Hic-5 drives epithelial mechanotransduction promoting a feed-forward cycle of bronchoconstriction.

Mechanical forces are essential for organ function, but excessive or dysregulated forces can promote pathologic conditions. In asthma, bronchoconstriction narrows the airway, compressing the airway epithelium and activating mechanotransduction, yet key regulators of mechanotransduction remain unclear. Here we show that Hic-5, a focal adhesion adaptor, is a key regulator of epithelial mechanotransduction. In human airway epithelial cells at air–liquid interface exposed to mechanical compression that mimics bronchoconstriction, we find that compression induces Hic-5 expression in airway basal cells. We further validated these in vitro findings by reanalyzing single-cell RNA-seq data from patients with asthma undergoing bronchoconstriction after allergen challenge, which revealed increased Hic-5 expression in airway basal cells. Hic-5 knockdown in human airway epithelial cells markedly attenuates mechanoresponses to compression, including stress fiber formation, differential gene expression, and increased secretion of endothelin-1 (ET-1). Through secretion of ET-1, a potent bronchoconstrictor, Hic-5 drives epithelial mechanotransduction and promotes a feed-forward cycle of bronchoconstriction, thereby highlighting dysregulated mechanical forces as active drivers of human disease. Overall design: To mimic bronchoconstriction, well-differentiated HBE cells were exposed to apical-to-basal pressure with a magnitude of 30cm H2O for 3 hrs, as previously described (11, 12, 49, 53). Time-matched control cells received 0cm H2O pressure. Cells and conditioned media were collected between 3 and 72 hrs. In experiments using recombinant human (rh) TGF-ß1 (Cell Signaling Technologies, Danvers, MA), we spiked 10 ng/ml into the basolateral media of HBE cells in ALI cultures. Cells were collected between 24 and 72 hrs for further analysis. To determine the signaling pathways that regulate mechanical compression-induced Hic-5 protein expression, we used pharmacological inhibitors of the TGF-ß receptor (SB431542; 10 µM, Tocris, Bristol, UK), MEK (U0126; 10 µM, Tocris), and EGFR (AG1478; 10 µM, Tocris). Each inhibitor was added to the basolateral media of HBE cells 1 hr prior to exposure to mechanical compression or TGF- ß. As a control, 0.1% DMSO was used.To knock down Hic-5 expression in HBE cells, we used antisense oligonucleotides designed and provided by Ionis Pharmaceuticals. We initially tested five ASOs targeting TGFB1I1(denoted as Hic-5 ASO). Then, we selected the most effective ASO and used for subsequent experiments. HBE cells maintained in ALI culture were treated with 10µM of either a non-targeting control (scrambled) ASO or Hic-5 ASO between ALI days 9 and 20 prior to the application of mechanical compression. We confirmed the knockdown efficiency of the Hic-5 ASO in well-differentiated HBE cells by RT-qPCR and western blot analysis.