IN-VIVO HYPEROXIC EXPOSURE INCREASES CULTURED LUNG FIBROBLAST PROLIFERATION AND C-HA-RAS EXPRESSION

Exposure to hyperoxia has been demonstrated to alter the cell number of lung fibroblasts in vivo The precise mechanism of lung fibroblast proliferation after hyperoxic exposure has not been elucidated, however, We examined the growth characteristics of lung fibroblasts isolated from 21-day-old rats exposed to air or 100% O-2 for 8 days. Cell proliferation was assessed by hemocytometry, [H-3]thymidine incorporation, and fractional labeling with the thymidine analog bromodeoxyuridine. Under all conditions: tested, fibroblasts isolated from O-2-exposed rats grew more rapidly than those from air-exposed rats. Conditioned medium from fibroblasts isolated from hyperoxia-exposed rats failed to increase the [H-3]thymidine incorporation of control cells to that observed in cells isolated from hyperoxia-exposed animals, suggesting that an autocrine growth factor was not responsible for the excess proliferation. Sensitivity to exogenous growth factors was assessed by measuring the response to increasing concentrations of insulin-like growth factor-1 (IGF-1). Relative to 1% fetal bovine serum (FBS), concentrations of IGF-1 between 3 and 30 ng/ml significantly increased the [H-3]thymidine incorporation of fibroblasts derived from hyperoxic animals, whereas control cells were unresponsive to IGF-1 stimulation. The apparent sensitivity to IGF-1 led us to assess the effect of in vivo hyperoxic exposure on the expression of c-Ha-ras, which encodes a membrane-bound, GTP-binding/hydrolyzing protein essential for progression through G(1) in the cell cycle. ras mRNA levels in quiescent, control cells were minimal but increased following serum stimulation. The c-Ha-ras expression of lung fibroblasts from hyperoxia-exposed animals, on the other hand, was substantial in quiescent cells and remained high after serum exposure. When normalized to levels of 7S cytoplasmic RNA, growth-arrested fibroblasts from O-2-exposed rats displayed a 38% greater expression of c-Ha-ras message compared with fibroblasts from air-exposed rats (P = 0.0013). The level of c-Ha-ras mRNA correlated positively with DNA synthesis in response to 10% FBS (r = 0.70, P < 0.05). We conclude that in vivo hyperoxic exposure increases the proliferation and c-Ha-ras expression of cultured lung fibroblasts. The precise relationship between these findings remains to be elucidated.