Abstract: TH-PO392

TGF-Beta Exposure Represses Fibroblast Transcription of the Anti-Fibrotic Molecule Slit2: A Novel Fibrogenic Mechanism?

Session Information

Category: Cell Biology

  • 203 Growth Factors, Chemokines, Autacoids

Authors

  • Tolosa, Monica F, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada
  • Yuen, Darren A., Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada
Background

Recent work has demonstrated that the molecular guidance ligand Slit2 may also serve as an important anti-fibrotic signal in the kidney. Specifically, we have previously shown that Slit2 is reduced following renal injury, and bioactive N-terminal Slit2 attenuates fibrosis following both ischemia-reperfusion injury and unilateral ureteral obstruction (UUO). While this provides early evidence that Slit2 could be a possible adjunct therapy for chronic kidney disease, the regulation of endogenous Slit2 during fibrosis—as well as its role in downstream injury—remains largely uncharacterized. Objective: To study the endogenous regulation of Slit2 expression by fibroblasts in fibrotic disease, and further characterize the role of Slit2 on fibroblast activation and renal injury.

Methods

Using real-time PCR and immunofluorescence, we measured Slit2 expression in primary human dermal fibroblasts in response to TGF-beta. In vitro tools, including siRNA-mediated knockdown and pharmacological inhibition, were used to further identify and characterize novel regulators of Slit2 expression.

Results

Here, we demonstrate that fibroblast exposure to TGF-beta, a master regulator of fibrosis, causes a downregulation of the anti-fibrotic factor, Slit2. Furthermore, we determined that TGF-beta mediated repression of Slit2 occurred at the level of transcription, by observing a corresponding downregulation of Slit2 pre-mRNA transcript levels. In silico analysis showed putative binding sites for TGF-beta-regulated transcription factors in the Slit2 promoter region, suggesting that repression may occur through proximal cis-acting elements. Finally, we determined that Slit2 repression occurs through a Smad- and YAP-dependent mechanism, as loss of each of these transcriptional regulators by siRNA reversed the repressive effect of TGF-beta.

Conclusion

While many forms of chronic kidney disease are characterized by fibrosis, few strategies exist to effectively suspend fibrotic progression. Here, we determined that fibroblast exposure to TGF-beta leads to a marked decrease in Slit2 expression, suggesting that loss of Slit2 may facilitate fibroblast activation in a disease context. Further studies will help to clarify the mechanism of Slit2 repression and how this may be targeted to potentially reduce fibrotic injury.

Funding

  • Government Support - Non-U.S.