O51
BMP-7 OPPOSES TGF BETA SIGNALLING VIA ALTERED DNA BINDING AND ID
EXPRESSION
Luo, D, Phillips, A O, Fraser, D J
Cardiff University
PROBLEM: Tubulointerstitial fibrosis is an important determinant of progression in chronic
kidney disease: Transforming Growth Factor Beta-1 (TGF beta) plays a central role in this
process. A number of factors serve to limit TGF beta signalling in the normal kidney. Bone
Morphogenetic Protein-7 (BMP-7) is a powerful inhibitor of TGF beta dependent fibrosis in
animal models of renal disease and has attracted substantial interest as a potential therapeutic
target. Surprisingly, there is little data on mechanisms by which BMP-7 opposes the effects of
TGF beta.
PURPOSE: To define the mechanisms by which BMP-7 ameliorates TGF beta dependent renal
fibrosis.
DESIGN: In vitro studies of the effects of BMP-7 on TGF beta signalling, and TGF beta
dependant changes in cell phenotype, using the human proximal tubular cell line HK-2.
FINDINGS: Downstream of the TGF beta receptor, signals are propagated by Smad2 and
Smad3, with TGF beta-induced fibrosis being Smad3 dependent. Transient transfection of
reporter gene constructs showed that BMP-7 inhibited Smad3 signalling response to TGF beta,
in contrast, Smad2 signalling was unchanged.
Smad signalling response to TGF beta is highly regulated at multiple points from receptorligand interaction to Smad DNA binding, and was assessed systematically. Immunoblotting
demonstrated that Smad3 phosphorylation and nuclear accumulation were unaffected by BMP7. Pulse-chase studies with TGF beta and the Alk5 Kinase inhibitor SB431542 showed that
Phospo-Smad3 half-life was unaffected by BMP-7. Chemical inhibition of MAP Kinase
pathways did not alter BMP-7 dependent effects, suggesting that Smad3 linker region
phosphorylation (which is p38/ERK mediated) was not relevant here. The known inhibitors of
Smad signalling Smad7, Ski and SnoN, were investigated with Immunoblotting, and small
interfering RNA knockdown, and were not involved in the effects of BMP-7.
Electrophoretic mobility shift assays, and subsequent supershift assays with specific antibodies,
showed that BMP-7 specifically inhibited DNA binding of Smad3/Smad4 complexes. Smad 5 is
a major signalling intermediary for BMP-7, and has been reported to bind to Smad3 consensus
sequences, but competitive binding of Smad5 downstream of BMP-7 was excluded by further
supershift experiments.
The Inhibitor of DNA Binding Proteins 1-3 (Id1-3) alter epithelial cell responses to TGF beta in
malignant contexts, leading to epithelial resistance to TGF beta-mediated growth arrest and
epithelial to mesenchymal transition. Quantitative RT-PCR demonstrated profound induction of
Id1-3 by BMP-7.
CONCLUSION: BMP-7 inhibits Smad3 signalling by inhibition of Smad3/4 complex DNA
binding, and by increased expression of Id proteins. Future work will address the relative
importance of Id proteins 1-3 in the effects of BMP-7.
RELEVANCE: Defining the mechanisms underlying modulation of TGF beta signalling by
BMP-7 may facilitate therapeutic use of BMP-7 or related agonists in renal disease, and may
also aid drug development, by enabling in vitro assessment of lead compounds.