KLF11 is a pancreas enriched member of the Sp1/KLF transcription factor family which plays an important role in mammalian gene regulation. Previously we demonstrated that human (h)KLF11) inhibits human insulin promoter (hInsP) activity in rodent beta-cell lines via an unknown mechanism. Here we present results from ongoing experiments in which we test the hypothesis that KLF11 interferes with the major hInsP transactivator PDX1. We performed transient cotransfections of INS-1E and beta-TC3 beta-cells and HEK293 non-beta-cells with hInsP-controlled SEAP (secreted alcaline phosphatase) reporter plasmids and CMV-driven expression plasmids for hKLF11 and/or hPDX1 at concentrations of 0.5 μg unless otherwise noted. DNA amounts were equilibrated by addition of empty plasmid (mock). As demonstrated before, hKLF11 dose-dependently inhibits -881hInsP activity in both beta-cell lines employed (0.5 μg, 30%; 1 μg, 50%). Ectopic PDX1 only marginally stimulates hInsP (a known effect resulting from the presence of endogenous PDX1 in beta-cells) and, similar to transfections without PDX1, KLF11 inhibits hInsP activation about 30%. In contrast to beta cells, KLF11 stimulates −881hInsP activity in HEK293 about 40%. This stimulation is abolished in shorter hInsP fragments lacking the GC box, a known KLF11 binding site (≤−355) and converted into 3040% inhibition in very short hInsP fragments (≤−101). In hInsP fragments ≤−101 also PDX1 stimulated activation was nearly abolished, while PDX1 enhances hInsP activation to about 300% in all fragments ≤−173. Interestingly, the combination of KLF11 and PDX1 superadditively stimulates -881hInsP activity about 10-fold. This superadditive stimulation is continuously reduced to levels of PDX1 alone by deletion of hInsP from −881 to −173. These results strongly indicate that KLF11-mediated inhibition of hInsP activity in beta-cells is closely related to PDX1 function. For further proof of concept we currently investigate physical interactions of KLF11 with PDX1 and its transcription complex partners, for example p300/CBP.
03 - 07 May 2008
European Society of Endocrinology