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GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Popsueva A, Poteryaev D, Arighi E, Meng X, Angers-Loustau A, Kaplan D, Saarma M, Sariola H - J. Cell Biol. (2003)

Bottom Line: However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable.The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not.Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.

ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

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GDNF induces branching of GFRα1-expressing MDCK cells in three-dimensional collagen gel. (A) Ret/GFRα1- and GFRα1-expressing MDCK cells were grown in collagen gel with GDNF (100 ng/ml), and wild-type MDCK cells were grown in collagen gel with HGF (50 ng/ml). BSA (100 ng/ml) was used as a negative control. Bar, 100 μm. (B) GDNF induces branching of GFRα1 and Ret/GFRα1 cells but not wild-type MDCK cells, which only respond to HGF. Persephin (PSPN; 100 ng/ml) does not induce branching of any MDCK cell line tested. From the total number of cysts in the field, the percentage of cysts with long branches was calculated. Only the branches with the length of more than two cyst diameters were counted. (C) Dose dependency of the GDNF-induced branching of GFRα1- and Ret/GFRα1-expressing MDCK cells. GDNF concentrations are marked per ml. Results are reported as fold of branching cysts over the noninduced control. Means ± SEM of five to eight counted fields are shown. The results are representative of five (A and B) and three (C) independent experiments. (B and C) GDNF significantly increases branching in GFRα1- and Ret/GFRα1-expressing MDCK and HGF increases branching of wild-type MDCK (B) compared with the control media (P < 0.001).
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fig2: GDNF induces branching of GFRα1-expressing MDCK cells in three-dimensional collagen gel. (A) Ret/GFRα1- and GFRα1-expressing MDCK cells were grown in collagen gel with GDNF (100 ng/ml), and wild-type MDCK cells were grown in collagen gel with HGF (50 ng/ml). BSA (100 ng/ml) was used as a negative control. Bar, 100 μm. (B) GDNF induces branching of GFRα1 and Ret/GFRα1 cells but not wild-type MDCK cells, which only respond to HGF. Persephin (PSPN; 100 ng/ml) does not induce branching of any MDCK cell line tested. From the total number of cysts in the field, the percentage of cysts with long branches was calculated. Only the branches with the length of more than two cyst diameters were counted. (C) Dose dependency of the GDNF-induced branching of GFRα1- and Ret/GFRα1-expressing MDCK cells. GDNF concentrations are marked per ml. Results are reported as fold of branching cysts over the noninduced control. Means ± SEM of five to eight counted fields are shown. The results are representative of five (A and B) and three (C) independent experiments. (B and C) GDNF significantly increases branching in GFRα1- and Ret/GFRα1-expressing MDCK and HGF increases branching of wild-type MDCK (B) compared with the control media (P < 0.001).

Mentions: To study the possible mechanism and mode of action of GDNF in GFRα1 and GFRα1/Ret signaling, MDCK cells were transfected with expression vectors encoding the human ret and rat gfrα1, or gfrα1 only with or without fused GFP. Multiple clones expressing GFRα1 with or without fused GFP and clones expressing Ret together with GFRα1 were identified by RT-PCR and Western blotting. The clonal cell lines expressing GFRα1 with (N3) or without GFP (N14) showed similar biological responses to GDNF (Fig. 2 and unpublished data).


GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase.

Popsueva A, Poteryaev D, Arighi E, Meng X, Angers-Loustau A, Kaplan D, Saarma M, Sariola H - J. Cell Biol. (2003)

GDNF induces branching of GFRα1-expressing MDCK cells in three-dimensional collagen gel. (A) Ret/GFRα1- and GFRα1-expressing MDCK cells were grown in collagen gel with GDNF (100 ng/ml), and wild-type MDCK cells were grown in collagen gel with HGF (50 ng/ml). BSA (100 ng/ml) was used as a negative control. Bar, 100 μm. (B) GDNF induces branching of GFRα1 and Ret/GFRα1 cells but not wild-type MDCK cells, which only respond to HGF. Persephin (PSPN; 100 ng/ml) does not induce branching of any MDCK cell line tested. From the total number of cysts in the field, the percentage of cysts with long branches was calculated. Only the branches with the length of more than two cyst diameters were counted. (C) Dose dependency of the GDNF-induced branching of GFRα1- and Ret/GFRα1-expressing MDCK cells. GDNF concentrations are marked per ml. Results are reported as fold of branching cysts over the noninduced control. Means ± SEM of five to eight counted fields are shown. The results are representative of five (A and B) and three (C) independent experiments. (B and C) GDNF significantly increases branching in GFRα1- and Ret/GFRα1-expressing MDCK and HGF increases branching of wild-type MDCK (B) compared with the control media (P < 0.001).
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Related In: Results  -  Collection

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fig2: GDNF induces branching of GFRα1-expressing MDCK cells in three-dimensional collagen gel. (A) Ret/GFRα1- and GFRα1-expressing MDCK cells were grown in collagen gel with GDNF (100 ng/ml), and wild-type MDCK cells were grown in collagen gel with HGF (50 ng/ml). BSA (100 ng/ml) was used as a negative control. Bar, 100 μm. (B) GDNF induces branching of GFRα1 and Ret/GFRα1 cells but not wild-type MDCK cells, which only respond to HGF. Persephin (PSPN; 100 ng/ml) does not induce branching of any MDCK cell line tested. From the total number of cysts in the field, the percentage of cysts with long branches was calculated. Only the branches with the length of more than two cyst diameters were counted. (C) Dose dependency of the GDNF-induced branching of GFRα1- and Ret/GFRα1-expressing MDCK cells. GDNF concentrations are marked per ml. Results are reported as fold of branching cysts over the noninduced control. Means ± SEM of five to eight counted fields are shown. The results are representative of five (A and B) and three (C) independent experiments. (B and C) GDNF significantly increases branching in GFRα1- and Ret/GFRα1-expressing MDCK and HGF increases branching of wild-type MDCK (B) compared with the control media (P < 0.001).
Mentions: To study the possible mechanism and mode of action of GDNF in GFRα1 and GFRα1/Ret signaling, MDCK cells were transfected with expression vectors encoding the human ret and rat gfrα1, or gfrα1 only with or without fused GFP. Multiple clones expressing GFRα1 with or without fused GFP and clones expressing Ret together with GFRα1 were identified by RT-PCR and Western blotting. The clonal cell lines expressing GFRα1 with (N3) or without GFP (N14) showed similar biological responses to GDNF (Fig. 2 and unpublished data).

Bottom Line: However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable.The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not.Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland.

ABSTRACT
Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

Show MeSH
Related in: MedlinePlus