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Title: Inducible expression and regulated processing of heparin-binding epidermal growth factor-like growth factor in an intestinal epithelial cell line
Author: Ellis, P. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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The HB-EGF gene has been shown to be highly inducible in a number of cell types in vitro but the mechanisms involved are not well defined. This question was addressed using an epithelial cell line derived from rat intestine (RIE-1). HB-EGF mRNA expression can be rapidly and transiently induced by agonists that act through distinct receptor types. HB-EGF mRNA expression could be induced independently of protein synthesis, thus defining the HB-EGF gene as a primary response gene in these cells. Agonist-induced HB-EGF mRNA expression was found to occur via both protein kinase C (PKC)-dependent and -independent pathways, and required activation of the Ras/Raf/mitogen-activation protein kinase (MAPK) kinase (MEK)/MAPK signalling cascade. Raised intracellular cyclic AMP levels were found to inhibit agonist-induced HB-EGF mRNA expression, although not by interfering directly with the activation of p42 MAPK. In addition, heparin-affinity chromatography and Western blot analysis of conditioned medium from agonist-stimulated RIE-1 cells revealed that these cells synthesise and release multiple forms of soluble HB-EGF (18-28kDa). Soluble HB-EGF is generated by proteolytic cleavage of a larger membrane-anchored precursor, proHB-EGF. In order to study the mechanisms that regulate the processing of proHB-EGF to soluble HB-EGF, RIE-1 cells were engineered to constitutively overexpress a full length rat HB-EGF cDNA. These cells were found to express multiple forms of proHB-EGF(21.5-32kDa) at the cell surface. In unstimulated cells, the processing of proHB-EGF to soluble HB-EGF was found to be slow with only small amounts of soluble HB-EGF released into the culture medium. However, in response to both PKC- and calcium dependent signals, proHB-EGF was rapidly and completely converted to soluble HB-EGF. In contrast with agonist-induced HB-EGF mRNA expression, processing of proHB-EGF to soluble HB-EGF was found to be independent of the activation of the Ras/Raf/MEK/MAPK signalling cascade.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available