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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 migration of BM-MSC through control of uPAR and β1-integrin association. a uPAR (green) and β1-integrin subunit (red) repartition in migrating cells. Images were acquired with a confocal microscope. uPAR and β1-integrin stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR-β1 subunit coimmunoprecipitation. Two hours after adherence (Adh) on type I dermal collagen, cells were grown in serum-free control medium (−) or treated with PDGF-AB (+) for 1–18 hours. β1 integrin was immunoprecipitated from cell lysates with anti-β1 monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for β1-integrin subunit (top western blot) and uPAR (bottom western blot). (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) uPAR blots quantification by densitometry analysis using ImageJ software. Data expressed as the uPAR fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). c Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Two hours after adherence, cells were grown in serum-free control medium or treated with PDGF-AB, supplemented or not with integrin blocking peptide β1P1 along with the corresponding scrambled control scβ1P1 or anti-uPAR neutralizing antibody. Results are expressed as percentages of wound closure at T = 22 h compared with T = 0. Mean of three independent experiments ± SEM are represented (one donor per experiment), each performed in triplicate. *P <0.05. PDGF-AB platelet-derived growth factor AB, uPAR urokinase-type plasminogen activator receptor
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Fig4: PDGF-AB enhances migration of BM-MSC through control of uPAR and β1-integrin association. a uPAR (green) and β1-integrin subunit (red) repartition in migrating cells. Images were acquired with a confocal microscope. uPAR and β1-integrin stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR-β1 subunit coimmunoprecipitation. Two hours after adherence (Adh) on type I dermal collagen, cells were grown in serum-free control medium (−) or treated with PDGF-AB (+) for 1–18 hours. β1 integrin was immunoprecipitated from cell lysates with anti-β1 monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for β1-integrin subunit (top western blot) and uPAR (bottom western blot). (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) uPAR blots quantification by densitometry analysis using ImageJ software. Data expressed as the uPAR fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). c Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Two hours after adherence, cells were grown in serum-free control medium or treated with PDGF-AB, supplemented or not with integrin blocking peptide β1P1 along with the corresponding scrambled control scβ1P1 or anti-uPAR neutralizing antibody. Results are expressed as percentages of wound closure at T = 22 h compared with T = 0. Mean of three independent experiments ± SEM are represented (one donor per experiment), each performed in triplicate. *P <0.05. PDGF-AB platelet-derived growth factor AB, uPAR urokinase-type plasminogen activator receptor

Mentions: We tested whether PDGF-AB promotes the association of uPAR with β1 integrin in BM-MSC. We first examined the localization of uPAR using confocal microscopy and its interaction with the cytoskeleton in migrating cells. BM-MSC underwent scratch and they were treated or not with PDGF-AB. uPAR and phalloidin stainings were performed 6 hours after the scratch (this is the time at which cell migration, cytoskeleton remodeling, and lamellipodia extension begin). Immunofluorescence analysis revealed that in resting cells uPAR was uniformly distributed on the cell surface and dispersed as small aggregates. In migrating cells, PDGF-AB treatment strongly modified cell morphology and uPAR distribution, with a clustering of uPAR at the leading edge of the cells in focal adhesions (Additional file 7). We next examined the distribution of uPAR and β1 integrin on migrating cells induced by PDGF-AB. Because uPAR staining appeared to be partially co-localized with β1 integrin as soon as 1 hour after PDGF-AB treatment of BM-MSC plated on type I dermal collagen (Fig. 4a) and on fibronectin (Figure S7A in Additional file 8), we used coimmunoprecipitation experiments to determine whether uPAR and β1 integrin were physically associated. Results shown in Fig. 4b demonstrated that the antibodies against the β1-integrin subunit (but not the control IgG) precipitated not only the β1-integrin subunit but also uPAR in nontreated cells, suggesting a physical association between uPAR and the β1-integrin subunit. The quantity of coimmunoprecipitated β1 integrin and uPAR was increased 1 and 3 hours post PDGF-AB treatment. The same trend was obtained at 3 hours on fibronectin (Figure S7B in Additional file 8).Fig. 4


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 migration of BM-MSC through control of uPAR and β1-integrin association. a uPAR (green) and β1-integrin subunit (red) repartition in migrating cells. Images were acquired with a confocal microscope. uPAR and β1-integrin stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR-β1 subunit coimmunoprecipitation. Two hours after adherence (Adh) on type I dermal collagen, cells were grown in serum-free control medium (−) or treated with PDGF-AB (+) for 1–18 hours. β1 integrin was immunoprecipitated from cell lysates with anti-β1 monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for β1-integrin subunit (top western blot) and uPAR (bottom western blot). (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) uPAR blots quantification by densitometry analysis using ImageJ software. Data expressed as the uPAR fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). c Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Two hours after adherence, cells were grown in serum-free control medium or treated with PDGF-AB, supplemented or not with integrin blocking peptide β1P1 along with the corresponding scrambled control scβ1P1 or anti-uPAR neutralizing antibody. Results are expressed as percentages of wound closure at T = 22 h compared with T = 0. Mean of three independent experiments ± SEM are represented (one donor per experiment), each performed in triplicate. *P <0.05. PDGF-AB platelet-derived growth factor AB, uPAR urokinase-type plasminogen activator receptor
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Fig4: PDGF-AB enhances migration of BM-MSC through control of uPAR and β1-integrin association. a uPAR (green) and β1-integrin subunit (red) repartition in migrating cells. Images were acquired with a confocal microscope. uPAR and β1-integrin stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR-β1 subunit coimmunoprecipitation. Two hours after adherence (Adh) on type I dermal collagen, cells were grown in serum-free control medium (−) or treated with PDGF-AB (+) for 1–18 hours. β1 integrin was immunoprecipitated from cell lysates with anti-β1 monoclonal antibodies or isotypic control IgG1. Immunoprecipitates were analyzed by SDS-PAGE and blotted for β1-integrin subunit (top western blot) and uPAR (bottom western blot). (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) uPAR blots quantification by densitometry analysis using ImageJ software. Data expressed as the uPAR fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). c Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Two hours after adherence, cells were grown in serum-free control medium or treated with PDGF-AB, supplemented or not with integrin blocking peptide β1P1 along with the corresponding scrambled control scβ1P1 or anti-uPAR neutralizing antibody. Results are expressed as percentages of wound closure at T = 22 h compared with T = 0. Mean of three independent experiments ± SEM are represented (one donor per experiment), each performed in triplicate. *P <0.05. PDGF-AB platelet-derived growth factor AB, uPAR urokinase-type plasminogen activator receptor
Mentions: We tested whether PDGF-AB promotes the association of uPAR with β1 integrin in BM-MSC. We first examined the localization of uPAR using confocal microscopy and its interaction with the cytoskeleton in migrating cells. BM-MSC underwent scratch and they were treated or not with PDGF-AB. uPAR and phalloidin stainings were performed 6 hours after the scratch (this is the time at which cell migration, cytoskeleton remodeling, and lamellipodia extension begin). Immunofluorescence analysis revealed that in resting cells uPAR was uniformly distributed on the cell surface and dispersed as small aggregates. In migrating cells, PDGF-AB treatment strongly modified cell morphology and uPAR distribution, with a clustering of uPAR at the leading edge of the cells in focal adhesions (Additional file 7). We next examined the distribution of uPAR and β1 integrin on migrating cells induced by PDGF-AB. Because uPAR staining appeared to be partially co-localized with β1 integrin as soon as 1 hour after PDGF-AB treatment of BM-MSC plated on type I dermal collagen (Fig. 4a) and on fibronectin (Figure S7A in Additional file 8), we used coimmunoprecipitation experiments to determine whether uPAR and β1 integrin were physically associated. Results shown in Fig. 4b demonstrated that the antibodies against the β1-integrin subunit (but not the control IgG) precipitated not only the β1-integrin subunit but also uPAR in nontreated cells, suggesting a physical association between uPAR and the β1-integrin subunit. The quantity of coimmunoprecipitated β1 integrin and uPAR was increased 1 and 3 hours post PDGF-AB treatment. The same trend was obtained at 3 hours on fibronectin (Figure S7B in Additional file 8).Fig. 4

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