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

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: EMT (epithelial mesenchymal transition) • growth factors/growth factor receptors • proliferative vitreoretinopathy