Knowledge of how hormones work and how genes are regulated at the molecular level is accumulating rapidly. As more is learned, however, it has become apparent that hormonal regulatory mechanisms are even more intricate and complex than originally imagined. This often makes it difficult to reconcile isolated molecular events with the hormone responses that occur in an intact organism. Understanding these processes in the biological context of human physiology is the challenge presented by the needs of the medical community. Therefore, the overall goal of this project is to show at the molecular level how a hormonal signal activates a specific gene in a specific tissue of an organism that serves as a model for human biology. This study focuses on the androgen stimulation of b-glucuronidase (GUS) gene expression in kidney proximal tubules of the mouse. This experimental model system is exceptional for several reasons: (1) The response of GUS to androgen is dramatic, as much as 100-fold. (2) The GUS response to androgen is highly tissue specific and does not occur in androgen target organs other than kidney. (3) GUS induction is progressive and remarkably slow for a steroid hormone response. This implies that the mechanism underlying the response has novel features and will provide new insights into how gene regulation is managed. (4) The GUS response to androgen is enhanced by growth hormone, providing the opportunity to dissect the mechanism of a multi-hormonal response. (5) GUS can be effectively studied in vivo using genetically variant or mutant mice and transgenic mice. (6) The GUS response to androgen can be studied in primary cultures of kidney epithelial cells. This is one of very few systems in which the in vivo response to androgen can be mimicked in vitro. (7) A number of genetic variants that specifically affect the hormonal regulation of GUS have been identified and extensively characterized. (8) There is a substantial foundation of physiological, biochemical, molecular and genetic information about GUS that makes it particularly amenable to experimental manipulation.
The specific aims of this proposal are to (A) determine if apparent androgen response elements, identified by sequence analysis and receptor binding studies, play an essential role in the response of GUS to androgen; (B) locate and identify mutated regulatory sites within or near the GUS gene; (C) determine how growth hormone modulates the response to androgen. Experimental approaches will include the following: comparing regulatory sequences among genetic variants that differ in their response to androgen; testing regulatory functions of DNA constructs in both transfected cultured cells and transgenic mice; studying binding between androgen response elements and androgen receptor protein; doing a cis/trans analysis of the growth hormone effect; and identifying factors that modify the androgen response in cell culture.
| Niermann, G L; Watson, G L (1999) Growth hormone and insulin-like growth factor-I enhance beta-glucuronidase gene activation by androgen in mouse kidney. Mol Cell Endocrinol 153:47-55 |
| Elliger, S S; Elliger, C A; Aguilar, C P et al. (1999) Elimination of lysosomal storage in brains of MPS VII mice treated by intrathecal administration of an adeno-associated virus vector. Gene Ther 6:1175-8 |
| Watson, G L; Sayles, J N; Chen, C et al. (1998) Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus. Gene Ther 5:1642-9 |
