significance (t-test) against None with p<0.001. B. THP-1 cells incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 30 minutes at 37°C and exposed to 10 nM FITC-fMLF for additional 30 minutes at 37°C. Scale bar: 5 μm. Original magnifications: 630x.

Mentions: Once documented that both SRSRY and [SRSRY] peptides compete with fMLF for binding to FPR1, we investigated the effect of [SRSRY] on the motility of monocytes. Preliminarily, we assessed whether [SRSRY] is a ligand of FPR1 on monocyte cell surface. Similarly to RBL-2H3/ETFR cells, THP-1 cells exposed to 10 nM FITC-fMLF at 4°C, revealed an appreciable binding which was dramatically reduced by an excess of unlabeled fMLF, SRSRY or [SRSRY], but not by the control peptide [RSSYR] (Fig 3A). When binding experiments were performed at 37°C, FPR1 appeared mainly localized within the cytoplasm, adjacent but outside the nucleus of THP-1 cells. Internalization of fluorescent agonist was dramatically reduced in all cell population when THP-1 cells were pre-incubated with an excess of unlabelled fMLF, or [SRSRY] while the control peptide [RSSYR] did not exert such effect (Fig 3B). Although we did not determine the binding affinity of [SRSRY] for FPR1, our findings indicate that the peptide [SRSRY] prevents fMLF/FPR1 interaction and inhibits agonist-induced FPR1 internalization in THP-1 cells.


Cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr Peptide generates a potent inhibitor of trans-endothelial migration of monocytes.

Yousif AM, Minopoli M, Bifulco K, Ingangi V, Di Carluccio G, Merlino F, Motti ML, Grieco P, Carriero MV - PLoS ONE (2015)

The peptide [SRSRY] binds to FPR1 on THP-1 cell surface.A. THP-1 cells (1.5 x106 cells/sample) were pre-incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 60 minutes at 4°C, then exposed to 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for additional 60 minutes at 4°C. Fluorimetric measurement of cell-associated fluorescence was assed using 485 nm excitation and 535 nm emission filters. Data are expressed as a percentage of the fluorescence associated to THP-1 cells exposed to FITC-fMLF alone (None = 100%) and represent a mean ± SD from triplicates. *Statistical significance (t-test) against None with p<0.001. B. THP-1 cells incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 30 minutes at 37°C and exposed to 10 nM FITC-fMLF for additional 30 minutes at 37°C. Scale bar: 5 μm. Original magnifications: 630x.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418665&req=5

pone.0126172.g003: The peptide [SRSRY] binds to FPR1 on THP-1 cell surface.A. THP-1 cells (1.5 x106 cells/sample) were pre-incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 60 minutes at 4°C, then exposed to 10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for additional 60 minutes at 4°C. Fluorimetric measurement of cell-associated fluorescence was assed using 485 nm excitation and 535 nm emission filters. Data are expressed as a percentage of the fluorescence associated to THP-1 cells exposed to FITC-fMLF alone (None = 100%) and represent a mean ± SD from triplicates. *Statistical significance (t-test) against None with p<0.001. B. THP-1 cells incubated with diluents (None), 100 nM fMLF, 100 nM SRSRY or 100 nM [SRSRY] peptides for 30 minutes at 37°C and exposed to 10 nM FITC-fMLF for additional 30 minutes at 37°C. Scale bar: 5 μm. Original magnifications: 630x.
Mentions: Once documented that both SRSRY and [SRSRY] peptides compete with fMLF for binding to FPR1, we investigated the effect of [SRSRY] on the motility of monocytes. Preliminarily, we assessed whether [SRSRY] is a ligand of FPR1 on monocyte cell surface. Similarly to RBL-2H3/ETFR cells, THP-1 cells exposed to 10 nM FITC-fMLF at 4°C, revealed an appreciable binding which was dramatically reduced by an excess of unlabeled fMLF, SRSRY or [SRSRY], but not by the control peptide [RSSYR] (Fig 3A). When binding experiments were performed at 37°C, FPR1 appeared mainly localized within the cytoplasm, adjacent but outside the nucleus of THP-1 cells. Internalization of fluorescent agonist was dramatically reduced in all cell population when THP-1 cells were pre-incubated with an excess of unlabelled fMLF, or [SRSRY] while the control peptide [RSSYR] did not exert such effect (Fig 3B). Although we did not determine the binding affinity of [SRSRY] for FPR1, our findings indicate that the peptide [SRSRY] prevents fMLF/FPR1 interaction and inhibits agonist-induced FPR1 internalization in THP-1 cells.

Bottom Line: The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility.FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis.PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY].

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University Federico II, Naples, Italy.

ABSTRACT
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus