17β-estradiol Can Suppress TGFβ2-induced Transdifferentiation And Collagen Production, But Does Not Inhibit Matrix Contraction

Abstract

Purpose: : Posterior capsule opacification (PCO) is the most common complication following cataract surgery and is associated with transdifferentiation of lens epithelial cells to myofibroblasts, matrix deposition and contraction. TGFβ is widely reported to promote these changes. Oestrogen (17β-estradiol) has been shown to suppress TGFβ actions. We therefore tested whether 17β-estradiol can suppress TGFβ induced cellular events during PCO development.

Methods: : Matrix contraction was assessed using a patch assay; following a 24hr period of serum starvation, human lens epithelial cells (FHL 124) were maintained in experimental conditions for 3 days. All areas covered by cells were measured using imaging techniques following fixation in 4% formaldehyde and cell staining with the total protein dye Coomassie Blue. Gene and protein expression of the transdifferentiation marker alpha smooth muscle actin was determined using Real-time PCR and western blotting respectively. Matrix deposition was assessed by expression of mColIa2 using Real-time PCR. To determine Smad2/3 distribution patterns immunocytochemistry was employed.

Results: : Real-time PCR analysis showed 10ng/ml TGFβ2 significantly increased expression of mαSMA to 596 ± 96% non-stimulated control levels. 10µM 17β-estradiol alone did not significantly affect baseline maSMA levels. However, 17β-estradiol did significantly inhibit TGFβ2 induced mαSMA expression. A similar pattern was also observed at the protein level. Expression of mColIa2 in response to TGFβ2 was increased to 236.9 ± 49.3% control levels. This induction was significantly inhibited by 17β-estradiol treatment. Patch assays cultured for 3 days in the presence of TGFβ showed significant matrix contraction relative to non-stimulated controls. This response was observed in the presence or absence of 10µM 17β-estradiol. Smad2/3 translocation to the nucleus was significantly increased following 2hr exposure to 10ng/ml TGFβ2; this distribution pattern was unaffected by the presence of 10µM 17β-estradiol.

Conclusions: : 17β-estradiol does not inhibit TGFβ induced matrix contraction, but can suppress TGFβ induced transdifferentiation and matrix production. 17β-estradiol can therefore target some of the clinical features of PCO, but not all.

Keywords: posterior capsular opacification (PCO) • growth factors/growth factor receptors • EMT (epithelial mesenchymal transition)