ABSTRACT: Background: Fibrosis, defined by the excessive accumulation of extracellular matrix (ECM) proteins, including type I collagen, results in a stiff scar tissue that impairs organ function and can cause death. The effector cells of fibrosis are myofibroblasts, an activated form of fibroblast. In gingiva (gums), fibrotic responses are predominantly hyperproliferative (hyperplasia), where transforming growth factor (TGF)-β is overexpressed, and via canonical signaling pathway acts on gingival fibroblasts to induce fibroproliferative genes such as cellular communications network factor 2 (CCN2). Targeting of TGFβ for therapeutic gain however is challenging due its pleotropic nature, and therefore identifying new and targetable alternatives is warranted. We have shown that mechanical tension as a result of stiff ECM increases TGFβ-driven expression of CCN2 in gingival fibroblasts. We further identified yes-activated protein (YAP1), a mechanosensitive transcription (co)factor that translocate to nucleus in response to mechanical tension in gingival fibroblasts. To test if YAP is the perpetuator of TGFβ-mediated fibroproliferative gene induction, I used celastrol, a recently identified YAP/TAZ (transcriptional co-activator PDZ-binding motif) inhibitor, whose anti-fibrotic potential is not yet elucidated -- projecting it as a potential treatment option for gingival hyperplasia. Methods: Human gingival fibroblasts (HGFs) in culture were serum starved overnight and pretreated with 500nM Celastrol or DMSO for 45 minutes followed by stimulation with or without TGFβ1 for 6 hours (RT-PCR) and 24 hours (Western blot). mRNA samples collected at 6 hours were also analysed using BulkRNAsequencing and functional cluster analysis. Celastrol’s capacity to prevent the TGFβ induced proliferation of HGFs was investigated with BrdU cell proliferation assay. Celastrol’s ability to block TGFβ-mediated YAP nuclear localization in HGFs was assessed using immunofluorescent staining of YAP protein. Results: In response to added TGFβ1, the gene and protein expression of fibrotic marker and mediator CCN2 (a known YAP1 target) was significantly downregulated in the presence of Celastrol. Similarly, of the 1364 genes induced by TGFβ1 (>2-fold), 657 genes showed a >50% decrease in the presence of Celastrol including genes involved in fibrotic responses such as type 4 collagen, interleukin 11, etc., Functional cluster analysis revealed ECM, Wnt/cytokine signaling, focal adhesion clusters induced by added TGFβ1, were downregulated in a Celastrol sensitive fashion. Also, basal level proliferation of HGFs was repressed in the presence of Celastrol. Additionally, Celastrol also successfully blocked TGFβ-mediated nuclear translocation of YAP as seen with immunofluorescence microscopy. Conclusion: These results are consistent with our hypothesis that targeting YAP/TAZ may be useful in blocking the profibrotic effects of TGFβ and that celastrol or other YAP inhibitors are eligible candidates as a treatment option for gingival hyperplasia.
