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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

uPA/uPAR mediate PDGF-AB-induced BM-MSC migration. a, b Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Pictures of scratches were taken immediately (T = 0) or 22 hours after culture (T = 22 h) in serum-free control medium or PDGF-AB supplemented medium in the presence or not of anti-uPAR or anti-uPA neutralizing antibodies. a An experiment representative of three experiments. b Quantification of BM-MSC migration. c, d Scratch test assay was performed on BM-MSC isolated from three different donors transfected with either control siRNA (si Neg) or uPAR siRNA (si uPAR) and grown on type I dermal collagen in serum-free control medium or treated with PDGF-AB. c An experiment representative of three experiments. d Migration quantification of transfected BM-MSC. b, d Results 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. Scale bars: 200 μm. PDGF-AB platelet-derived growth factor AB, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
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Fig3: uPA/uPAR mediate PDGF-AB-induced BM-MSC migration. a, b Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Pictures of scratches were taken immediately (T = 0) or 22 hours after culture (T = 22 h) in serum-free control medium or PDGF-AB supplemented medium in the presence or not of anti-uPAR or anti-uPA neutralizing antibodies. a An experiment representative of three experiments. b Quantification of BM-MSC migration. c, d Scratch test assay was performed on BM-MSC isolated from three different donors transfected with either control siRNA (si Neg) or uPAR siRNA (si uPAR) and grown on type I dermal collagen in serum-free control medium or treated with PDGF-AB. c An experiment representative of three experiments. d Migration quantification of transfected BM-MSC. b, d Results 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. Scale bars: 200 μm. PDGF-AB platelet-derived growth factor AB, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor

Mentions: As PDGF-AB stimulated both uPA and uPAR expression and BM-MSC migration in vitro, we tested whether these proteins were required for PDGF-AB-induced BM-MSC migration using two loss-of-function strategies. We first investigated the effect of neutralizing anti-uPAR antibodies in a scratch test assay. Neutralizing antibodies displayed no effect on BM-MSC basal migration but significantly decreased the capacity of PDGF-AB-treated BM-MSC to reconstitute the scratched area (Fig. 3a, b). The same results were obtained using anti-uPA antibodies (Fig. 3a, b) independently of the matrix tested (Additional file 5). To confirm these results, we used a siRNA to reduce uPAR expression in BM-MSC and the scratch test assay was performed on type I dermal collagen. BM-MSC were transfected with control siRNA (si Neg) or uPAR targeted siRNA (si uPAR) and the knocking down efficiency was evaluated by quantitative PCR and flow cytometry approaches. A 77 % decrease in uPAR mRNA expression and a 90 % decrease in uPAR protein synthesis were observed (Figure S5A, B in Additional file 6). BM-MSC wound closure capacity was increased by 29 % after 22 hours of treatment with PDGF-AB and this effect was inhibited when BM-MSC were transfected with uPAR siRNA (Fig. 3c, d). On transwell dishes, the effect of PDGF-AB was significantly reduced by uPAR siRNA (81 % reduction) (Figure S5C in Additional file 6). All together these results demonstrate that uPAR is required for PDGF-AB-induced migration of BM-MSC.Fig. 3


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)

uPA/uPAR mediate PDGF-AB-induced BM-MSC migration. a, b Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Pictures of scratches were taken immediately (T = 0) or 22 hours after culture (T = 22 h) in serum-free control medium or PDGF-AB supplemented medium in the presence or not of anti-uPAR or anti-uPA neutralizing antibodies. a An experiment representative of three experiments. b Quantification of BM-MSC migration. c, d Scratch test assay was performed on BM-MSC isolated from three different donors transfected with either control siRNA (si Neg) or uPAR siRNA (si uPAR) and grown on type I dermal collagen in serum-free control medium or treated with PDGF-AB. c An experiment representative of three experiments. d Migration quantification of transfected BM-MSC. b, d Results 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. Scale bars: 200 μm. PDGF-AB platelet-derived growth factor AB, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588680&req=5

Fig3: uPA/uPAR mediate PDGF-AB-induced BM-MSC migration. a, b Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Pictures of scratches were taken immediately (T = 0) or 22 hours after culture (T = 22 h) in serum-free control medium or PDGF-AB supplemented medium in the presence or not of anti-uPAR or anti-uPA neutralizing antibodies. a An experiment representative of three experiments. b Quantification of BM-MSC migration. c, d Scratch test assay was performed on BM-MSC isolated from three different donors transfected with either control siRNA (si Neg) or uPAR siRNA (si uPAR) and grown on type I dermal collagen in serum-free control medium or treated with PDGF-AB. c An experiment representative of three experiments. d Migration quantification of transfected BM-MSC. b, d Results 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. Scale bars: 200 μm. PDGF-AB platelet-derived growth factor AB, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
Mentions: As PDGF-AB stimulated both uPA and uPAR expression and BM-MSC migration in vitro, we tested whether these proteins were required for PDGF-AB-induced BM-MSC migration using two loss-of-function strategies. We first investigated the effect of neutralizing anti-uPAR antibodies in a scratch test assay. Neutralizing antibodies displayed no effect on BM-MSC basal migration but significantly decreased the capacity of PDGF-AB-treated BM-MSC to reconstitute the scratched area (Fig. 3a, b). The same results were obtained using anti-uPA antibodies (Fig. 3a, b) independently of the matrix tested (Additional file 5). To confirm these results, we used a siRNA to reduce uPAR expression in BM-MSC and the scratch test assay was performed on type I dermal collagen. BM-MSC were transfected with control siRNA (si Neg) or uPAR targeted siRNA (si uPAR) and the knocking down efficiency was evaluated by quantitative PCR and flow cytometry approaches. A 77 % decrease in uPAR mRNA expression and a 90 % decrease in uPAR protein synthesis were observed (Figure S5A, B in Additional file 6). BM-MSC wound closure capacity was increased by 29 % after 22 hours of treatment with PDGF-AB and this effect was inhibited when BM-MSC were transfected with uPAR siRNA (Fig. 3c, d). On transwell dishes, the effect of PDGF-AB was significantly reduced by uPAR siRNA (81 % reduction) (Figure S5C in Additional file 6). All together these results demonstrate that uPAR is required for PDGF-AB-induced migration of BM-MSC.Fig. 3

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