TGF(31 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGF(3. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGF(31-induced EDA + fibronectin and to prevent the cascade of events initiated by TGF(31 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGF(31 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGF(3, aSMA (a smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGF(31 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGF(31 induced endogenous TGF(3, aSMA, MMP2, MMP9 and Col I mRNA. TGF(31 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGF(31-induced profibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGF(3, further increases in aSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGF(31 was confirmed by the use of a TGF(3 receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGF(3 driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.
