Lysozyme expression during metaplastic squamous differentiation of retinoic acid-deficient human tracheobronchial epithelial cells

We previously reported (Gray, T. E., K. Guzman, C. W. Davis, L. H. Abdullah , and P. Nettesheim. 1996. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. B iol. 14:104-112) that retinoic acid (RA)-deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells became squamous, failed to produc e mucin, and instead secreted or released large amounts of lysozyme (LZ). T he purpose of the studies reported here was to elucidate the relationship b etween RA deficiency-induced squamous differentiation and increased LZ, and to determine what mechanisms were involved. We found that intracellular LZ began to accumulate in RA-deficient NHTBE cultures early during squamous d ifferentiation. Between Days 10 and 18 of culture, cellular LZ levels were more than 10 times higher in RA-deficient than in RA-sufficient cultures. O n Day 12, large numbers of cells began to exfoliate in RA-deficient culture s and extracellular LZ appeared at the apical surface, presumably released from the exfoliated cells. Metabolic labeling studies showed that the rate of LZ synthesis was not increased in RA-deficient cultures over that in RA- sufficient cultures; however, intracellular LZ half-life was much longer in RA-deficient cultures. We concluded that the increased accumulation of bot h intra- and extracellular LZ in RA-deficient cultures was due to increased LZ stability and was not the result of increased LZ synthesis. When RA-def icient cultures were treated on Day 7 with 10(-6) M RA, intracellular LZ le vels did not substantially decrease until 3 d later, coinciding with a mark ed increase in mucin secretion. LZ messenger RNA levels were unchanged at 2 4 h, but were modestly increased (rather than decreased) at all subsequent time points. We concluded that RA does not directly regulate LZ, and that t he excessive accumulation of LZ in RA-deprived NHTBE cells is a consequence of vitamin A deficiency-induced abnormal differentiation.