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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2504
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 8 3128-3135
Copyright © 2008 by The Endocrine Society

Genetic Analysis of Krüppel-Like Zinc Finger 11 Variants in 5864 Danish Individuals: Potential Effect on Insulin Resistance and Modified Signal Transducer and Activator of Transcription-3 Binding by Promoter Variant –1659G>C

Ruth Gutiérrez-Aguilar, Philippe Froguel, Yasmin H. Hamid, Yamina Benmezroua, Torben Jørgensen, Knut Borch-Johnsen, Torben Hansen, Oluf Pedersen and Bernadette Neve

Centre National de la Recherche Scientifique Unit Mixté de Recherche 8090-Institute of Biology (R.G.-A., P.F., Y.B., B.N.), F-59021 Lille, France; Steno Diabetes Center and Hagedorn Research Institute (Y.H.H., K.B.-J., T.H., O.P.), DK-2820 Gentofte, Denmark; Research Centre for Prevention and Health (T.J.), Glostrup University Hospital, DK-2600 Glostrup, Denmark; Department of Genomic Medicine (P.F., B.N.), Hammersmith Hospital, Imperial College London, London W12 OHS, United Kingdom; and Faculty of Health Science (K.B.-J., O.P.), University of Aarhus, DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: B. Neve, Department of Genomic Medicine, Hammersmith Hospital, Imperial College London, London W12 OHS, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The transcription factor Krüppel-like zinc finger 11 (KLF11) has been suggested to contribute to genetic risk of type 2 diabetes (T2D). Our previous results showed that four KLF11 variants, in strong linkage disequilibrium (LD block including +185 A>G/Gln62Arg and –1659 G>C) were associated with T2D in a north European case-control study. Here we further analyzed these variants for T2D association in a general Danish population and assess their possible effect on gene function.

Methods: We genotyped Gln62Arg variant, representative for the LD block, in 5864 subjects of the INTER99 study to assess association to T2D and glucose metabolism-related quantitative traits. We studied effects of LD-block variants on KLF11 function and in particular, the effect of –1659G>C on transcriptional regulation of KLF11 using EMSA, chromatin immunoprecipitation, gene reporter assays, and small interfering RNA transfection.

Results: We could not confirm T2D association of the KLF11 LD block, however, in glucose-tolerant subjects; it was significantly associated with higher fasting serum insulin and C-peptide levels and increased homeostasis model assessment insulin resistance indexes (P = 0.00004, P = 0.006, and P = 0.00002, respectively). In addition, binding of signal transducer and activator of transcription (STAT)-3 to the wild-type (–1659G>C) allele stimulated gene transcription, whereas STAT3 did not bind onto the mutant allele.

Conclusions: We showed that KLF11 may interfere with glucose homeostasis in a Danish general population and that STAT3-mediated up-regulation of KLF11 transcription was impaired by the –1659G>C variant. Overall, KLF11 variants may have a deleterious effect on insulin sensitivity, although that may not be sufficient to lead to T2D.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Transcription factor Krüppel-like zinc finger 11 (KLF11) has been implicated in type 2 diabetes (T2D) and pancreatic cancer (1, 2), two diseases that are closely related to pancreatic endocrine, and exocrine compartments, respectively. KLF11 regulates exocrine cell growth and is a tumor suppressor in pancreatic cancer (2). In addition, KLF11 may play a role in endocrine β-cell function, in which it can regulate insulin expression (1, 3). However, its exact role remains to be established because it is not clear whether KLF11 stimulates or inhibits the insulin gene transcription (1, 3). KLF11 mRNA is highly expressed in the pancreas but is also present in multiple tissues like muscle and liver (4). Moreover, KLF11 is up-regulated in fasting conditions in mouse skeletal muscles (5), and its promoter can be bound by hepatocyte nuclear factor-1{alpha} in hepatocytes (6). Thus, these data suggest that KLF11 may have an impact on glucose homeostasis in both pancreas and peripheral tissues.

We previously reported four frequent variants in a strong linkage disequilibrium (LD) block that were associated with T2D in a pooled north European population (1). Those variants [+185 A>G Gln62Arg (rs35927125), AS+343 G>T (rs4444493), IVS3 + 1398 del(A), and –1659 G>C] are in strong LD in both black African and European populations (unpublished results and Ref. 1). The Gln62Arg KLF11 variant may have functional implications (1), and here we analyzed whether the other variants may influence regulation of KLF11 expression and function.

Recently a case-control study in additional European populations did not confirm association of Gln62Arg variant with T2D (7). Therefore, we further analyzed contribution of this KLF11 locus to T2D and associated phenotypes in a large cohort representative of the Danish general population.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study population

The INTER99 cohort is a well-established Danish general population recruited at the Research Centre for Prevention and Health from Glostrup University Hospital (Glostrup, Denmark) (8). All participants underwent a series of anthropometric, physiological, and biochemical examinations. Studies were approved by the Ethical Committee of Copenhagen and also consent was obtained (Helsinki Declaration II).

Subjects from INTER99 were classified according to their oral glucose tolerance test (OGTT) data. We genotyped 4443 glucose-tolerant, 491 subjects with impaired fasting glucose, 679 subjects with impaired glucose tolerance, and 369 T2D subjects.

Variant genotyping and statistical analysis

PCR fragments covering KLF11 variants were amplified using genomic DNA isolated from leukocytes and the following probes: +185A>G, 5'-CTCGGTGTTTGTTGCTATAGACT, 5'-CAGGGAATCTTCTCACAAGTTCT; AS+343G>T, 5'-GAACAGCAAGTGCGAGGAC, 5'-GTTTAAGACGAAAGAACCGTGATA; IVS3 + 1,398 del(A), 5'-TTTACAACCTTTTCTATTGA, 5'-TTGGTGACAGGAATGATTGC; and –1659G>C, 5'-GAACAGCAAGTGCGAGGAC, 5'-GTTTAAGACGAAAGAACCGTGATA. Genotypes were determined using LightTyper assays, with the Gln62Arg-probes 5'-LCRed640-ATCCCAGAAAGGTGACCT and 5'-TCTTGTTTGTATGAGCTCCT GGGGTCA-fluorescine (Roche Diagnostics, Meylan, France). Genotype distributions were tested for Hardy-Weinberg equilibrium, and Fisher’s exact tests were used to assess significant differences in allele frequencies and genotype distributions in case-control analysis. Quantitative traits were analyzed and, if needed, transformed to a normal distribution, where after differences between groups were determined by ANOVA (SPSS software; SPSS, Chicago, IL).

Cell culture

HepG2 was cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin), and 1x nonessential amino acids (Life Technologies, Inc.-Invitrogen, Cergy Pontoise, France) at 37 C in a humidified atmosphere of 5% CO2. Two similar HeLa cell lines, but originally independently maintained in different laboratories [HeLaS was a gift from Dr. Ralph Jockers (Institut Cochin, Unité Mixte de Recherche 8104, Centre National de la Recherche Scientifique, Université Paris Descartes, 75014 Paris, France) and HeLa-S from Dr. Yves Rouillé (Unité Mixte de Recherche 8161, Centre National de la Recherche Scientifique, Institut de Biologie de Lille, Institut Pasteur de Lille, 59021 Lille, France)] were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotics. The pancreatic βTC3 cell line was cultured in DMEM supplemented with 2.5% heat-inactivated FBS, 10% heat-inactivated bovine newborn calf serum, and antibiotics.

EMSA

Nuclear protein extracts were prepared using hypotonic buffer [1 mM dithiothreitol (DTT), 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1x protease inhibitors (Complete, Roche)]. After centrifugation, the protein extracts were resuspended in 20 mM HEPES buffer (with 0.4 M NaCl, 1 mM EDTA, 1 mM MgCl2, 1 mM PMSF, and protease inhibitors). Biotin-labeled double-stranded probes [biotin-3'-end DNA labeling kit; Pierce, Rockford IL); –1659G, 5'-GTGCTGAGTGGG AAGAGGCTCTG; –1659G, 5'-GTGCTGACTGGGAAGAGGCTCTG; +1,398A, 5'-CACAGCTTCCAAATGTTGGAGGTGA; +1,398 Del(A), 5'-CACAGCTTCCAATGTTGGAGGTGA] were incubated with 8 µg nuclear proteins for 30 min at room temperature following recommendations of the LightShift chemiluminescent EMSA kit (Pierce). Unlabeled probes were used for DNA-binding competition. For supershift analysis, extracts were incubated with 2 µg signal transducer and activator of transcription (STAT)-3-antibody (H-190/sc-7179X; Santa Cruz Biotechnology, Santa Cruz, CA) during 20 min before the incubation with biotin probes. The mixtures were subjected to 6% nondenaturing PAGE, transferred to Hybond N+ membrane (Amersham Biosciences, Buckinghamshire, UK), and visualized by chemiluminescence (Pierce).

Chromatin immunoprecipitation

HepG2 cells were cross-linked with 1% formaldehyde and harvested in 50 mM Tris buffer (pH 8.1) containing 1% sodium dodecyl sulfate (SDS) and 10 mM EDTA. The samples were sonicated to approximately 500 bp DNA fragments; diluted in a 16.7 mM Tris (pH 8.1) buffer with 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, and protease inhibitors; and then incubated overnight at 4 C with or without STAT3 antibodies (sc-7179x; Santa Cruz Biotechnology) in combination with Protein A-Dynal beads (DynaI Biothech ASA, Oslo, Norway). The immunoprecipitations were washed twice with 20 mM Tris-buffer (pH 8.1) containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 150 mM NaCl and twice in this buffer with 500 mM NaCl. Eluates were obtained with 1% SDS and 0.1 M NaHCO3, and cross-links were removed overnight at 65 C. Immunoprecipitated DNA was purified by phenol-chloroform-isoamilyc extraction and ethanol precipitation. Then a 398-bp region of the KLF11 promoter region (–1864 to –1470 bp) was amplified by PCR using primers 5'-GAACAGCAAGTGCGAGGAC and 5'-GTTTAAGACGAAA GAACCG.

Western blot analysis

HepG2, HelaS, and Hela-S cells were lysed in a 10 mM HEPES buffer containing 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1x Triton X-100, 0.5 mM PMSF, 0.1 mM DTT, 0.1 mM Na3VO4, and 1x protease inhibitors. Proteins (30 µg/lane) were separated by 10% SDS-PAGE followed by Western blotting. Immunoreactive bands were visualized using STAT3 antibody diluted 1:2,000 (sc-482; Santa Cruz Biotechnology) and antirabbit IgG-horseradish peroxidase 1:10,000 (NA934V,; Amersham Biosciences) with the ECL-plus Western blot detection system (Amersham). To verify equal quantities of protein loading on the blot, β-actin protein was visualized with β-actin antibodies diluted to 1:2,000 (Sigma, St. Louis, MO), antirabbit IgG-horseradish peroxidase 1:10,000 (NA934V; Amersham), and enhanced chemiluminescence.

Reporter constructs and expression plasmids

The whole KLF11 promoter is hard to clone, probably due to its high GC content. Therefore, a 700-bp fragment covering –1659G>C variant was generated from genomic DNA using the GeneAmp PCR system (Applied Biosystems, Foster City, CA) and primers 5'-TATACGCGTTCACCTACGTGGACTTA (containing a MluI site) and 5'-ATATCTAGAGGGGTTGACCAGGCTGA (with a BglII site). The amplified fragment (–1983 to –1284 bp relative to KLF11 transcription start site) was cloned into a pGL3-basic vector (Promega, Madison, WI) upstream of a cloned thymidine kinase (TK) promoter, into pGL3-promoter [Simian virus 40 (SV40)] vector, and pGL3-basic vector. The –1659C sequence was introduced using primer 5'-TAAAGACCCCTGGGGGTGCTGACTGGGAAGAGGCTCTGGGAGC and the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequences of all constructs were confirmed by automated sequencing (Applied Biosystems).

Luciferase reporter gene assay

HepG2, HelaS, and Hela-S cells, cultured to 70% confluence on 6-well plates, were transiently transfected with 500 ng of –1659G KLF11, – 1659C KLF11, or empty reporter constructs using FuGENE 6 transfection reagent (Roche). To normalize for transfection efficiency, 1 ng of pRL-CMV vector (Promega) was cotransfected. In several experiments with HeLa-S cells, thereafter 1 µg of STAT3-expressing plasmid was transfected. After 48 h of transfection, luciferase activities were measured using the Dual-Glo luciferase assay system (Promega). Data of at least triplicate experiments (mean ± SD) are expressed as relative luciferase activity with the activity measured from control construct transfected cells set to 100%.

Small interfering RNA (siRNA) transfection and quantitative mRNA expression

Cells were transfected at a density of 3 x 105 cells/well (6-well plates) with 80 nM STAT3 siRNA (no. 42861) or negative control siRNA (no. 1) using SiPORT NeoFX (all from Ambion, Austin, TX). After 48 h of transfection, RNA was isolated using the NucleoSpin RNAII kit (Macherey-Nagel, Duren, Germany) and then cDNA prepared using the high-capacity cDNA archive kit and random hexamers (Applied Biosystems). Real-time PCR analyses were performed using the 7900HT real-time PCR system (Applied Biosystems), TaqMan universal PCR master mix, and the gene-specific assays for human (Hs) glycerhaldehyde-3-phosphate dehydrogenase (HsGAPDH; 99999905), human KLF11 (Hs00231614), and human STAT3 (Hs00234174) that were all purchased from Applied Biosystems.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Genetic analysis of the LD block KLF11 variants in the Danish INTER99 cohort

To confirm the previously described KLF11 LD block we genotyped variants +185A>G Gln62Arg, AS+343G>T, IVS3 + 1,398del(A), and –1659G>C in 44 Danish subjects. Because we observed complete LD [with identical minor allele frequencies (MAF) for +185A>G Gln62Arg and –1659G>C], we decided to further genotype Gln62Arg variant in the INTER99 as representative for the LD block.

No overall association with T2D was found when comparing Gln62Arg MAF between glucose-tolerant and impaired fasting glucose, impaired glucose-tolerant, or T2D subjects (supplementary Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). Because the previously reported odds ratio for Gln62Arg was 1.29 (1), this study was underpowered to detect the expected differences in MAF [statistical power calculated by Power for Association With Error (PAWE) (9) is 8–13%, with {alpha} = 0.05 and a MAF of 0.10–0.20 in control subjects]. Next, we analyzed T2D-related quantitative traits in individuals who were classified as glucose tolerant according to their OGTT. We observed a significant trend for association with higher fasting serum insulin levels, higher fasting serum C-peptide levels, and increased homeostasis model assessment (HOMA) insulin resistance indexes (P = 0.00004, P = 0.006, and P = 0.00002; Table 1Go).


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  		TABLE 1. Insulin-related variables in Danish white glucose-tolerant subjects classified in accordance to KLF11’s LD block representative Gln62Arg genotype

 
Effects of variants on KLF11’s function

We previously showed that Gln62Arg effects transcriptional regulation activity of KLF11 protein (1). KLF11 binds to the preproinsulin promoter and regulates its activity in vitro. However, it is reported to both increase and decrease insulin transcription (1, 3). Due to this discrepancy, it is unclear how Gln62Arg may affect insulin gene expression. In the present study, we analyzed whether other variants present in the LD block [AS+343G>T; IVS3 + 1,398del(A), and –1659G>C] influence the regulation of KLF11 expression and function. Because the AS+343G>T variant is located in the predicted 3'-untranslated region, we performed in silico analysis of mRNA properties in the presence of this variant. No significant differences in predicted RNA stability was observed using the mfold program (http://mfold.bioinfo.rpi.edu/) or in predicted microRNA binding (T-ScanS and PicTar UCSC tracks at http://genome.ucsc.edu).

Next, we analyzed nuclear protein binding onto the –1659G>C promoter variant and IVS3 + 1,398del(A) intronic variant of the LD block in βTC3 and HepG2 cells by EMSA. For the IVS3 + 1,398del(A) variant, we observed no specific nuclear complex binding onto the variant sequences (supplementary Figure 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). For the –1659G>C variant (promoter variant), we identified a complex that bound only onto WT but not onto MT allele (βTC3, data not shown; HepG2, Fig. 1AGo, lane 2 vs. lane 12). Competition with unlabeled WT probe resulted in a decrease of signal (lanes 3–5), whereas presence of an excess of unlabeled MT or nonspecific probe did not alter the observed band shift (with a 1- to 10-fold excess (Fig. 1AGo, lanes 6–8 and 9–10, respectively).


Figure 1
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  		FIG. 1. Nuclear protein binding of –1659G>C KLF11 variant in HepG2 cells. A, Nuclear extracts from HepG2 cells were incubated with biotin-labeled wild-type (WT, –1659G; lane 1–10) or mutant (MT, –1659C) double-stranded oligonucleotide probes (lane 11–15) and with or without an excess of either WT (lanes 3, 1-fold; 4, 2.5-fold; 5 and 14, 10-fold excess), MT (lanes 6, 1-fold; 7, 2.5-fold; 8 and 13, 10-fold excess), or nonspecific, unlabeled probe (lanes 9, 2.5-fold; 10 and 15, 10-fold excess). B, Nuclear extracts from HepG2 cells were incubated with biotin-labeled wild-type probe (WT, –1659G) with or without either unlabeled WT probe at a 50- and 100-fold excess (lanes 3 and 4), nonspecific unlabeled probe at a 50-fold and 100-fold excess (lanes 5 and 6), and 2 µg STAT3 antibodies (lane 7). C, Chromatin immunoprecipitation of KLF11 with STAT3 antibodies in HepG2 cells. The cross-linked nuclear complex of DNA/protein was immunoprecipitated (IP) with STAT3 antibodies and analyzed by specific PCR of the –1659 KLF11 variant region. Initial lysate (input) and {alpha}-STAT3 immunoprecipitation with protein A-agarose beads (+) and without antibody (–). Figures are representative for at least three independent experiments.

 
In silico analysis, using TESS (http://www.cbil.upenn.edu) and MatInspector (http://www.genomatix.de) software, predicted that transcription factor STAT3 could bind onto the WT allele and not onto the MT allele sequence. Indeed, when we incubated nuclear proteins of HepG2 cells with an STAT3 antibody, we observed a decreased signal at the original position and a supershifted nuclear complex (Fig. 1BGo, lane 7). In addition, Fig. 1BGo shows inhibition of complex formation with an excess of unlabeled WT probe (lanes 3 and 4) but not with increasing excess (up to 100-fold) of nonspecific unlabeled probe (Fig. 1BGo, lanes 5 and 6). Figure 1Go presents data from HepG2 cells, but similar results were obtained in the pancreatic cell-line βTC3. Because STAT3 is highly expressed and studied in HepG2 and the liver is a major insulin sensitive tissue, we decided to continue our experiments in this hepatic cell line.

To determine STAT3 binding to KLF11’s endogenous promoter, we realized chromatin immunoprecipitation in HepG2 cells (homozygous WT). Indeed, KLF11’s promoter DNA precipitated with STAT3 antibody but not in the negative control (Fig. 1CGo, lanes (+) and (–), respectively).

To further explore STAT3 binding onto the WT allele, we used two HeLa cells lines that differentially expressed STAT3. First, we analyzed the quantity of STAT3 protein by Western blot in the three different cell lines (HepG2, HeLaS, and HeLa-S, Fig. 2AGo). The HepG2 cell line expressed more STAT3 protein, compared with the HeLa cell line containing STAT3 (HeLaS), and we established that HeLa-S cells do not contain detectable protein levels of STAT3 (Fig. 2AGo, lanes 1–3, respectively). Then we performed EMSAs using HeLaS and HeLa-S nuclear extracts. We observed that in HeLaS, a complex fixes onto WT allele and not onto MT allele sequences (Fig. 2BGo, lanes 2, and 5). This complex is specific as it disappeared in presence of excess unlabeled WT probe and remains in presence of nonspecific unlabeled probe (Fig. 2BGo, lanes 3 and 4). Moreover, in HeLa-S cells, the complex was completely absent (Fig. 2BGo, lane 9). These results confirm that STAT3 is the transcriptional factor bound onto the WT allele KLF11 promoter sequence, without defining the effect on the transcriptional regulation.


Figure 2
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  		FIG. 2. Expression of STAT3 and binding onto the –1659G>C KLF11 variant. A, Representative Western blot with nuclear extracts of HepG2 cells (1 ), HeLaS cells (2 ), and HeLa-S cells (3 ) analyzed with STAT3 and β-actin antibodies. B, Nuclear extracts from HeLaS (lane 1–7) or HeLa-S (lane 8–11) cells were incubated with biotin-labeled wild-type (WT, –1659G; lanes 1–4 and 8–11) or mutant (MT, –1659C) probe (lanes 5–7) and with or without either unlabeled probe at a 100-fold excess (lanes 3, 6, and 10) or nonspecific probe at a 100-fold excess (lanes 4, 7, and 11).

 
Transcriptional regulation is affected by –1659G>C KLF11 promoter variant

To analyze effects of KLF11 promoter variant on transcriptional regulation of the KLF11 gene, we cloned 700 bp of the variant region in luciferase reporter plasmids. Transfection of WT (–1659G) TK-pGL3 construct in HepG2 cells showed a significantly increased luciferase activity when compared with empty construct, suggesting the cloned region enhances transcriptional activation (Fig. 3AGo, solid bar, HepG2). On the other hand, enhancer activity of MT construct was significantly lower than the WT (Fig. 3AGo, striped bar, HepG2). Similarly to HepG2 cells, when HeLaS cells were transfected with TK-pGL3 constructs, a significantly different transcriptional activation between WT and MT allele was observed. On the other hand, in the HeLa-S cells, this activation difference was not perceptible (Fig. 3AGo, solid and striped bar, HeLaS, solid and striped bar, HeLa-S, respectively). Thus, these results show that only the STAT3-containing cell lines (HepG2 and HeLaS) have a different transcriptional enhancer-like activity between WT and MT allele and that this difference was not observed in the cell line without STAT3 (HeLa-S). In light of the distant position of –1659G>C, compared with the transcription initiation site, we expect that this region has a native enhancer-like activity. Indeed transfection of pGL3-promoter constructs (SV40 promoter) showed a similar decrease of enhancer activity between WT and MT alleles as upstream of the TK promoter (TK-pGL3, supplementary Fig. 2). In contrast, without a minimal promoter (pGL3-basic), the constructs’ transcriptional activities were very low, demonstrating that this region has an enhancer-like activity (supplementary Fig. 2). Moreover, transcriptional activity of the MT allele in pGL3-basic remains lower than the enhancer activity in the presence of TK promoter. In addition, the KLF11 region downstream of the variant contains several predicted transcription factor binding sites present in the TK and SV40 promoter as well (see supplementary Fig. 2). Additional validation of STAT3-mediated transcriptional regulation of KLF11’s WT allele promoter was obtained by luciferase assays in the STAT3-negative HeLa-S cell line. Without STAT3 transfection, no difference in luciferase activity was observed between transfection with the WT and MT TK-pGL3 constructs (Fig. 3BGo, solid and striped bar, –STAT3, respectively). However, when we cotransfected the STAT3 expression construct with the KLF11 promoter-luciferase plasmid in these HeLa-S cells, we observed a significant increase of transcriptional activity of the WT TK-pGL3 but not the MT allele construct (Fig. 3BGo, solid and striped bar, +STAT3, respectively).


Figure 3
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  		FIG. 3. Luciferase activity of –1659G>C KLF11 promoter variant reporter constructs. A, HepG2, HeLaS, and HeLa-S cells were transfected with empty, wild-type –1659G (solid bars), or mutant –1659C (striped bars) KLF11 promoter variant reporter constructs pGL3-TK. Luciferase activity was standardized by renilla activity (cotransfection with pCMV-RL) and expressed as mean relative units of the empty construct (100%) of five experiments. B, HeLa-S cells transfected with empty, wild-type –1659G, or mutant –1659C KLF11 promoter variant reporter constructs (pGL3-TK) and pCMV-RL, followed by a transfection with 1 µg of a STAT3 expression construct. After 48 h the relative luciferase activity was measured (percent of WT reporter construct, n = 3). Asterisks indicate significant differences (P < 0.05).

 
STAT3 stimulates KLF11 transcription

To analyze STAT3’s role over the WT (–1659G) KLF11 promoter in an endogenous environment, we measured KLF11 mRNA expression levels in HepG2 cells after STAT3 down-regulation by siRNA. Quantitative mRNA measurements after 48 h showed at best a 60% reduction of the STAT3 transcript in the presence of Ambion siRNA 42861 (Fig. 4Go). Under these conditions, the expression levels of KLF11 mRNA are decreased up to 40%. No diminution of STAT3 or KLF11 transcript was observed in the control transfection without siRNA (Fig. 4Go) or with control siRNA (data not shown). The consistency of all presented results suggests that, among the different transcription factors that may bind the endogenous KLF11 promoter, STAT3 regulates the transcription of KLF11 in hepatic cells.


Figure 4
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  		FIG. 4. Transcriptional regulation of STAT3 over KLF11. HepG2 cells were transfected with STAT3 siRNA (80 nM). After 48 h of transfection, total RNA was purified and KLF11 (solid bars), STAT3 (striped bars), and GAPDH expression levels were measured by quantitative PCR (triplicate). Results were normalized by GAPDH levels and expressed as percentage of transfection without STAT3 siRNA. (n = 3). *, P < 0.05, significant differences.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study addressed two questions. First, we aimed to further analyze genetic contribution of the KLF11 LD block to T2D and its associated phenotypes in a large, well-defined general Danish population. First, we confirmed the strong linkage disequilibrium between the reported variants and the genotyped Gln62Arg as representative for all LD block variants. Our present study does not further support our previous data showing strong association of KLF11 variants with T2D. Another study by Florez et al. (7) was also unable to replicate our initial findings. One possible explanation for these contradictory results could be the difference in T2D onset, which was earlier for cases of the initial study (1), compared with T2D patients of the present study and to the T2D/severe impaired glucose tolerance subjects studied by Florez et al. Moreover, it is worth indicating that the initial French T2D cases analyzed in our previous study (1) are highly selected for familial T2D history with closely matched glucose-tolerant lean controls (spouses). Indeed, an enriched familial T2D background increases the chance of finding genetic association to T2D and associated phenotypes in case-control studies (10, 11). Another limitation of our present study is the low power for case-control analysis (low number of T2D cases). On the other hand, KLF11 is at best a minor contributor to T2D genetic background because this locus has not been reported as a hit in whole genome association studies (12, 13, 14).

Nevertheless, our quantitative trait analysis showed that KLF11 variants may contribute to modulate fasting insulin, C-peptide levels, and HOMA insulin resistance index. Previous association reports that included only a limited number of glucose-tolerant homozygous 62Arg-allele carriers [19 of 791 normoglycemic subjects with OGTT data (7) and 0 of 70 subjects subjected to OGTT (1)] did not observe this association. Among the 4443 glucose-tolerant subjects analyzed in the present study were 76 GG-allele carriers, which indeed gave at least a 85% statistical power to retrieve a potential significant recessive effect of at least 0.1% of the total variance for both fasting insulin levels and the HOMA insulin resistance indexes [P < 0.05; determined by Quanto V (15)]. To unequivocally evaluate the role of KLF11 variants in glucose metabolism, a replication study of a similar well-phenotyped, uniform population sample with sufficient OGTT data (allowing the analysis of more then 4000 glucose tolerant subjects) will be needed. From our data we may suggest that KLF11’s at-risk genotype decreases insulin sensitivity in the Danish population, although it is insufficient to increase risk for T2D by itself.

The second question addressed is the potential functional properties of the four variants in the KLF11 Gln62Arg LD block associated with insulin resistance indexes. We previously showed that the nonsynonymous Gln62Arg affects KLF11’s transcriptional regulation activity (1). In addition, we here showed that –1659G>C promoter variant affects transcriptional regulation of KLF11 gene. In particular, STAT3 binds only to the wild-type G allele of this promoter variant and after binding stimulates the transcription of the KLF11 gene, having an enhancer-like activity. In fact, KLF11 overexpression was also observed in a previous study with constitutively active STAT3 in lung epithelial cells (16).

The potential implication of KLF11 in insulin resistance may be interesting with regard to regulation of KLF11 by STAT3. In fact, general STAT3 knockout (KO) mouse revealed an early embryonic lethality (17) and several tissue-specific KO mouse models show implication of STAT3 in diabetes and obesity (18, 19, 20, 21). Interestingly, liver STAT3 KO mice show insulin resistance associated with increased hepatic expression of gluconeogenic genes as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (22). Moreover, phosphoenolpyruvate carboxykinase has also been reported to be regulated by KLF15 via a specificity protein-1-like binding element (23), suggesting this may present a putative binding site for KLF11. It is possible that KLF11 is contributing to the STAT3-mediated effect on the expression of some gluconeogenic genes. Therefore, additional studies are needed to analyze effects of KLF11 on transcriptional regulation of gluconeogenic genes.

Thus, the insulin-resistant phenotype observed in our genetic study for minor allele KLF11 promoter carriers could be related to the putative loss of STAT3 regulation over KLF11. The loss of transcriptional regulation of STAT3 over KLF11 promoter variant (–1659G>C), which may influence the quantity of KLF11 protein, may be more important than an altered transcriptional regulation activity of 62Arg-KLF11 protein. The exact role of KLF11 in glucose metabolism remains unclear. KLF11 may regulate the insulin gene, although controvert effects (stimulating/inhibiting) were shown (1, 2). Further studies are needed to clarify this discrepancy. In addition, it is still unknown whether KLF11 could affect in vivo insulin gene transcription in the presence of important regulators like PDX-1.

The KLF11–/– mice under basal conditions did not present a T2D phenotype (24), but further studies are needed to further analyze its phenotype after fasting or under diabetogenic conditions. Moreover, it will be a good model to study KLF11 action in adipose tissue, skeletal muscle, and hepatocytes to define the exact role of KLF11 in these peripheral tissues that are implicated in insulin-regulated glucose metabolism.

In conclusion, the KLF11 LD block variants are associated with insulin sensitivity in middle-aged Danish people. This phenotype association may result from an impaired STAT3 binding to KLF11 promoter sequences because STAT3 is an important regulator of KLF11 transcription.


   Acknowledgments
 
We are indebted to all individuals who participated to this study. We thank Dr. Ralph Jockers (Cochin Institute, Paris, France) and Dr. Yves Rouillé (Biology Institute of Lille, Lille, France) for their kind gift of HeLaS and HeLa-S, respectively.


   Footnotes
 
This work was supported by the European Regional Development Fund and Region Nord-Pas de Calais. B.N. was supported by a Value in People grant from the Imperial College. R.G.-A. is supported by a Société française d’exportation des ressources éducatives-Consejo Nacional de Ciencia y Technologia-Secretaría de Educaión Pública grant.

Present address for B.N.: Centre National de la Recherche Scientifique 8090, Pasteur Institute, 1 Rue du Professeur Calmette B.P.447, F-59021 Lille Cedex, France. E-mail: bneve{at}good.ibl.fr.

Disclosure statement: K.B.-J. is employed as director and professor of the Steno Diabetes Center, a hospital providing public health care but owned by Novo Nordisk A/S. Novo Nordisk strongly subsidizes the research activities of the Steno Diabetes Center. K.B.-J. also holds shares in companies related to health care and has received fees for invited lectures by Novo Nordisk, Bristol-Myers Squibb, Novartis, Pfizer, Hermedico, and AstraZeneca. R.G.-A., P.F., Y.H.H., Y.B., T.J., T.H., O.P., and B.N. have nothing to declare.

First Published Online May 27, 2008

Abbreviations: DTT, Dithiothreitol; FBS, fetal bovine serum; GAPDH, glycerhaldehyde-3-phosphate dehydrogenase; HOMA, homeostasis model assessment; Hs, human; KLF11, Krüppel-like zinc finger 11; KO, knockout; LD, linkage disequilibrium; MAF, minor allele frequencies; OGTT, oral glucose tolerance test; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; STAT, signal transducer activator of transcription; SV40, Simian virus 40; T2D, type 2 diabetes; TK, thymidine kinase.

Received November 9, 2007.

Accepted May 16, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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