Background/Purpose: Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by excessive collagen production, leading to the thickening and hardening of skin and internal organs. Although its exact cause remains unknown and no cure exists, current treatments only alleviate symptoms without addressing the underlying disease mechanisms. Our research investigates the Hippo pathway, a key regulator of cell proliferation, apoptosis, and stemness. Dysregulation in this pathway within SSc results in increased YAP/TAZ transcriptional activity in fibroblasts via transcription factor binding, promoting fibrosis. Our previous work identified that the YAP inhibitor Verteporfin can reduce fibrosis. By targeting specific transcription factors and coactivators in the Hippo pathway, specifically TEADs and VGLL3, we aim to clarify its role in SSc fibrosis and develop more targeted therapies.

Methods: We examined TEAD1, TEAD2, TEAD3, TEAD4, and VGLL3 in SSc fibroblasts to assess their roles in fibrosis. Using Accell siRNA, we knocked down TEADs and VGLL3 individually in dermal fibroblasts from SSc patients. We evaluated changes in gene expression and cell functions including proliferation, migration, and contractility. Knockdown cells underwent single-cell RNA-sequencing. To induce pro-fibrotic phenotypes, healthy dermal fibroblasts were stimulated with TGF-β (10 ng/ml) for 1 or 72 hours. Statistical analysis was performed using GraphPad Prism v.10, with significance set at p< 0.05.

Results: Knocking down TEADs and VGLL3 achieved over 50% gene downregulation in SSc dermal fibroblasts. Knockdown of TEAD1, TEAD3, or VGLL3 consistently reduced pro-fibrotic gene expression, whereas TEAD2 and TEAD4 did not. Protein-level confirmation by immunofluorescence showed significant decreases in alpha-smooth muscle actin, a key marker of fibroblast activation and myofibroblast differentiation, following TEAD1, TEAD3, or VGLL3 knockdown in SSc fibroblasts. VGLL3 downregulation consistently reduced cell proliferation and migration, while TEAD1 and TEAD3 downregulation impaired gel contraction. Single-cell RNA sequencing identified genes and pathways impacted by TEADs or VGLL3 knockdown. Interestingly, long-term TGF-β stimulation in healthy fibroblasts, which resulted in myofibroblast transformation in these cells, led to downregulation of TEAD1, 2, 3 and VGLL3. In contrast, transient activation of TGF-β, which resulted in increased p-SMAD2 in the nucleus, led to enhanced nuclear staining of TEAD1 or TEAD3, compared to non-treated fibroblasts.

Conclusion: TEAD1, TEAD3, and VGLL3 play crucial roles in mediating the pro-fibrotic effects of the Hippo pathway in SSc fibroblasts. These findings align with our prior research suggesting that disrupting YAP or VGLL3 binding to TEADs in the Hippo pathway could serve as a viable therapeutic strategy. Continued research will focus on the genes and pathways influenced by these transcription factors and coactivator, potentially benefiting treatment approaches for other fibrotic diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/unraveling-the-role-of-the-hippo-pathway-in-systemic-sclerosis-a-focus-on-teads-and-vgll3/