Abstract
Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G0/G1 cell cycle inhibition (IC50 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27Kip1 expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27Kip1 expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis.
Highlights
The type III intermediate filaments (IFs) are a family of cytoskeletal proteins that display dynamic and complex expression as both soluble and polymeric proteins [1,2,3,4]
withaferin A (WFA) targets soluble vimentin and inhibits cell cycle progression in rabbit Tenon’s capsule fibroblast (RbTCF) mediated by the skp2-p27kip1 pathway Vimentin staining has been employed as a marker for Tenon’s capsule fibroblasts [40], which is illustrated here in tissue sections showing the abundant expression of this cytoskeletal protein (Figure 1A)
We had identified that the cyclin-dependent kinase inhibitor p27kip1 is critical to WFA’s inhibition of cell proliferation [7]; we investigated the expression of p27kip1 in RbTCFs by western blot analysis (Figure 2B)
Summary
The type III intermediate filaments (IFs) are a family of cytoskeletal proteins that display dynamic and complex expression as both soluble and polymeric proteins [1,2,3,4]. WFA was shown to bind and downregulate the soluble forms of vimentin in human vascular endothelial cells, and interestingly, this finding led further to the illumination that the WFA-binding site in tetrameric vimentin was conserved evolutionarily from humans to sharks [7] This revelation drew our attention to WFA as a potent pharmacological agent and to it’s use as a chemical probe for interrogating biological signaling pathways that are linked to vimentin functions. Vimentin-deficient (Vim KO) mice were protected against corneal fibrosis in the alkali injury model lending evidence that vimentin is the druggable target [7] Together, these studies, along with several other findings made in other systems have illustrated vimentin involvement in epithelial mesenchymal transition [12,13] and point to a central pathological role for vimentin overexpression in fibrotic events
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
