The physiologic mediators of inflammation in the bladder, and the mechanisms by which the bladder responds to pathologic injury, are largely unknown. The experiments outlined in this proposal will test the hypothesis that two recently-discovered members of the EGF family, amphiregulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are physiologic regulators of inflammatory processes in the bladder. We have recently shown that normal human bladder epithelial cells cultured under serum-free conditions express heparin-binding EGF- like factors with the molecular properties of HB-EGF and amphiregulin. HB-EGF and amphiregulin are potent stromal and epithelial regulatory factors which induce biologic effects via activation of the EGF receptor. HB-EGF is expressed by a variety of inflammatory cells, including T lymphocytes, eosinophils and macrophages, and is an extremely potent smooth muscle cell mitogen. Expression of high levels of HB-EGF by normal epithelial cells is a novel finding, and suggests a role for bladder epithelial cells in inflammatory processes. We hypothesize that these HB- EGF and amphiregulin may participate in mechanisms of bladder epithelial and smooth muscle hyperplasia, and possibly immunoregulatory responses, associated with inflammatory conditions and would healing responses in the human bladder.
The specific aims are:
Aim 1. (a) To determine whether human bladder epithelial and smooth muscle cells cultured in vitro express HB-EGF and amphiregulin, and to characterize the biochemical properties of the bladder forms of these factors; (b) to identify cell type-specific effects of these factors on growth, motility and cellular differentiation of bladder cells. These include potential paracrine activities and effects induced by heparin sulfate-dependent activation of the EGF receptor.
Aim 2. To identify the sites of cellular localization and spatial and temporal characteristics of expression of HB-EGF and amphiregulin in the bladder in the following conditions: (a) in the normal urinary tract in humans and rodents; (b) in pathologic conditions related to interstitial cystitis in humans; (c) in a laser-induced model of bladder would healing in Lewis rats; and (d) in experimentally-induced autoimmune interstitial cystitis in Balb/cAN mice. Further, we will determine whether HB-EGF and AR accumulate in extracellular matrices secreted by bladder epithelial and smooth muscle cells.
Aim 3. To modify HB-EGF and amphiregulin expression in human bladder cells using recombinant techniques: (1) to determine potential cell type-specific function(s) of the precursor and mature forms of the factors in bladder epithelial and smooth muscle cells; and (2) to elucidate bladder-specific mechanisms of regulated secretion of membrane- bound HB-EGF and amphiregulin, mediated by inflammatory cytokines and growth factors; and (3) to identify conditions under which the membrane- anchored precursors of HB-EGF and amphiregulin bind to membrane glycolipids as a test for potential juxtacrine activity of the precursor forms of the growth factors.
