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Darrell C. Andrews, John Browne, Helen O' Donovan, Noel Faherty, Noelynn Oliver, Deborah Wallace, Emily Hughes, Colm O' Brien, John Crean; Integrated Signalling Between CTGF And TGF-β2 Leads To Divergent Activation Of Canonical And Non-canonical Pathways Controlling EMT In ARPE-19 Cells: Implications For The Pathogenesis Of Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1150.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) is considered the result of erroneous wound healing, characterised by disruption of the epiretinal membrane. Here we describe the interaction between CTGF and TGF-β2, signaling and transcriptional outcomes leading to transdifferentiation, suggesting a requirement for co-operation between these factors in PVR.
Human retinal pigment epithelial cells (ARPE-19) were cultured and stimulated with TGF-β 2, CTGF and both combined. Activation of canonical (Smad 2, Smad 3 Smad 1/5/8) and non-canonical (erk, p38, Akt, cdc42) pathways was assessed by immunoblot. Transcriptional activation was assessed by promoter reporter (3TP-Luc) assay. EMT was determined by immunocytochemistry and characterized by staining for vimentin, smooth muscle actin and ZO-1. Migratory capacity was assessed by wound healing assay. Gene expression profiles were determined using DNA microarrays.
Direct binding of CTGF to TGF-β2 was demonstrated using a solid-phase binding assay. CTGF had no effect on Smad signalling but antagonised TGF-β2 induced phosphorylation of Smad2 and Smad3. Both CTGF and TGF-β2 phosphorylated erk and p38; phosphorylation was significantly enhanced upon co-stimulation, suggesting cooperative effect and a shift from canonical to non-canonical signaling. 3TP-luciferase promoter reporter activity in response to TGF-β was inhibited by CTGF. These responses were reversed by the anti CTGF monoclonal antibody FG-3019. The consequences of this signaling were inhibition of migratory capacity and increased cell differentiation. Microarray studies identified a number of genes that were co-regulated by CTGF and TGF-β including several implicated in epithelial to mesenchymal transition.
These findings suggest that a CTGF-enriched permissive cellular environment regulates the initiation and promotion of TGF-β signaling networks, identifying a hitherto undescribed "switch". The consequences of this switch are altered phenotypic behaviour, representing a significant advance in our understanding of the patho-mechanisms of PVR. Therapeutic strategies targeting CTGF in PVR have the potential for significant therapeutic gain.
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