Abstract 1875 Poster Session IV, Tuesday, 5/4 (poster 309)

In the lung, retinoids (vitamin A and related molecules) participate in diverse biological processes, including pattern formation, cellular differentiation, growth and maintenance. Recently, retinoic acid (RA) has been identified as a potential regulator of both branching morphogenesis and alveolarization. To determine potential downstream targets of RA signaling during lung development, we utilized differential display (DD) to identify genes whose RNA expression is altered by RA in a murine lung epithelial cell line (MLE-12), which represents distal bronchiolar and alveolar epithelial cells. Duplicate flasks of MLE-12 cells were cultured either with 10-6 M all-trans RA (final concentration) in DMSO or with DMSO alone (final concentration 0.03%) for 48 hours and total RNA was isolated. Northern analysis showed that RA-treatment of MLE-12 cells resulted in a 3-4 fold induction of retinoic acid receptor-beta, thereby demonstrating that MLE-12 cells are RA responsive. Differential display analysis was performed with the RNAimage System (GenHunter, Nashville, TN) using duplicate samples of RNA from RA-treated and untreated MLE-12 cells, a single oligo dT primer (H-T11G) and seven arbitrary primers (AP-1-AP-7). In this analysis, nine bands were identified that displayed a consistent pattern of either up-regulation or down-regulation of a gene in response to RA. One hand, hereafter designated 8-1/1A, was significantly up-regulated in response to RA. Northern analysis using RNA from RA-treated and untreated MLE-12 cells demonstrated a 7-fold increase in 8-1/1A transcript abundance in response to RA treatment. Initial sequence analysis of 9-1/1A yielded 194 nucleotide (nt) sequence that did not produce a match in database searches. 5′ RACE extended this cDNA by 950 nt. The sequence of the 5′ RACE product, which now represents approximately 15% of the transcript, shows no homology to existing databases and thus may represent a novel RA-induced gene. Northern analysis and in situ hybridization have been utilized to study the temporal and spatial expression of this DD product. Preliminary data show that this cDNA is not expressed in the postnatal lung during the period of septation but appears to be expressed in the early developing lung, during the pseudoglandular period.

(Supported by HL58753)