The studies proposed in this application describe my continuing efforts to determine the function of cytochrome P450s being expressed in the human lung. It is now well established that cytochrome P450s perform a fundamental function in detoxifying numerous xenobiotics including airborne contaminants. In this application, we are proposing a novel and unique role for the human cytochrome P450 4B1 gene expression in regulating the developing lung. We have shown in our previous assays that the human 4B1 protein displays dramatic differences in its substrate preferences when compared to homologues in the other animals species. Thus, the human 4B1 protein has only a fraction of carcinogenic activating properties as compared to rodent counterparts. However, the human 4B1 enzyme has high 6beta-testosterone hydroxylase activity while the rabbit 4B1 enzyme lacks this activity. This androgen activity of the human enzyme could be of critical importance to the developing lung by reducing the deleterious effects of androgens on this organ. Our recent results show that dexamethasone and other steroid hormones which accelerates lung development, promotes the 4B1 gene expression. This effect could further reduce the detrimental effects of androgens on growth and cellular differentiation of the lung. The studies contained in this proposal will determine the effects of steroid hormones, particularly those hormones that play a fundamental role in the lung maturation process on the expression and function of the human 4B1 gene. The information obtained from our studies will elucidate the potential novel of 4B1 protein in controlling lung development.
The specific aims of the project are: (1) To determine the effect of dexamethasone and other steroid hormones on the expression of 4B1 mRNA in human lung cell model: (2) To determine the mechanisms responsible the for the stimulatory effect of dexamethasone and other steroid hormones on the expression of 4B1 mRNA; (3) To determine the effects of hormones on the expression and function of human 4B1 protein. We will use Northern blot analysis to determine the effects of hormones on the expression and function of human 4B1 protein. We will use Northern blot analysis to determine the effects of hormone on 4B1 mRNA levels, and we will employ nuclear run-off and pulse labeling assays to determine the effects of hormones on the rate of transcription of the 4B1 gene and on the rate of degradation of the 4B1 mRNA, respectively. In order to produce anti-4B1 antibodies which will be used in the immunochemical analysis to assess the effects of hormones on 4B1 protein levels and P450 activity mediated by the 4B1 protein, we have expressed the human 4B1 cDNA in prokaryotes. The 4B1 protein will be purified and will be used as immunogen in rabbits and mice. By determining the effects of steroid hormones which are known to control the maturation process of the lung, on the 4B1 gene expression, we believe our studies will further clarify the role of 4B1 protein in regulating the developing and normal lung function. The identification of endogenous factors which regulate the 4B1 gene expression could also be relevant to mechanisms responsible for chemically-induced damage of the lung.
