Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C025

Oral Communications

Activator protein 1 complex controls Claudin 1 expression in response to air-liquid-interface conditions in NCI-H441 epithelia

R. Lochbaum1, C. Schilpp1, M. Frick1, P. Dietl1, O. Wittekindt1

1. Institute of General Physiology, Ulm University, Ulm, Germany.

Tight junctions (TJ) are crucial for the barrier function of lung epithelia. They are essential for establishing and maintaining air-liquid-interface (ALI) conditions in the airways and alveoli and hence they are prerequisite for an appropriate lung function. Breakdown of TJ and ALI results in respiratory distress syndrom. Most important for the specific function of TJ are proteins called claudins, the activator protein 1 complex (AP-1), a dimer consisting of c-Jun and c-Fos, is shown to modulate different claudins in tissues other than lung. However, the role of AP-1 for regulating claudin expression in human airway epithelium in response to ALI is sparsely elaborated. To elaborate the changes in functional properties of TJ while ALI conditions get established we investigated NCI-H441 cells as a model of the distal lung epithelium cultivated either at ALI or liquid-liquid interface (LLI) conditions. Paracellular permeability was quantified by measuring transepithelial electrical resistance (TEER). Semi-quantitative RT-PCR, western blot- and immunofluorescence experiments were performed to identify differentially regulated TJ-proteins and the changes in AP-1 expression. Promoter regions were analyzed in-silico for putative binding sites of AP-1 and AP-1 activation of cldn1 promoter was verified using luciferase assays. Formation of AP-1 was detected by proximity ligation assays. To uncover AP-1 controlling of claudin expression, c-Fos was overexpressed in cells using recombinant adenoviruses. In comparison to ALI cultivated NCI-H441 epithelia, LLI conditions resulted in TEER reduction by approximately 40%. This agreed with the observed downregulation of Claudin 1 (Cldn1), which is known as a claudin that tightens TJ. Based on the in silico analysis of Cldn1 promoter region we identified putative binding sites for c-Fos and c-Jun. While there is an upregulation of AP-1 components in LLI versus ALI cultivated epithelia on mRNA level, we observed reduced protein levels for c-Fos and c-Jun and reduced AP-1 formation in LLI cultivated cells. Inhibition of AP-1 with SR11302 reduced Cldn1 promoter activity in luciferase assay experiments. Adenoviral overexpression of c-Fos in ALI and LLI cultivated NCI-H441 epithelia resulted in upregulation of Cldn1 expression levels. Our results underscore the role of AP-1 as an activating factor for Cldn1 expression in NCI-H441 cells. It modulates Cldn1 expression in response to ALI conditions and therefore it adjusts functional properties of TJ to maintain ALI conditions. The herein identified mechanisms may contribute to disturbance of epithelial barrier function during lung edema and may facilitate respiratory distress. Supported by: Ministry of Science, Research and the Arts of Baden-Württemberg (Az: 32-7533-6-10/15/5) to O. H. Wittekindt.

Where applicable, experiments conform with Society ethical requirements