Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

Abstract

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR ‫؍‬ 1.29, P ‫؍‬ 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.insulin ͉ polymorphisms ͉ TGF-␤ ͉ type 2 diabetes C omponents of both the exocrine and endocrine pancreas are affected by diseases, e.g., pancreatic cancer and type 2 diabetes mellitus (T2DM), which severely compromise both the quality and span of human life. Both glandular compartments share the same cellular origin and early morphogenetic pathways, suggesting a close functional and pathophysiological relationship. For instance, the exocrine-specific transcription factor p48 and the endocrine-specific pancreatic duodenal homeobox gene 1 (PDX-1) are both expressed in the common cell precursor (1); and, under pathological conditions their compartmentalization may be lost, as exemplified by the detection of PDX-1 in pancreatic cancer (2). In fact, T2DM is both a common feature and a risk factor for the subsequent development of pancreatic cancer (3, 4). The TGF-␤-inducible transcription factor KLF11 regulates exocrine cell growth and behaves as a tumor suppressor in pancreatic cancer (ref. 5 and M.E.F.-Z. and R.U., unpublished observation). Because the TGF-␤ signaling pathway is also a major regulator of endocrine cell fate (1, 6), the current study has been designed to define the role of the Sp1-like transcription factor KLF11 in the biology of ...

Human Krüppel-like factor 11 inhibits human proinsulin promoter activity in pancreatic beta cells

“…This opposing observation may be explainable by possible differences between native INS-1E beta cells used in our study and the INS832/13 beta cell line, which is derived from a highly selected INS-1 subclone stably transfected with a plasmid containing the human proinsulin gene [16]. More importantly, Neve et al [10] demonstrated activation of hInsP by cotransfected FLAG-tagged KLF11 in beta-TC3 beta cells. To be sure that this conflicting finding is not caused by the use of different beta cell lines, we confirmed the hKLF11-induced inhibition of cotransfected hInsP activity observed in INS-1E beta cells also in beta-TC3 beta cells.…”

“…Sequence analysis of hInsP not only retrieved the previously described GC box but also identified a CACCC box element, both putative binding sites of KLF11. Since our results are in contrast to those of Neve et al [10] and to better understand how hKLF11 regulates human insulin gene expression, we here further studied the functional role of the GC and CACCC box by electrophoretic mobility shift analysis (EMSA), mutation and 5′-deletion constructs of the hInsP in INS-1E and beta-TC3 beta cells.…”

“…In line with this inhibitory function KLF11 mRNA levels in INS-1E beta cells were repressed by high glucose conditions in which insulin production and secretion are enhanced. Interestingly, in a recent study Neve et al [10] reported a high glucoseinduced stimulation of KLF11 mRNA expression in INS832/13 beta cells. This opposing observation may be explainable by possible differences between native INS-1E beta cells used in our study and the INS832/13 beta cell line, which is derived from a highly selected INS-1 subclone stably transfected with a plasmid containing the human proinsulin gene [16].…”

“…The requirement of GC box sequences for functional KLF11-induced repression of promoter activity has been evaluated by several studies [6,13,14]. Based on this established concept Neve et al [10] predicted, although they demonstrate activation and not repression, that KLF11 influenced hInsP via an identified GC box sequence, which they have tested by EMSA. We also verified binding of hKLF11 to the GC box and further investigated the functional relevance of this interaction, but unexpectedly, neither 5′-deletion nor mutation of the GC box altered hKLF11 overexpression-induced inhibition of hInsP activity.…”

“…The role of KLF11 within the endocrine pancreas remained unestablished until recently Neve et al [10] reported that, in beta cell lines, high glucose conditions stimulate KLF11 mRNA expression and cotransfected human (h)KLF11 can activate the human proinsulin promoter (hInsP). In our own experiments, however, endogenous KLF11 mRNA levels were reduced by high glucose in INS-1E beta cells.…”