Androgens interact with certain growth factors to influence the proliferation and differentiation of the prostate. A primary step in the mechanism of action of some growth factors is the expression of early growth response genes which include fos, cjun, Egr-1, Egr-2 and Egr-3. The Egr-1 gene is induced after mitogenic stimulation of diverse cell types, and in turn Egr-1 regulates gene transcription suggesting a potential role in the process of mitogenic stimulation. Our laboratory is interested in determining the mechanism of action of androgens in the prostate and their interaction with growth factors. We have utilized the differential display PCR technique to isolate and characterize a novel androgen-responsive gene whose cDNA encodes a protein of 469 amino acids. It is an early growth response gene (named Egralpha) and belongs to the family of Egr-1 genes. Egralpha mRNA is negatively regulated by inhibitory levels of androgens and positively regulated by EGF and the phorbol ester, TPA. The hypothesis that we plan to test is that androgens influence prostate cell proliferation in conjunction with growth factors which in turn affect the prostate through the early growth response genes. We will investigate the regulation of the novel Egralpha and Egr-1 genes.
In specific aim I, we will determine the regulation of these genes by androgens, anti- androgens, EGF, KGF, TPA and forskolin. Northern blots will demonstrate the expression of the genes, and nuclear run-on experiments will indicate their transcriptional regulation. Experiments measuring the half-life of mRNA will demonstrate whether the stability of mRNA is affected by the treatments. Western blot analysis will determine the effect of treatment on protein expression.
In specific aim II, we will isolate and analyze the 5'-flanking region of the Egr alpha and Egr-1 genes to identify key regulatory elements such as androgen response elements. We will utilize PCR-based techniques to construct deletion and point mutations in the 5'- flanking region. The wild type and mutant constructs will be used in transient transfections assays to determine the presence of functional response elements. Footprinting and gel retardation assays will be used to investigate the protein-DNA interactions which are important for regulating transcriptional activity. These experiments should help elucidate the mechanisms through which androgens and growth factors might regulate the growth of prostate cells through early growth response genes.
