Akt Activity is a Critical Regulator of Airway Epithelial Cell Fate Decisions

Lalit Gautam; Erik Quiroz; Brett Wineinger; Jason Babcock; Beck Fitzpatrick; Amy Ryan

doi:10.1152/physiol.2026.41.S1.2298191

Abstract only Mucociliary clearance is a fundamental defense mechanism of the airway epithelium, and its disruption underlies chronic airway disease pathogenesis. Multicilin, encoded by MCIDAS, functions as a transcriptional co-activator, forming the EDM complex with E2F4/5 and DP1 to activate genes required for multiciliated cell differentiation. However, MCIDAS activation alone is insufficient to induce multiciliogenesis in all cells, and upstream mechanisms regulating its function remain poorly understood. We hypothesized that Notch inhibition creates a permissive cellular state enabling MCIDAS to drive multiciliogenesis. To test this, primary human airway basal cells (BCs) were infected with a doxycycline-inducible MCIDAS lentivirus. Using a submerged culture system to examine early multiciliogenesis, we pharmacologically inhibited Notch and induced MCIDAS, comparing four conditions via bulk RNA sequencing: (1) MCIDAS induction alone, (2) Notch inhibition alone, (3) combined MCIDAS induction and Notch inhibition, and (4) vehicle control. Transcriptomic profiling revealed that canonical cilia-related genes, including FOXJ1, CCDC20B, MYB, and DRC1, were upregulated expressed upon combined MCIDAS induction and Notch inhibition. MCIDAS activation specifically altered pathways related to DNA replication, cilia assembly and organization, and cell-cycle regulation. Pathway enrichment analysis identified PI3K–Akt signaling as one of the most negatively enriched pathways jointly influenced by Notch and MCIDAS. Biochemical analysis showed that Notch inhibition reduced phospho-Akt-T308 and S473, while T450 increased with concomitant increased in total Akt protein, a pattern recapitulated within the first two days of air–liquid interface (ALI) differentiation. Functional studies using the Akt inhibitor MK2206 (1 µM) revealed that Akt inhibition in submerged culture could substitute for Notch inhibition, enhancing early ciliogenesis markers (Foxj1, Cep63, DNAAF7) and P73. In ALI culture, where Notch is intrinsically low, Akt inhibition promoted club cell differentiation, but reversibly blocked progression to MCCs, whereas Akt activation, via increased p-Akt-S473, allowed for early multiciliated cell lineage commitment and continued differentiation intermediary club cells. Confocal imaging and gene expression analyses confirmed that Akt modulation shapes mucociliary differentiation trajectories. Our data support a two-phase Akt regulatory mechanism in multiciliogenesis. Early Notch inhibition suppresses PDK1-mediated p-Akt-T308, creating a transient low-Akt state that permits MCIDAS to activate the transcriptional program driving centriole amplification and multiciliogenesis. Subsequent Akt activation via increased p-Akt-S473, potentially downstream of DNA-PK, is essential for progression from the club-cell state to fully differentiated ciliated and goblet cells. Indeed, DNA-PK inhibition with NU7441 (1 µM) mirrored Akt inhibition, reducing ciliated cell differentiation and SC79, a PH-domain–binding Akt activator, restored Akt phosphorylation and activity, demonstrating mechanistic specificity. In conclusion, early p-Akt-T308 inhibition establishes a permissive state for MCIDAS-driven multiciliogenesis, whereas prolonged Akt inhibition blocks differentiation, arresting cells at the club-cell stage. These findings reveal a novel mechanistic framework linking Notch, Akt, and MCIDAS in airway epithelial differentiation. This research was funded by NIH-NHLBI (5R01HL139828), Cystic Fibrosis Foundation (RYAN21XX0), both awarded to ALR and (GAUTAM25F0) awarded to LKG This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Akt Activity is a Critical Regulator of Airway Epithelial Cell Fate Decisions

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