Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RAR alpha (CD336), RAR beta (CD2019), RAR gamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RAR beta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARa and beta agonists or RAR panagonist, but not the RAR gamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RAR alpha/beta and RXR agonists. The RAR gamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, The RAR beta gamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G(1)-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RAR alpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RAR gamma ligand appears to be inefficient in metaplasia inhibition, but the selective RAR gamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.