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Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

Chabot V, Dromard C, Rico A, Langonné A, Gaillard J, Guilloton F, Casteilla L, Sensebé L - Stem Cell Res Ther (2015)

Bottom Line: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration.This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration.

View Article: PubMed Central - PubMed

Affiliation: EFS Centre-Atlantique, BP 40661, 37 206, Tours, Cedex 3, France. valerie.chabot@efs.sante.fr.

ABSTRACT

Introduction: Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.

Methods: Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.

Results: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Conclusions: We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

No MeSH data available.


Related in: MedlinePlus

PDGF-AB enhances uPAR/P-FAK interactions in migrating BM-MSC. a uPAR (green) and P-FAK (red) repartition in migrating cells. BM-MSC were seeded in a Labtek chamber coated with type I dermal collagen. After the scratch was performed, cells were grown in serum-free control medium or treated with PDGF-AB for 3 hours. uPAR and P-FAK stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR–P-FAK coimmunoprecipitation. Two hours after adherence on type I dermal collagen, cells were seeded in serum-free control medium (−) or treated with PDGF-AB (+) for 1 hour. uPAR was immunoprecipitated from cell lysates with anti-uPAR monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for uPAR and P-FAK. (Left) Western blot representative of three experiments. T0, lysate of cells before seeding; Adh., lysate of cells allowed to adhere for 2 hours on type I dermal collagen. (Right) P-FAK blots quantification by densitometry analysis using ImageJ software. Data expressed as the P-FAK fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). PDGF-AB platelet-derived growth factor AB, P-FAK autophosphorylated focal adhesion tyrosine kinase, uPAR urokinase-type plasminogen activator receptor
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Fig5: PDGF-AB enhances uPAR/P-FAK interactions in migrating BM-MSC. a uPAR (green) and P-FAK (red) repartition in migrating cells. BM-MSC were seeded in a Labtek chamber coated with type I dermal collagen. After the scratch was performed, cells were grown in serum-free control medium or treated with PDGF-AB for 3 hours. uPAR and P-FAK stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR–P-FAK coimmunoprecipitation. Two hours after adherence on type I dermal collagen, cells were seeded in serum-free control medium (−) or treated with PDGF-AB (+) for 1 hour. uPAR was immunoprecipitated from cell lysates with anti-uPAR monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for uPAR and P-FAK. (Left) Western blot representative of three experiments. T0, lysate of cells before seeding; Adh., lysate of cells allowed to adhere for 2 hours on type I dermal collagen. (Right) P-FAK blots quantification by densitometry analysis using ImageJ software. Data expressed as the P-FAK fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). PDGF-AB platelet-derived growth factor AB, P-FAK autophosphorylated focal adhesion tyrosine kinase, uPAR urokinase-type plasminogen activator receptor

Mentions: The focal adhesion tyrosine kinase (FAK), when autophosphorylated at Tyr397 (P-FAK), is known to be involved in the β1-integrin signaling pathway induced by uPAR. We thus examined uPAR and P-FAK expression in migrating cells on type I dermal collagen in response to PDGF-AB treatment. As for uPAR, P-FAK immunofluorescent staining was poorly detected in resting control cells, while it was clearly enhanced in PDGF-AB-induced migrating cells (Fig. 5a). To assess whether uPAR and P-FAK were physically associated in focal complexes, coimmunoprecipitation experiments were performed on cell lysates from BM-MSC induced to migrate on type I dermal collagen. As shown in Fig. 5b, the anti-uPAR antibodies (but not the control IgG) immunoprecipitated uPAR isoforms as well as P-FAK in nontreated cells, suggesting a physical association between uPAR and P-FAK. This effect appeared to take place as soon as the cells adhered to the coated plastic and was enhanced after 1 hour of treatment with PDGF-AB.Fig. 5


Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

Chabot V, Dromard C, Rico A, Langonné A, Gaillard J, Guilloton F, Casteilla L, Sensebé L - Stem Cell Res Ther (2015)

PDGF-AB enhances uPAR/P-FAK interactions in migrating BM-MSC. a uPAR (green) and P-FAK (red) repartition in migrating cells. BM-MSC were seeded in a Labtek chamber coated with type I dermal collagen. After the scratch was performed, cells were grown in serum-free control medium or treated with PDGF-AB for 3 hours. uPAR and P-FAK stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR–P-FAK coimmunoprecipitation. Two hours after adherence on type I dermal collagen, cells were seeded in serum-free control medium (−) or treated with PDGF-AB (+) for 1 hour. uPAR was immunoprecipitated from cell lysates with anti-uPAR monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for uPAR and P-FAK. (Left) Western blot representative of three experiments. T0, lysate of cells before seeding; Adh., lysate of cells allowed to adhere for 2 hours on type I dermal collagen. (Right) P-FAK blots quantification by densitometry analysis using ImageJ software. Data expressed as the P-FAK fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). PDGF-AB platelet-derived growth factor AB, P-FAK autophosphorylated focal adhesion tyrosine kinase, uPAR urokinase-type plasminogen activator receptor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4588680&req=5

Fig5: PDGF-AB enhances uPAR/P-FAK interactions in migrating BM-MSC. a uPAR (green) and P-FAK (red) repartition in migrating cells. BM-MSC were seeded in a Labtek chamber coated with type I dermal collagen. After the scratch was performed, cells were grown in serum-free control medium or treated with PDGF-AB for 3 hours. uPAR and P-FAK stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR–P-FAK coimmunoprecipitation. Two hours after adherence on type I dermal collagen, cells were seeded in serum-free control medium (−) or treated with PDGF-AB (+) for 1 hour. uPAR was immunoprecipitated from cell lysates with anti-uPAR monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for uPAR and P-FAK. (Left) Western blot representative of three experiments. T0, lysate of cells before seeding; Adh., lysate of cells allowed to adhere for 2 hours on type I dermal collagen. (Right) P-FAK blots quantification by densitometry analysis using ImageJ software. Data expressed as the P-FAK fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). PDGF-AB platelet-derived growth factor AB, P-FAK autophosphorylated focal adhesion tyrosine kinase, uPAR urokinase-type plasminogen activator receptor
Mentions: The focal adhesion tyrosine kinase (FAK), when autophosphorylated at Tyr397 (P-FAK), is known to be involved in the β1-integrin signaling pathway induced by uPAR. We thus examined uPAR and P-FAK expression in migrating cells on type I dermal collagen in response to PDGF-AB treatment. As for uPAR, P-FAK immunofluorescent staining was poorly detected in resting control cells, while it was clearly enhanced in PDGF-AB-induced migrating cells (Fig. 5a). To assess whether uPAR and P-FAK were physically associated in focal complexes, coimmunoprecipitation experiments were performed on cell lysates from BM-MSC induced to migrate on type I dermal collagen. As shown in Fig. 5b, the anti-uPAR antibodies (but not the control IgG) immunoprecipitated uPAR isoforms as well as P-FAK in nontreated cells, suggesting a physical association between uPAR and P-FAK. This effect appeared to take place as soon as the cells adhered to the coated plastic and was enhanced after 1 hour of treatment with PDGF-AB.Fig. 5

Bottom Line: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration.This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration.

View Article: PubMed Central - PubMed

Affiliation: EFS Centre-Atlantique, BP 40661, 37 206, Tours, Cedex 3, France. valerie.chabot@efs.sante.fr.

ABSTRACT

Introduction: Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.

Methods: Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.

Results: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Conclusions: We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

No MeSH data available.


Related in: MedlinePlus