GROWTH-FACTORS AND DEXAMETHASONE REGULATE HOXB5 PROTEIN IN CULTURED MURINE FETAL LUNGS

Studies on lung morphogenesis have indicated a role of homeobox (Hox) genes in the regulation of lung development. In the present study, we attempted to modulate the synthesis of Hoxb5 protein in cultured murin e fetal lungs after mechanical or chemical stimuli. Murine fetuses at gestational day 14 (GD14) were removed from pregnant CD-1 mice, and lu ngs were excised and cultured for 7 days in BGJb media. The experiment al groups were 1) untreated, unligated; 2) tracheal ligation; 3) suppl emented media with either epidermal growth factor (EGF; 10 ng/ml), tra nsforming growth factor (TGF)-beta 1 (2 ng/ml), dexamethasone (10 nM), EGF+TGF-beta 1, or EGF+TGF-beta 1+dexamethasone. After 3 or 7 days, t he cultured lungs were compared with in vivo lungs. Immunoblotting sig nals at 3 days in culture were stronger than those at 7 days. Western blot analyses showed that ligation, EGF, TGF-beta 1, and EGF+TGF-beta 1 downregulated Hoxb5 protein to similar to 20-70% of Hoxb5 protein le vels in unligated, untreated cultured lungs. Furthermore, dexamethason e alone or in combination with EGF and TGF-beta 1 downregulated Hoxb5 protein by >90% (P < 0.05) signal strength, similar to that seen in GD 19 or in neonatal lungs. Immunostaining showed that Hoxb5 protein was expressed strongly in the lung mesenchyme at early stages in gestation . However, by GD19 and in neonates, it was present only in specific ep ithelial cells. A persistent level of Hoxb5 protein in the mesenchyme after EGF or TGF-beta 1 treatments or tracheal ligation was noted. Hox b5 protein was significantly downregulated by EGF+TGF-beta 1, and it w as least in lungs after dexamethasone or EGF+TGF-beta 1-dexamethasone treatment. The decrease in Hoxb5 protein was significant only in the g roups with dexamethasone added to the media. Thus immunostaining resul ts parallel those of immunoblotting. The degree of Hoxb5 downregulatio n by dexamethasone or EGF+TGF-beta 1+ dexamethasone was similar to tha t seen in vivo in very late gestation, which correlated to the advanci ng structural development of the lung.