Upregulation of IGSF3 expression by iron chelation leads to the inhibition of epithelial cell proliferation

Abstract

Background/Significance We recently demonstrated that the Immunoglobulin Superfamily-3 (IGSF3), a transmembrane tetraspanin interacting protein, is required for scratch wound repair, cell migration, and barrier function, suggesting a potential key role in lung injury repair. However, the role of IGSF3 in epithelial cell proliferation remain unknown. Furthermore, it is unclear how IGSF3 expression is affected in acute respiratory distress syndrome (ARDS), characterized by hypoxia, loss of lung epithelial barrier, and insufficient cell repair. We hypothesized that hypoxia alters IGSF3 expression and that IGSF3 is mechanistically involved in the effects of hypoxia on human bronchial epithelial cell proliferation. Methods To mimic hypoxia, we exposed human bronchial epithelial cells (Beas2B) submerged in cell culture media to iron chelator deferoxamine (DFO, 100uM, 16-24hr). Proliferation was measured using CCK8 assay. We stably knocked down IGSF3 in Beasw2B using CRISPR-CAS9. Tetraspanin enriched microdomains (TEMs) were isolated using density gradient ultracentrifugation. Single cell RNA-seq (scRNA-seq) was performed on deidentified human ARDS (n=1) and non-diseased (n=3) lungs. Results DFO caused a significant decrease in epithelial cell proliferation (by~ 25%, p<0.001) concomitant to a markedly increased IGSF3 gene and protein expression (by 2- and 2.5-fold, respectively, p<0.01) that was prevented by treatment with the HIF-1a inhibitor, KCF72 (p<0.01). IGSF3-deficient Beas2B exhibited increased cell proliferation at baseline (by~20-50% vs control cells, 24hr; p<0.01) and following treatment with DFO (by ~ 200% vs DFO-treated control cells; 24hr, p<0.01). Compared to non-diseased lungs, scRNA-seq of ARDS lungs revealed marked enrichment of IGSF3 in basal epithelial cell. Interestingly, a similar pattern was observed in Tspan5, an IGSF3 binding tetraspanin induced by iron chelation and in CD147, a hypoxia-inducible Immuhnoglobulin Superfamily protein (Basigin, EMMPRIN). We confirmed that IGSF3 co-localized with Tspan5 within TEMs in Beas2B and, interestingly found that both proteins co-localized with CD147. Conclusions IGSF3 may be induced by hypoxia in lung epithelial cells such as basal cells where it may inhibit cell proliferation and therefore repair. Interestingly, loss of IGSF3 has also a detrimental effect on processes involved in repair such as cell migration and barrier function. This suggests that IGSF3 homeostasis is important in epithelial cell function, possibly through interactions with membrane complexes such as tetraspanins and other transmembrane proteins such as the matrix-regulating CD147.