Abstract Introduction/Rationale During asthma exacerbations, bronchial epithelial cells experience compressive mechanical stress, triggering mechanotransduction linked to asthma pathogenesis. In our previous study, we identified Hic-5, a focal adhesion scaffold protein, as a key regulator of compression-induced mechanotransduction that drives mechanoresponsive transcription and cytoskeletal organization in bronchial epithelial cells. Here, we tested the hypothesis that Hic-5-dependent cytoskeletal organization is required for compression-induced release of extracellular vesicles (EVs) from airway epithelial cells. Methods We used primary human bronchial epithelial cells (HBECs) differentiated at air-liquid interface for 21 days (n = 3 HBEC donors). To mimic bronchoconstriction-induced mechanical stress, we applied mechanical compression (30 cm H2O, 3 hours) and collected cells and basolateral media at the indicated time points. To test the role of Hic-5, we used antisense oligonucleotide-mediated Hic-5 knockdown (KD). We first assessed the effect of Hic-5 KD on EV release by quantifying the number of EV particles using Nano flow cytometry. We then isolated EVs from the whole basolateral medium by ultracentrifugation and detected secreted proteins by Western blot, including CD9 and ARRDC1 (canonical EV markers), tissue factor (compression-induced EV protein), and YKL-40 (compression-induced non-EV protein). Results Mechanical compression induced both Hic-5 expression and EV release from HBECs (Fig. 1). In wild-type (WT) cells, compression increased expression of Hic-5, which was predominantly localized in the cytosol of basal cells. Compression also significantly increased the number of released EV particles by 4.6-fold at 3 hours (p 0.05) and 7.8-fold at 24 hours (p 0.0005) vs. each time-matched control. We also qualitatively confirmed compression-induced EV release by detection of canonical EV markers, CD9 and ARRDC1. We also detected secretion of two asthma-relevant, compression-induced proteins: tissue factor in the EV fraction and YKL-40 in the non-EV fraction. However, in Hic-5 KD cells, compression-induced EV release was markedly attenuated to near the baseline level as determined by EV particle counts and no detection of EV proteins, including EV markers and tissue factor, indicating Hic-5-dependent EV release. By contrast, in Hic-5 KD cells, YKL-40 remained detectable, but the compression-induced increase was abolished, indicating Hic-5-independent secretion and possible Hic-5-dependent expression. Importantly, in Hic-5 KD cells, compression-induced cytoskeletal organization was abolished. Conclusions Our data demonstrate that Hic-5 is required for compression-induced EV release but not for EV-independent protein secretion from HBECs. This study identifies Hic-5 as a key mechanoregulator that links mechanical compression to EV release, potentially through the coordination of cytoskeletal rearrangement. This abstract is funded by: NIEHS: P30ES000002; NHLBI: P01HL120839, R01HL148152, T32HL007118; Chemical Insights Research Institute Grant
Kim et al. (Fri,) studied this question.