Heparin-binding EGF-like growth factor (HB-EGF), is synthesized as an insoluble membrane-anchored protein that is processed to release soluble mature HB-EGF. Both forms are biologically active. HB-EGF is multifunctional in that mature HB-EGF is a potent mitogen and chemotactic factor for smooth muscle cells (SMC), fibroblasts and keratinocytes, while transmembrane HB-EGF (HB-EGF/TM) mediates cell-cell contact as a juxtacrine signaling and adhesion molecule acting via the EGF receptor. HB-EGF/TM is also the receptor for diphtheria toxin (DT). Expression of HB_EGF has been linked to normal physiological processes such as wound healing, blastocyst implantation and myogenesis, and to pathological processes such as SMC hyperplasia. It is proposed that HB-EGF expression and activities are regulated in several ways. One is a regulatory post- translational processing mechanism that converts a juxtacrine factor into a potent paracrine mitogen. Normal processing could allow for rapid mobilization of mature HGB-EGF, for example, as a response to injury, while abnormal processing could lead to undesired cell proliferation such as SMC hyperplasia. HB-EGF activity is also regulated at the level of gene expression by specific transcription factors that bind to the HB-EGF promoter such as MyoD and NFkB, and by external inducers of HB-EGF transcription such as thrombin, phorbol ester and lysoPC. The major focus of the proposal is to gain insights into the regulation of HB-EGF activity by analyzing in depth the biology of HB-EGF/TM, juxtacrine interactions, the mechanisms of HB-EGF processing, the consequences of aberrant release of mature HB-EGF, and the control of HB-EGF gene expression.
The Specific Aims of this proposal are: 1. To analyze the structural properties and juxtacrine activities of HB-EGF/TM in vitro and in vivo; including: A) expression and localization of HB-EGF/TM in differentiating systems in culture and in vivo: B) analysis of HB-EGF/TM juxtacrine signaling and adhesion activities, and the mechanisms that regulate these activities such as modulation by HSPG and CD9; and C) analysis of HB-EGF/TM internalization and interactions with membrane and cellular proteins; 2. To analyze the processing of HB-EGF/TM in vitro and in vivo; including: A) mechanisms of HB-EGF/TM processing, identification of the HB-EGF/TM C-terminal cleavage site(s) and preparation of non-cleavable HB-EGF/TM mutants; and B) examining the phenotypic consequences in transgenic mice of expressing mature HB-EGF under control of the HB-EGF promoter, and of removal of the cytoplasmic and transmembrane domains by homologous recombination; 3. To analyze the regulation of HB-EGF gene expression and growth factor activity in vitro and in vivo with an emphasis on HB-EGF activity in proliferative SMC disease; including: A) regulation of HB-EGF transcription in cells associated with vascular SMC hyperplasia; and B) expression and localization of HB-EGF in normal myometrial and leiomyoma (uterine fibroids) SMC.
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