SP090
THE LYSINE METHYLTRANSFERASE SETD7 IS CRITICAL FOR TGF B-1 DRIVEN FIBROSIS

Introduction and Aims: Numerous studies have demonstrated the importance of TGF B-1 signalling in the development of fibrosis, the histopathological hallmark of Chronic Kidney Disease (CKD). Additionally, deposition of this Collagen rich fibrotic tissue is inhibited in mice that are deficient in SMAD3, the principle downstream transcription factor responsible for mediating TGF B-1 dependent changes in gene expression. There is a relative paucity of studies that have examined the precise mechanism of SMAD3 activation.

In our search for SMAD3-interacting proteins that could enhance transcriptional activity, and that might represent therapeutic targets in the treatment of CKD, we identified the lysine methyltransferase enzyme SETD7.

Methods: Immunoprecipitation was performed using whole cell line extracts from promixal tubular epithelial cells (PTECs) using either SETD7 or SMAD3 antisera. SMAD3 stability was determined by immunoblotting after addition of cycloheximide onto either SETD7 deficient cells or PTECs depleted of SETD7 by siRNA. SMAD3 subcellular localisation was confirmed by immunofluorescence or detergent mediated nuclear / cytoplasmic separation followed by immunoblotting, in SETD7 deficient cells or PTECs depleted of SETD7 by siRNA. Luciferase reporter gene assays were performed according to standard methodology in PTECs by transient co-transfection of SETD7 or a dominant negative mutant with either the CAGA or PAI reporter genes prior to treatment with TGF B-1. To examine markers of fibrosis, SETD7 deficient cells cultured on glass cover slips were subject to immunofluorescence with antisera towards Collagen I, III and Fibronectin-1.

Results: We demonstrate the following:

1. SMAD3 interacts with SETD7 in response to TGF B-1, by immunoprecipitation, resulting in enhanced SMAD3 stability in cells.

2. TGF B-1 driven SMAD3 nuclear import is dependent upon SETD7, as determined by immunofluorescence and subcellular fractionation.

3. SETD7 overexpression enhances TGF B-1 stimulated SMAD3 transcriptional activity on natural (PAI-1) and synthetic (CAGA) cis regulatory elements, as demonstrated in reporter gene assays. Additionally SETD7 depletion results in a blunted TGF B-1 response.

4. SETD7 deficient cells fail to undergo myofibroblast transformation in response to TGF B-1 and these cells express lower levels of key fibrotic proteins including Collagens and Fibronectin-1.

Conclusions: We conclude that SETD7 is critical in the activation of SMAD3 in response to TGF B-1. As such, we propose that SETD7 is required for the development of TGF B-1 driven renal fibrosis (and other fibrotic disorders driven by TGF B-1) opening a new direction for targeted therapy in Chronic Kidney Disease.