title: Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element.
abstract: The expression of members of the Reg family of secreted lectin-like proteins is increased in response to stress, inflammation and damage in many tissues. In the stomach, Reg is located in enterochromaffin-like cells, where its expression is stimulated by the gastric hormone gastrin. We have examined the mechanisms by which gastrin stimulates expression of Reg-1. Deletional mutations of 2.1 to 0.1 kb of the rat Reg-1 promoter in a luciferase reporter vector were transiently transfected into gastric cancer AGS-G(R) cells. All promoter fragments tested showed similar relative increases in luciferase expression in response to gastrin (1 nM). The response to gastrin of the smallest (104 bp) construct was 4.2+/-0.4-fold over basal. These responses were reduced by Ro-32-0432, a protein kinase C inhibitor, by C3-transferase, a Clostridium botulinum toxin and a selective inhibitor of the Rho family GTPase RhoA, and by co-transfection with a dominant negative form of RhoA. Co-transfection with a constitutively active form of RhoA stimulated expression 11.6+/-1.7-fold over basal. Mutations through the 104 bp construct identified a C-rich element (C-79CCCTCCC-72) required for responses to gastrin, PKC (protein kinase C) and L63RhoA (the constitutively active form of human RhoA protein containing a glutamine-to-leucine substitution at position 63). EMSAs (electrophoretic-mobility-shift assays) using nuclear extracts of control and gastrin-stimulated AGS-G(R) cells and a probe spanning -86 to -64 bp revealed multiple binding proteins. There was no effect of gastrin on the pattern of binding. Supershift assays indicated that transcription factors Sp1 and Sp3 bound the C-rich sequence. We conclude that gastrin stimulates Reg expression via activation of PKC and RhoA, that a C-rich region (-79 to -72) is critical for the response and that Sp-family transcription factors bind to this region of the promoter.
authors.affiliation: Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
authors.forename: Felicity J
authors.initials: FJ
authors.lastname: Ashcroft
authors.forename: Andrea
authors.initials: A
authors.lastname: Varro
authors.forename: Rod
authors.initials: R
authors.lastname: Dimaline
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Dockray
mesh_terms: D000818:Animals; D001483:Base Sequence / Q000235:genetics / Q000502:physiology; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D016207:Cytokines / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D005755:Gastrins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000502:physiology; D051705:Sp3 Transcription Factor; D013274:Stomach Neoplasms; D014157:Transcription Factors / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; D005755:Gastrins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; C106354:flotillins; D051705:Sp3 Transcription Factor; D003596:Cytosine; D008156:Luciferases; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein