Transforming growth factor-β signaling pathway activation in Keratoconus.

Keratoconus is a bilateral progressive corneal disease, leading to thinning, scarring, and protrusion of the central cornea.1 The origin and the pathogenesis of this disorder are not well understood. Although most often an isolated disease, it has been associated with several accompanying factors such as Down syndrome, contact lens wear, connective tissue disease, atopy, and eye rubbing, and it can occur in a familial setting. Keratoconus most likely is caused by multiple genes and may result from complex interactions between genes and environmental factors.2 Therapeutic measures focus first on the correction of refractive errors. Although preliminary results on riboflavin/ultraviolet-A-induced collagen-crosslinking suggest a favorable outcome, in the advanced stages, corneal transplantation is still the most effective treatment to date.3,4 Keratoconus in fact is the most common indication for keratoplasty.5 Gaining more insight into the mechanisms of keratoconus to find ways to prevent disease progression or to discover new treatment options therefore would be an important accomplishment. Histologically, in the course of the disease, breaks in Bowman membrane and subepithelial scarring can be observed. Furthermore, the affected areas have marked alterations in the components of the extracellular matrix and show apoptotic cells, which, along with the thinning of the corneal stroma, suggest an increased activation of degrading enzymes and cell death resulting from oxidative stress.6 However, the exact mechanisms of the tissue breakdown remain unclear. The signaling pathway of transforming growth factor-β (TGFβ) is a complex, multibranched signal transduction cascade that may modulate ECM alterations in keratoconus. TGFβ, with its 3 isoforms, TGFβ1, TGFβ2, and TGFβ3, is only one of numerous ligands of the TGFβ superfamily that bind to the TGFβ receptors that exist in 3 different isoforms. Binding of ligands to the TGFβ2 receptor, which has an intrinsic serin/threonine kinase activity, leads to recruitment and phosphorylation of the TGFβ1 receptor, which subsequently phosphorylates the mothers against decacentaplegic homolog (Smad) 2 and Smad3 proteins intracellularly. The Smad proteins are homologs of the Drosophila protein mothers against deca-pentaplegic and the C. elegans protein SMA. Phosphorylated Smad2 (pSmad2) forms a complex with the mediator Smad4 and is translocated into the nucleus, where it acts as a transcription factor for multiple TGFβ-dependent genes. Smad2 and Smad3 can be activated as well by non-TGFβ growth factors, which are capable of activating mitogen-activated protein kinases. These multiple growth factors include fibroblast growth factor, insulin-like growth factor-1, hepatocyte growth factor, and endothelial growth factor. Many of the cellular effects of the TGFβ pathway have in common their involvement in the restoration of normal tissue after injury by induction of both extracellular matrix and matrix-degrading enzymes.7–13 The involvement of the TGFβ pathway in the modulation and production of extracellular matrix suggests involvement in the pathogenesis of keratoconus, either in a causative role or a secondary repair response leading to structural changes in keratoconus. However, previous reports linking the TGFβ pathway with the pathogenesis of keratoconus have been inconclusive. Although Maier and associates found TGFβ2 levels to be elevated in the aqueous humor in keratoconus cases, immunofluorescence studies on TGFβ2 in patients with keratoconus did not show an increase in staining as compared with normal controls.14,15 This work attempts to elucidate the role of the TGFβ signaling pathway in keratoconus by focusing on the extracellular receptor ligands TGFβ and its isoforms, as well as the intracellular activation of Smad2, by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) of keratoconus epithelium.