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The RhoA activator GEF-H1/Lfc is a transforming growth factor-beta target gene and effector that regulates alpha-smooth muscle actin expression and cell migration.

Tsapara A, Luthert P, Greenwood J, Hill CS, Matter K, Balda MS - Mol. Biol. Cell (2010)

Bottom Line: GEF-H1 inhibition counteracted alpha-SMA up-regulation and cell migration.In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo.Our data indicate that GEF-H1 is a target and functional effector of TGF-beta by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-beta critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of alpha-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-beta responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-beta signaling, but how TGF-beta-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-beta. GEF-H1 was the only detectable Rho exchange factor increased by TGF-beta1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted alpha-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-beta by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

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TGF-β1 induces junctional disruption and GEF-H1 up-regulation in RPE cells. RPE cells were stimulated with TGF-β1 (A–E for 3 d; F–I as indicated) and processed for immunofluorescence (A–E) or immunoblot (F–H) analysis. (A–C) Samples were stained for either α-SMA (A) and ZO-1 (B), occludin (C), or GEF-H1 (D) and cingulin (E). (F–H) Immunoblots of total RPE cell extracts stimulated with TGF-β1 for the indicated time were probed with antibodies against ZO-1 and occludin (by densitometry, both proteins were decreased by >50% after 3 and 5 d of TGF-β treatment; F), GEF-H1 and α-SMA (the numbers indicate the ratios of TGF-β–treated divided by control samples obtained by densitometry; all values were normalized by those obtained for tubulin in each sample; G), cingulin (H); α-tubulin was used as loading control. (I) Immunoblot of RPE cell extracts was probed for phosphorylated (p-MYPT1) and total myosin light chain phosphatase (MYPT1) (the numbers indicate the relative increase in p-MYPT1 in TGF-β–treated samples). Shown are representative results from at least two experiments.
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Figure 1: TGF-β1 induces junctional disruption and GEF-H1 up-regulation in RPE cells. RPE cells were stimulated with TGF-β1 (A–E for 3 d; F–I as indicated) and processed for immunofluorescence (A–E) or immunoblot (F–H) analysis. (A–C) Samples were stained for either α-SMA (A) and ZO-1 (B), occludin (C), or GEF-H1 (D) and cingulin (E). (F–H) Immunoblots of total RPE cell extracts stimulated with TGF-β1 for the indicated time were probed with antibodies against ZO-1 and occludin (by densitometry, both proteins were decreased by >50% after 3 and 5 d of TGF-β treatment; F), GEF-H1 and α-SMA (the numbers indicate the ratios of TGF-β–treated divided by control samples obtained by densitometry; all values were normalized by those obtained for tubulin in each sample; G), cingulin (H); α-tubulin was used as loading control. (I) Immunoblot of RPE cell extracts was probed for phosphorylated (p-MYPT1) and total myosin light chain phosphatase (MYPT1) (the numbers indicate the relative increase in p-MYPT1 in TGF-β–treated samples). Shown are representative results from at least two experiments.

Mentions: We used primary porcine RPE cells as a model to analyze TGF-β signaling because they form well-differentiated monolayers in culture and respond to TGF-β (Lee et al., 2001; Ablonczy and Crosson, 2007). As expected, addition of TGF-β stimulated dissolution of cell–cell adhesion structures, such as adherens and tight junctions, correlating with altered cell morphology and reduced expression of junctional proteins, such as ZO-1 and occludin (Figure 1, A–F).


The RhoA activator GEF-H1/Lfc is a transforming growth factor-beta target gene and effector that regulates alpha-smooth muscle actin expression and cell migration.

Tsapara A, Luthert P, Greenwood J, Hill CS, Matter K, Balda MS - Mol. Biol. Cell (2010)

TGF-β1 induces junctional disruption and GEF-H1 up-regulation in RPE cells. RPE cells were stimulated with TGF-β1 (A–E for 3 d; F–I as indicated) and processed for immunofluorescence (A–E) or immunoblot (F–H) analysis. (A–C) Samples were stained for either α-SMA (A) and ZO-1 (B), occludin (C), or GEF-H1 (D) and cingulin (E). (F–H) Immunoblots of total RPE cell extracts stimulated with TGF-β1 for the indicated time were probed with antibodies against ZO-1 and occludin (by densitometry, both proteins were decreased by >50% after 3 and 5 d of TGF-β treatment; F), GEF-H1 and α-SMA (the numbers indicate the ratios of TGF-β–treated divided by control samples obtained by densitometry; all values were normalized by those obtained for tubulin in each sample; G), cingulin (H); α-tubulin was used as loading control. (I) Immunoblot of RPE cell extracts was probed for phosphorylated (p-MYPT1) and total myosin light chain phosphatase (MYPT1) (the numbers indicate the relative increase in p-MYPT1 in TGF-β–treated samples). Shown are representative results from at least two experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2836967&req=5

Figure 1: TGF-β1 induces junctional disruption and GEF-H1 up-regulation in RPE cells. RPE cells were stimulated with TGF-β1 (A–E for 3 d; F–I as indicated) and processed for immunofluorescence (A–E) or immunoblot (F–H) analysis. (A–C) Samples were stained for either α-SMA (A) and ZO-1 (B), occludin (C), or GEF-H1 (D) and cingulin (E). (F–H) Immunoblots of total RPE cell extracts stimulated with TGF-β1 for the indicated time were probed with antibodies against ZO-1 and occludin (by densitometry, both proteins were decreased by >50% after 3 and 5 d of TGF-β treatment; F), GEF-H1 and α-SMA (the numbers indicate the ratios of TGF-β–treated divided by control samples obtained by densitometry; all values were normalized by those obtained for tubulin in each sample; G), cingulin (H); α-tubulin was used as loading control. (I) Immunoblot of RPE cell extracts was probed for phosphorylated (p-MYPT1) and total myosin light chain phosphatase (MYPT1) (the numbers indicate the relative increase in p-MYPT1 in TGF-β–treated samples). Shown are representative results from at least two experiments.
Mentions: We used primary porcine RPE cells as a model to analyze TGF-β signaling because they form well-differentiated monolayers in culture and respond to TGF-β (Lee et al., 2001; Ablonczy and Crosson, 2007). As expected, addition of TGF-β stimulated dissolution of cell–cell adhesion structures, such as adherens and tight junctions, correlating with altered cell morphology and reduced expression of junctional proteins, such as ZO-1 and occludin (Figure 1, A–F).

Bottom Line: GEF-H1 inhibition counteracted alpha-SMA up-regulation and cell migration.In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo.Our data indicate that GEF-H1 is a target and functional effector of TGF-beta by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-beta critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of alpha-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-beta responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-beta signaling, but how TGF-beta-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-beta. GEF-H1 was the only detectable Rho exchange factor increased by TGF-beta1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted alpha-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-beta by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.

Show MeSH
Related in: MedlinePlus