Abstract: FR-PO368

The Lysine Methyltransferase SETDB1 Inhibits Renal Myofibroblast Differentiation

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

  • Shuttleworth, Victoria Gemma, Newcastle University, UK, Newcastle Upon Tyne, United Kingdom
  • Sheerin, Neil S., Newcastle University, Newcastle upon Tyne, United Kingdom
  • Logan, Ian, newcastle hospitals, Newcastle----, United Kingdom
Background

Renal fibrosis is characterised by accumulation of myofibroblasts expressing alpha-SMA. These cells deposit extracellular matrix that replaces normal kidney tissue, leading to chronic kidney disease and organ failure.
TGF β-1 is critical to myofibroblast transdifferentiation, signalling through transmembrane receptors, resulting in SMAD3 phosphorylation. Phosphorylated SMAD3 undergoes nuclear translocation to regulate transcription of transdifferentiation genes, such as alpha-SMA. Regulatory mechanisms governing these events are unknown, but SMAD3 may utilise co-regulators from other pathways. For this reason, we screened for methyltransferases involved in TGF β-1 signaling.
We identify SETDB1 as a new repressor of SMAD3 and TGF β-1 induced transdifferentiation.

Methods

siRNA Screen. siRNAs targeting 48 human methyltransferases were transfected into cells harboring a TGF β-1-responsive pCAGA12-luc reporter gene. These were treated with TGF β-1, prior to reporter gene assay.
Immunoprecipitation. HKC-8 cells were starved in serum-free medium and treated with TGF β-1. SETDB1 immunoprecipitation was performed on nuclear fractions.
Immunofluorescence. HKC-8 cells starved in serum-free medium were treated with TGF β-1 prior to immunofluorescence with SMAD3 and SETDB1 antibodies. Human primary renal fibroblasts were transfected with either control or SETDB1 siRNA then treated with TGF β-1 and subjected to immunofluorescence for alpha-SMA, or light microscopy to evaluate morphology.

Results

1 siRNA screening identified SETDB1 as a strong corepressor of SMAD3, even in the presence of TGF β-1
2 Immunoprecipitation showed an interaction between SETDB1 and SMAD3 in nuclear fractions. No interaction was observed without TGF β-1 or control immunoglobulins
3 TGF β-1 promoted both SMAD3 and SETDB1 nuclear translocation, demonstrating co-localisation
4 Primary renal fibroblasts transfected with SETDB1 siRNA exhibited markedly more alpha-SMA expression and myofibroblastic changes in morphology, compared to control cells

Conclusion

1 SETDB1 is a new SMAD3 co-repressor
2 TGF β-1 treatment promotes a specific interaction between with SMAD3 and SETDB1, in the nucleus
3 SETDB1 inhibits myofibroblast differentiation in human renal fibroblasts

The conclusions are explained by SETDB1 acting as a brake on TGF β-1, preventing myofibroblast transdifferentiation, which may prevent progressive CKD.