Human Krüppel-like transcription factor 11 (KLF11) and CACCC box mutation inhibit activity of the human proinsulin promoter in pancreatic beta-cells

K. Laubner; X. Niu; C. Limbert; M. D. Brendel; R. G. Bretzel; G. Päth; J. Seufert

doi:10.1055/s-2007-972211

Experimental and Clinical Endocrinology & Diabetes, Table of Contents

Human Krüppel-like transcription factor 11 (KLF11) and CACCC box mutation inhibit activity of the human proinsulin promoter in pancreatic beta-cells

K Laubner ¹, X Niu ¹, C Limbert ¹, MD Brendel ², RG Bretzel ², G Päth ¹, J Seufert ¹

¹University Hospital of Freiburg, Division of Endocrinology and Diabetology, Department of Internal Medicine II, Freiburg, Germany
²University Hospital of Gießen and Marburg, Department of Internal Medicine III, Gießen, Germany

Abstract

Objectives: KLF11 (TIEG2), a pancreas enriched Sp1-like transcription factor, is a negative regulator of pancreatic exocrine cell growth. Since a recent study indicated KLF11-induced activation of the human proinsulin promoter, we further investigated the functional role of KLF11 in pancreatic beta-cells.

Methods: RT-PCR, quantitative PCR, Western blot, in vitro translation, EMSA, transient transfections of INS-1 and beta-TC3 beta cells with human proinsulin promoter reporter plasmids and human KLF11 expression plasmids.

Results: Endogenous KLF11 mRNA expression was found in whole rat pancreas, human pancreatic islets, and INS-1 β-cells and was not affected by high glucose stimulation in INS-1. Cotransfections in INS-1 and beta-TC3 beta-cells of a human (h)KLF11 expression plasmid and a human proinsulin promoter driven reporter plasmid resulted in a substantial dose-dependent and glucose-independent inhibition of proinsulin promoter activity. 5-deletion of the human proinsulin promoter demonstrated that KLF11 acts via upstream DNA sequences above -173 and requires the β-cell specific transcription machinery since hKLF11-mediated inhibition of promoter activity was abolished in HEK293. As putative binding sites of KLF/Sp1-like proteins we identified both a previously described GC box and a CACCC box within the human proinsulin promoter. EMSA verified binding of in vitro translated hKLF11 to the GC box, but neither hKLF11-induced inhibition nor basal human proinsulin promoter activity was altered by GC box mutation or 5-deletion. In contrast, CACCC box mutation substantially reduced basal promoter activity thereby possibly explaining partially diminished hKLF11 inhibition. This effect may be indirect through corepressor function, because binding of in vitro translated hKLF11 to the CACCC box could not be verified by EMSA.

Conclusions: Our data characterize hKLF11 as a glucose- and GC box-independent negative regulator of human proinsulin gene expression and demonstrate a prominent role for the CACCC box in maintaining basal proinsulin promoter activity.