RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis

A core pathophysiology underlying many respiratory diseases is multiciliated cell dysfunction leading to inadequate mucociliary clearance. Due to the prevalence and high variability of etiologies of MCC dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelium, however this does not apply to all cell types nor occur evenly across all cells in the same cell population. In this study we investigated we further investigate how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells we identify RBL2 as an inhibitor of the transcriptional activity of Multicilin. Both knockdown of RBL2 and phosphorylation of RBL2 driven by exposure to an air-liquid interface in the presence of Multicilin activated multiciliogenesis demonstrating a dynamic interaction regulating the differentiation of human airway epithelial cells. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases where mucociliary clearance is impeded. Primary MEFs were plated with 10% FBS-DMEM for overnight then treated with 2% FBS-DMEM. Six hours after serum-reduced treatment, siRNAs were transfected into MEFs using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions. Eighteen hours later, the cells were washed with 2% FBS-DMEM then infected with pAd/CMV/V5 vector expressing pAd/CMV/V5 vector encoding 3xFLAG-Multicilin, NLS-6xmyc-E2f4ΔTA-VP16-T2A-3xFLAG-Multicilin, and NLS-6xmyc-E2f4WT-T2A-3x-FLAG-Multicilin. Adenovirus infection was performed by adding the adenovirus crude lysates to the MEF cells for four hours, washing with pre-warmed PBS once, then growing infected cells with 2% FBS-DMEM. The cells were subjected to RNA extraction 2 days after infection.