Dulin, N. O. | bioRxiv Science

bioRxiv2023

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-{beta}) is one of the most established drivers of fibrotic processes. TGF-{beta} promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-{beta} robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-{beta}-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-{beta} signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-{beta} treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-{beta}-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-{beta}-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-{beta}; and (ii) ANO1 mediates TGF-{beta}-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity. NEW & NOTEWORTHYThis study describes a novel mechanism of differentiation of human lung fibroblasts (HLF) to myofibroblasts - the key process in the pathogenesis of pulmonary fibrosis. TGF-{beta} drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive With-No-Lysine (K) kinase (WNK1) to activate pro-fibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

cell biology↗

bioRxiv2022

Identification of a cardiac glycoside exhibiting favorable brain bioavailability and potency for reducing levels of the cellular prion protein

Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4-dehydro-oleandrin to engage with its cognate binding pocket within the NKA subunit. The improved brain bioavailability of C4-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases. AUTHOR SUMMARYPrion diseases are fatal neurodegenerative diseases for which there is no effective treatment. An abundance of data indicates that reducing the levels of a specific protein, termed the cellular prion protein (PrPC), would not only be safe but would delay disease onset and extend prion disease survival. This project builds on our recent discovery that PrPC binds to NKAs, specific cellular transport proteins that use energy to electrify cellular membranes by pumping charged potassium and sodium metals in and out of cells. We showed that targeting NKAs with their natural inhibitors, cardiac glycosides (CGs), causes brain cells to internalize and degrade NKAs, and that PrPC, on account of residing next to NKAs, gets co-degraded. Natural CGs act primarily on the heart. Here, we used computational modeling to identify a CG, termed KDC203, that is predicted to have favorable characteristics for brain applications. We show that KDC203 reduces PrPC levels by 84% in immortalized human brain-like cells grown in the dish. Moreover, we show that KDC203 exhibits relatively low toxicity, predominantly targets the brain when subcutaneously injected into mice, and has other promising pharmacological characteristics that recommend it for further evaluation for the treatment of prion diseases.

neuroscience↗