Tight Junction Protein Expression and Junction Development During Differentiation of Normal Human Bronchial Epithelial Cells

Abstract

Normal human bronchial epithelial cells (NHBE) possessing a basal cell phenotype (p63+, KRT5/6a+) were differentiated on Transwell polyester membranes under air‐liquid interface (ALI) conditions. After 24 days of differentiation, NHBE cells had developed sufficient transepithelial resistance (TER) to perform voltage clamp experiments and expressed mRNAs associated with airway surface cells (Muc5b, FOXJ1). Additionally, mRNA transcripts for ENaC, CFTR and ATP1A1 all increased during differentiation and the corresponding proteins were appropriately locatized as confirmed by immunohistochemistry. Transcripts associated with epithelial adhesions (adherens and tight junctions) were also measured. Upregulation of Cldn3, Cldn8, and Cldn10 correlated with high TER, while ZO1/2, Jam1 did not exhibit differentiation‐dependent increases in mRNA expression. Examination of paracellular ionic permeability demonstrated sodium selectivity over chloride (PCl/PNa ∼ 83%). Furthermore, comparison of NHBE mRNA to an immortalized human airway cell line (HBEC) unable to develop TER revealed loss of claudin, ZO1, and E‐Cad transcripts as measured by qRT‐PCR. These results suggest that TER development in NHBE cells requires constitutive expression of ZO1, E‐Cad and differentiation induced increases in expression of Cldn3, Cldn8, and Cldn10.