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

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 (I 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 for-m 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 Q 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-GR 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.