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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 regulates uPAR and uPA expression in BM-MSC. BM-MSC were grown in serum-free control medium or treated with PDGF-AB. a Quantitative PCR analysis of uPAR mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as the uPAR mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). b Flow cytometry analysis of uPAR cell surface expression; 24 hours after treatment, cells were stained with anti-uPAR monoclonal antibody (dark curves) or its isotypic control (grey curves). One representative experiment of one donor of the five assessed is shown (left). Quantification data (right) expressed as mean ± SEM of uPAR fluorescence intensity relative to the isotype of five independent experiments (one donor per experiment). *P <0.05. c Western blot analysis of uPAR in cellular membranes 24 hours after treatment.***P <0.001 d Quantitative PCR analysis of uPA mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as uPA mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). e Western blot analysis of uPA in cellular membranes 24 hours after treatment. f Zymographic analysis of uPA activity in cellular membranes 24 hours after treatment. Purified high molecular weight human urokinase was used as standard (ST). c, e, f (left) Data represent one representative experiment, (right) bands were quantified by densitometry analysis using ImageJ software, normalized to β-actin (c, e). Data expressed as mean ± SEM of three to five independent experiments (one donor per experiment). MFI mean fluorescence intensity, PDGF-AB platelet-derived growth factor AB, PE phycoerythrin, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
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Fig2: PDGF-AB regulates uPAR and uPA expression in BM-MSC. BM-MSC were grown in serum-free control medium or treated with PDGF-AB. a Quantitative PCR analysis of uPAR mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as the uPAR mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). b Flow cytometry analysis of uPAR cell surface expression; 24 hours after treatment, cells were stained with anti-uPAR monoclonal antibody (dark curves) or its isotypic control (grey curves). One representative experiment of one donor of the five assessed is shown (left). Quantification data (right) expressed as mean ± SEM of uPAR fluorescence intensity relative to the isotype of five independent experiments (one donor per experiment). *P <0.05. c Western blot analysis of uPAR in cellular membranes 24 hours after treatment.***P <0.001 d Quantitative PCR analysis of uPA mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as uPA mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). e Western blot analysis of uPA in cellular membranes 24 hours after treatment. f Zymographic analysis of uPA activity in cellular membranes 24 hours after treatment. Purified high molecular weight human urokinase was used as standard (ST). c, e, f (left) Data represent one representative experiment, (right) bands were quantified by densitometry analysis using ImageJ software, normalized to β-actin (c, e). Data expressed as mean ± SEM of three to five independent experiments (one donor per experiment). MFI mean fluorescence intensity, PDGF-AB platelet-derived growth factor AB, PE phycoerythrin, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor

Mentions: To examine whether uPAR was involved in PDGF-AB-induced migratory capacities, we first quantified uPAR gene expression in BM-MSC and its regulation by PDGF-AB. Kinetic experiments revealed that uPAR mRNA levels reached a twofold increase after treatment with PDGF-AB as soon as 5 hours post treatment (Fig. 2a). At the protein level, both flow cytometry analysis on intact cells and western blotting on isolated cell membrane fractions revealed that control BM-MSC weakly expressed uPAR at their surface, whereas 24 hours post PDGF-AB treatment the cell surface expression of uPAR was significantly increased (Fig. 2b, c), indicating that mRNA and protein expressions are regulated in a similar manner. Three major isoforms of uPAR were detected in BM-MSC cellular membranes at 55, 45, and 40 kDa and each isoform’s expression was increased by the PDGF-AB treatment (Fig. 2c).Fig. 2


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 regulates uPAR and uPA expression in BM-MSC. BM-MSC were grown in serum-free control medium or treated with PDGF-AB. a Quantitative PCR analysis of uPAR mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as the uPAR mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). b Flow cytometry analysis of uPAR cell surface expression; 24 hours after treatment, cells were stained with anti-uPAR monoclonal antibody (dark curves) or its isotypic control (grey curves). One representative experiment of one donor of the five assessed is shown (left). Quantification data (right) expressed as mean ± SEM of uPAR fluorescence intensity relative to the isotype of five independent experiments (one donor per experiment). *P <0.05. c Western blot analysis of uPAR in cellular membranes 24 hours after treatment.***P <0.001 d Quantitative PCR analysis of uPA mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as uPA mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). e Western blot analysis of uPA in cellular membranes 24 hours after treatment. f Zymographic analysis of uPA activity in cellular membranes 24 hours after treatment. Purified high molecular weight human urokinase was used as standard (ST). c, e, f (left) Data represent one representative experiment, (right) bands were quantified by densitometry analysis using ImageJ software, normalized to β-actin (c, e). Data expressed as mean ± SEM of three to five independent experiments (one donor per experiment). MFI mean fluorescence intensity, PDGF-AB platelet-derived growth factor AB, PE phycoerythrin, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: PDGF-AB regulates uPAR and uPA expression in BM-MSC. BM-MSC were grown in serum-free control medium or treated with PDGF-AB. a Quantitative PCR analysis of uPAR mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as the uPAR mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). b Flow cytometry analysis of uPAR cell surface expression; 24 hours after treatment, cells were stained with anti-uPAR monoclonal antibody (dark curves) or its isotypic control (grey curves). One representative experiment of one donor of the five assessed is shown (left). Quantification data (right) expressed as mean ± SEM of uPAR fluorescence intensity relative to the isotype of five independent experiments (one donor per experiment). *P <0.05. c Western blot analysis of uPAR in cellular membranes 24 hours after treatment.***P <0.001 d Quantitative PCR analysis of uPA mRNA synthesis 5, 10, or 24 hours after treatment. Data from four donors are presented as uPA mRNA fold change relative to nonstimulated control cells for each donor (1 = no stimulation). e Western blot analysis of uPA in cellular membranes 24 hours after treatment. f Zymographic analysis of uPA activity in cellular membranes 24 hours after treatment. Purified high molecular weight human urokinase was used as standard (ST). c, e, f (left) Data represent one representative experiment, (right) bands were quantified by densitometry analysis using ImageJ software, normalized to β-actin (c, e). Data expressed as mean ± SEM of three to five independent experiments (one donor per experiment). MFI mean fluorescence intensity, PDGF-AB platelet-derived growth factor AB, PE phycoerythrin, uPA urokinase-type plasminogen activator, uPAR urokinase-type plasminogen activator receptor
Mentions: To examine whether uPAR was involved in PDGF-AB-induced migratory capacities, we first quantified uPAR gene expression in BM-MSC and its regulation by PDGF-AB. Kinetic experiments revealed that uPAR mRNA levels reached a twofold increase after treatment with PDGF-AB as soon as 5 hours post treatment (Fig. 2a). At the protein level, both flow cytometry analysis on intact cells and western blotting on isolated cell membrane fractions revealed that control BM-MSC weakly expressed uPAR at their surface, whereas 24 hours post PDGF-AB treatment the cell surface expression of uPAR was significantly increased (Fig. 2b, c), indicating that mRNA and protein expressions are regulated in a similar manner. Three major isoforms of uPAR were detected in BM-MSC cellular membranes at 55, 45, and 40 kDa and each isoform’s expression was increased by the PDGF-AB treatment (Fig. 2c).Fig. 2

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