Human Krüppel-like factor 11 inhibits human proinsulin promoter activity in pancreatic beta cells
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- Niu, X., Perakakis, N., Laubner, K. et al. Diabetologia (2007) 50: 1433. doi:10.1007/s00125-007-0667-3
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The Krüppel-like factor 11 (KLF11; TIEG2), a pancreas-enriched Sp1-like transcription factor, is a known negative regulator of pancreatic exocrine cell growth. A recent study indicated KLF11-induced activation of the human proinsulin promoter (hInsP).
Materials and methods
We investigated the functional role of KLF11 in pancreatic beta cells.
Endogenous KLF11 mRNA expression was found in whole rat pancreas, human pancreatic islets and INS-1E beta cells and was profoundly reduced by high glucose in INS-1E. Cotransfections of INS-1E and beta-TC3 beta cells with a human (h)KLF11 expression plasmid and an hInsP-driven reporter plasmid resulted in a substantial dose-dependent and glucose-independent inhibition of proinsulin promoter activity. 5′-deletion of hInsP demonstrated that hKLF11 acts via DNA sequences upstream of −173 and requires the beta cell-specific transcription machinery, since hKLF11-mediated inhibition of promoter activity was abolished in HEK293 cells. Besides a previously described GC box, we further identified a CACCC box within the hInsP, both putative KLF11-binding motifs. Electrophoretic mobility shift analysis (EMSA) verified binding of in vitro translated hKLF11 to the GC box, but neither hKLF11-induced inhibition nor basal hInsP activity was altered by mutation or 5′-deletion of the GC box. In contrast, CACCC box mutation substantially reduced basal promoter activity and partially diminished hKLF11 inhibition, although binding of in vitro translated hKLF11 to the CACCC box could not be verified by EMSA.
In rodent beta cell lines, we demonstrate hKLF11-overexpression of human proinsulin gene expression and characterise a prominent role for the CACCC box in maintaining basal proinsulin promoter activity.
KeywordsBeta-TC3 CACCC box GC box Human proinsulin promoter HEK293 INS-1 KLF11
electrophoretic mobility shift analysis
human proinsulin promoter
monoamine oxidase B
pancreatic duodenal homeobox protein-1
quantitative real-time PCR
secreted alkaline phosphatase
TGF-β-inducible early response gene
Krüppel-like transcription factor (KLF)11 is a member of the Sp1-like transcription factor family, which is defined by the presence of three conserved DNA-binding C-terminal zinc finger domains and variant N-terminal domains [1, 2, 3]. KLF/Sp1-like proteins bind with different selectivity to GC box or CACCC box promoter elements and take part as activators or repressors in virtually all aspects of cellular function, including cell proliferation, apoptosis, differentiation and neoplastic transformation.
Within the KLF/Sp1-like family KLF9, KLF10, KLF11, KLF13 and KLF16 are characterised by the existence of a repressor domain which interacts with the scaffold corepressor mammalian (m)Sin3A (SID or SID/R1 for KLF10 and KLF11) as a common structural feature . SID/R1 mediates chromatin modification and other repressor mechanisms via recruitment of histone deacetylases and the nuclear receptor corepressor N-CoR, respectively . Among the above-mentioned KLF proteins, KLF10 and KLF11 are further characterised by the existence of two additional repressor domains R2 and R3  and TGF-β-induced expression [6, 7]. For this reason KLF10 and KLF11 form the TGF-β-inducible early response gene (TIEG) subfamily and were alternatively named TIEG1 and TIEG2, respectively. Due to parallel cloning, KLF11 was also named FKLF .
In the adult organism, KLF11 is ubiquitously produced, but enriched in muscle and pancreas . Within the pancreas KLF11 has been described as a negative regulator of exocrine cell proliferation in transgenic mice with acinar cell-specific KLF11 overproduction in vivo and PANC1 epithelial cancer cells overexpressing KLF11 in vitro . As a result, KLF11 transgenic mice develop a significantly smaller exocrine pancreas than controls due to reduced proliferation and enhanced apoptosis. Albeit a smaller exocrine pancreas, only mild changes in tissue architecture could be observed and levels of the acinar enzymes remained normal. In vivo and in vitro reduced proliferation is accompanied by enhanced apoptosis. This is, at least in part, explainable by KLF11-mediated downregulation of the scavengers superoxide dismutase 2 and catalase 1, resulting in increased susceptibility of cells to oxidative stress.
The role of KLF11 within the endocrine pancreas remained unestablished until recently Neve et al.  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. Furthermore, cotransfected hKLF11 dose-dependently and glucose-independently inhibits the activity of hInsP in INS-1E and 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.  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.
Materials and methods
Cell lines were routinely cultured as follows. Rat INS-1E: RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 1 mmol/l sodium pyruvate, 2 mmol/l l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μmol/l β-mercaptoethanol. Media contained 11.1 mmol/l glucose unless otherwise stated. Mouse beta-TC3 beta cells and human HEK293 cells: DMEM containing 10% FBS, 25 mmol/l glucose, 2 mmol/l l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. INS-1E beta cells were kindly provided by C. B. Wollheim (University Medical Center, Geneva, Switzerland).
RT-PCR detection of KLF11 mRNA expression in pancreatic tissues and cells
Total RNA extraction was performed using Trizol (Invitrogen, Karlsruhe, Germany) for tissue or RNeasy Mini Kit (Qiagen, Hilden, Germany) for cells. RNA samples were reverse transcribed into cDNA in the presence (RT+) or absence (RT−) of Superscript II Plus using oligo-(dT) primers (both Invitrogen). RT− samples served as controls for the absence of genomic DNA contamination. Primers: rat (r)KLF11, forward 5′-GAAGCGGCACGACAGCGAAAG-3′ and reverse 5′-AGCTCTGGGCTCTGAGGAGGAGTT-3′ [annealing temperature (Ta), 64°C; product length, 250 bp]; hKLF11, forward 5′-GGTGACCTGTTGCGGATAAG-3′ and reverse 5′-CACAGGGATCATCTGGCAAAGGA-3′ (Ta, 60°C; product length, 687 bp). PCR conditions: 94°C for 2 min; 35 cycles of 94°C for 30 s, primer-specific Ta for 30 s and 72°C for 30 s; 72°C for 2 min.
The hKLF11 coding sequence was amplified from cDNA derived from human islets of Langerhans by using high fidelity Pwo Master (Roche, Mannheim, Germany). Primers: forward 5′-CACGATGCACACGCCGGACTTC-3′ and reverse 5′-GCTAGCAAAATCCCATGAGTGATGTCCTAATGG-3′. PCR conditions: 94°C for 2 min; 30 cycles, 94°C for 30 s−54°C for 30 s-72°C for 3 min. The resulting hKLF11 coding sequence was subcloned into pcR2.1-TOPO by using a TOPO TA Cloning kit (both Invitrogen) and subcloned into the CMV promoter-driven pcDNA3.1+ (Invitrogen) to obtain the hKLF11-pcDNA3.1+ expression plasmid. The −881 to +54 hInsP fragment was amplified with a BD Advantage HF PCR Kit (BD Biosciences Clontech, Heidelberg, Germany) from human genomic DNA which was extracted from whole blood using a QIAamp DNA Blood Mini Kit (Qiagen). Primers: forward 5′-TCCCTCACTCCCACTCTCCCAC-3′ and reverse 5′-TTCGAATTGGAACAGACCTGCTTGATGGCC-3′. PCR conditions: 94°C for 3 min; 28 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 90 s; 68°C for 2 min. The resulting -881+54hInsP fragment was subcloned into pcR2.1-TOPO plasmid using a TOPO TA Cloning kit (both Invitrogen) and subcloned into pSEAP2-Basic (BD Biosciences Clontech) to obtain the −881hInsP-pSEAP reporter plasmid (SEAP, secreted alkaline phosphatase). −881hInsP-pSEAP was used for generation of 5′-deleted hInsP fragments (−387, −355, −323, −254, −173, −101 and −85) with an identical 3′-end (+54) by PCR, which were then subcloned via pcR2.1-TOPO into pSEAP2-Basic. GC box and CACCC box mutations were created using a QuickChange Site-Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany). The GC box was mutated from 5′-CCCGCCCT-3′ into 5′-CCCGAACT-3′ and the CACCC box from 5′-CCCACCCC-3′ into 5′-CCCGATTCC-3′. Accuracy of all sequences and mutations was checked by sequencing.
Protein extraction and in vitro translation
For generation of whole cell extracts, freshly prepared RIPA buffer (Upstate/Biomol, Hamburg, Germany) containing one tablet per 10 ml Complete Mini EDTA-free protease inhibitor cocktail (Roche) was used. Nuclear extracts were acquired according to Schreiber et al. . hKLF11 was in vitro translated from hKLF11-pcDNA3.1+ using a TNT Quick Coupled Transcription/Translation System (Promega, Mannheim, Germany). Control reactions were performed in the absence of hKLF11-pcDNA3.1+.
Western blot analysis
Ten micrograms of protein from whole cell extract and nuclear extract or 1 μl in vitro translated reactions were separated on 10% PAGE-SDS gels. Proteins were transferred to polyvinylidene difluoride membranes by semi-dry blotting using Towbin transfer buffer. KLF11 was detected using a goat polyclonal antiserum (TIEG2 C-12, sc-23162; Santa Cruz, Heidelberg, Germany). Visualisation was achieved by a horseradish peroxidase-linked rabbit anti-goat secondary antibody (DAKO, Hamburg, Germany) and ECL Western Blotting Detection Reagent (Amersham, Freiburg, Germany).
SEAP reporter gene experiments
INS-1E, beta-TC3 and HEK293 cells were seeded in six-well plates at a density of 3 × 105 cells per well. Next day, cells were transiently transfected using Metafectene (Biontex, Martinsried/Planegg, Germany). The proportion of DNA (μg) to Metafectene (μl) was 1:2. Each plasmid was transfected at a concentration of 0.5 μg unless otherwise stated. After 48 h, supernatants were collected for measurement of SEAP using a BD Great EscAPe SEAP Chemiluminescence Detection Kit (BD Biosciences Clontech).
Quantification of insulin gene expression in INS-1E beta cells in response to KLF11 overexpression
INS-1E cells were seeded in six-well plates at a density of 3 × 105 cells per well in the presence of 11.1 mmol/l glucose. Next day, cells were transiently transfected with either rKLF11-pcDNA3.1+ or pcDNA3.1+ (mock control level) using Metafectene Pro (Biontex). The proportion of DNA (μg) to Metafectene Pro (μl) was 1:2. Each plasmid was transfected at a concentration of 1 μg/well. After 48 h, cells were harvested for analysis of monoamine oxidase B gene (MAOB), insulin-1 (Ins1) gene and insulin-2 (Ins2) gene mRNA expression levels using quantitative real-time PCR (qPCR) (see below).
Quantification of KLF11 mRNA levels in INS-1E beta cells in response to high glucose stimulation
INS-1E cells were seeded in six-well plates at a density of 3 × 105 cells per well in the presence of 11.1 mmol/l glucose. Next day, medium was replaced by medium containing 2.8 mmol/l glucose. After 24 h, cells were washed with PBS and exposed to medium containing either 2.8 or 25 mmol/l glucose for 6–48 h. Finally, cells were harvested for analysis of KLF11 mRNA expression levels using qPCR (see below).
Total RNA was isolated using the RNeasy Mini Kit (Qiagen), treated with Turbo DNA-free Kit (Ambion/Applied Biosystems, Darmstadt, Germany) and checked for contamination with genomic DNA by PCR with primers for rat hypoxanthine phosphoribosyltransferase gene (HPRT) (Quantitect Rn_HPRT1; Qiagen). RNA free from genomic DNA was reverse transcribed with Superscript III and oligo-(dT) primers (both Invitrogen). cDNA samples were investigated for mRNA levels by qPCR using Lightcycler 2.0 (Roche) and Faststart DNA Master+ CYBR Green I kit (Roche). Concentrations of mRNA were calculated from standard curves for each specific primer pair (dilution series: 1:1, 1:10, 1:100, 1:1,000 and 1:10,000) by Light Cycler Software 3.5 (Roche). All samples and standards were analysed in triplicates. Primer pairs for rat HPRT (Quantitect Rn_HPRT1), rat Ins1 (Quantitect Rn_INS1), rat Ins2 (Quantitect Rn_INS2) and rat MAOB (Quantitect Rn_MAOB) were obtained from a commercial supplier (Qiagen). Product length of all Quantitect primer pairs is 125 bp. See above for primers for rKLF11. qPCR conditions were: 95°C for 10 min; 40 cycles of 95°C for 15 s, 55°C (Quantitect primers) or 64°C (rKLF11 primers) for 10 s and 72°C for 20 s; followed by melting curve analysis for specificity of qPCR products.
Glucose-induced insulin secretion from INS-1E beta cells
INS-1E cells were seeded in six-well plates at a density of 3 × 105 cells per well in the presence of 11.1 mmol/l glucose. Next day, medium was replaced by medium containing 2.8 mmol/l glucose. After 24 h cells were washed twice with PBS and exposed to serum-free medium containing either 2.8 or 25 mmol/l glucose. After 1 h, medium samples were collected and centrifuged for 5 min at 16,000 g. Supernatant fractions were transferred to new tubes and stored at −20°C until measurement of secreted insulin using the High Range Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden).
One microlitre of in vitro translated reactions was added to 5 × EMSA buffer (100 mmol/l KPO4, pH 7.9, 5 mmol/l EDTA, 5 mmol/l dithiothreitol and 20% glycerol) and additionally supplemented with 50 mmol/l KCl, 1 μg poly-dIdC and 40,000 cpm/μl 32P-labelled double-stranded oligonucleotide. The following DNA probes were generated by annealing two oligonucleotides, followed by a fill-in reaction with Klenow polymerase and dGTP, dCTP, dTTP and α-32P-labelled dATP: GC box, 5′-GATCAAAGAGCCCCGCCCTGCAGCC-3′; GC box mutant, 5′-GATCAAAGAGCCCCGAACTGCAGCC-3′; CACCC box, 5′-GATCCGACCCCCCCACCCCAGGCCC-3′; CACCC box mutant, 5′-GATCCGACCCCCCGATTCCAGGCCC-3′. After incubation on ice for 15 min, samples were loaded onto a 5% polyacrylamide gel and run in 0.5 × Tris-borate-EDTA buffer for 2 h. For supershift assays, 2 μl of KLF11 antiserum (see above) were added and incubated on ice for 20 min before samples were loaded onto the gel.
Inhibition of insulin gene expression by KLF11 overexpression, glucose-induced insulin secretion and regulation of endogenous KLF11 mRNA expression by glucose
Binding of KLF11 to the GC and CACCC box
Inhibition of hInsP 5′-deletion constructs by hKLF11
Effects of GC box and CACCC box mutation on hKLF11-inhibited and basal hInsP activity
The TIEG subfamily, consisting of KLF10 and KLF11, is defined by the three N-terminal repressor domains SID/R1, R2 and R3. These domains have been demonstrated to repress transcription activity [4, 5]. In this sense, KLF11 acts as a dominant repressor of the caveolin-1 gene  and, besides its negative regulation of cell growth in the exocrine pancreas , KLF11 overexpression also inhibits cell proliferation in Chinese hamster ovary cells . KLF11 further suppressed oncogene-induced neoplastic transformation in mouse NIH-3T3 cells, and consequently KLF11 mRNA expression was found to be significantly downregulated in a substantial amount (50%) of investigated pancreatic, breast and kidney tumours . In this context, TGF-β inhibits growth of epithelial cells by activation of Smad signalling, which is potentiated through TGF-β-induced KLF11 by termination of the negative feedback loop imposed by Smad7 . In pancreatic cancer cells with an oncogenic Ras mutation this function is inhibited by ERK/mitogen-activated protein kinase phosphorylation of KLF11, leading to disruption of KLF11-mSin3a interaction, and thereby ends silencing of the Smad7 promoter [14, 15].
In line with these transcription-repressing properties of KLF11, our results demonstrate a dose-dependent inhibition of hInsP activity by cotransfected hKLF11 in INS-1E beta cells which is similar in standard and high glucose conditions. The observed hKLF11-mediated inhibition of hInsP activity is underlined by the reduction of Ins2 gene expression by rKLF11 overexpression. 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.  reported a high glucose-induced 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 . More importantly, Neve et al.  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. Noteworthy, the obtained inhibition was very similar in both beta cell lines demonstrating stable performance of our experimental approach in rodent beta cells independently of the species (rat and mouse). Since the hKLF11-induced inhibition of wild-type and mutated hInsP constructs was completely abolished in HEK293 cells, this function seems to be strictly dependent on the beta cell-specific transcription machinery.
Sp1-like proteins are known to bind with different selectivity to CGCCC or CACCC core sequences in GC-rich sites . 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.  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. This demonstrates that, at least in the context of hInsP, the inhibitory function of hKLF11 is GC box-independent. Moreover, the GC box is also dispensable for the maintenance of basal hInsP activity.
Searching for alternative KLF11-binding sites within hInsP we identified a CACCC box located from −88 to −96. The CACCC sequence was initially reported to be required for KLF11-mediated activation of -globin gene promoter , whereas a later study failed to induce significant alterations of -globin gene promoter activity by cotransfected KLF11 . However, KLF11 seems to be expendable for globin gene expression since KLF11−/− mice display normal haematopoiesis at all stages of development . Interestingly, Ou et al.  confirmed binding of KLF11 to both Sp1/GC and CACCC sites, thereby influencing the human MAOB promoter as an activator via its Sp1/GC site or as a repressor via its CACCC site. In contrast, we could not detect binding of in vitro translated hKLF11 to the CACCC box sequence within hInsP. This indicates that the observed decrease in hKLF11 inhibitory function due to CACCC box mutation may be caused by indirect interactions. Nevertheless, an involvement of the CACCC box in KLF11 action is supported by the specific KLF11-mediated inhibition of Ins2 gene expression while Ins1 gene expression remains unaffected. Of note, only the Ins2 promoter sequence contains a CACCC box.
Interestingly, mutation of the CACCC box substantially downregulated basal activity of hInsP in beta-TC3 beta cells and also HEK293 cells to about 25 and 50% of wild-type control levels, respectively. These results suggest that the CACCC box is mainly a target for general transcription factors independently of the beta cell-specific transcription machinery and demonstrates the requirement of a functional CACCC box for the maintenance of normal human proinsulin gene expression. From our results we conclude that this function is independent of the involvement in hKLF-mediated inhibition of hInsP.
Considering all findings we presume that hKLF11 may mainly act via another yet unknown site of hInsP or, more likely, indirectly by interfering with beta cell-specific transcription factors. This reasoning was initiated by the described interaction of KLF1 [19, 20], KLF2 , KLF4 [22, 23] and KLF13 [24, 25] with the transcriptional cofactors CBP and p300, which also interact with the pancreatic duodenal homeobox protein (PDX)-1, a major transactivator of proinsulin gene expression in pancreatic beta cells [26, 27]. Of note, KLF13 and KLF11 belong to the same KLF subfamily that is functionally characterised by the SID motif. The speculation of interactions between KLF11 and PDX-1 is supported by the results from 5′-deletion of hInsP demonstrating stepwise decrease of KLF11-induced inhibition. PDX-1 is known to bind to A elements present in the proinsulin promoter region. Although KLF11 function is unaffected by deletion of A5 (−254hInsp fragment), further deletion of A3 (-173hInsp fragment) reduced and deletion of both A3 and A2 (−101hInsP fragment) completely abolished KLF11-mediated inhibition.
In summary, high glucose conditions stimulating insulin production and secretion repress endogenous KLF11 expression in INS-1E beta cells. This is in line with our results characterising hKLF11 as a glucose-independent negative regulator of hInsP in INS-1E and beta-TC3 beta cells. Interestingly, KLF11 specifically reduced Ins2 but not Ins1 gene expression in INS-1E beta cells and only the Ins2 promoter contains a CACCC box. Although we could not verify KLF11 interaction with the CACCC box, KLF11-mediated inhibition of human proinsulin promoter activity depends on a functional CACCC box, thereby indicating an indirect mechanism. In contrast, and regardless of observed specific binding, the GC box was dispensable for this KLF11 function. Moreover, we demonstrate a new and substantial role for the CACCC box in maintaining basal hInsP activity. In conclusion, these findings may contribute to a better understanding of the complex regulation of proinsulin gene expression and may suggest that dysregulation of KLF11 function and CACCC box mutation have an impact on the development and clinical manifestation of diabetes mellitus.
This study was supported by the Bundesministerium für Bildung und Forschung (BMBF), Germany (grants FKZ 01GN0114 and FKZ 01GN0115). X. Niu was a recipient of an academic exchange fellowship by the German Academic Exchange Service (DAAD). G. Päth was supported by the Interdisciplinary Center of Clinical Research (IZKF) at the University of Würzburg, Germany (grant Z-4/57 and Z-4/66). C. Limbert is recipient of a fellowship by the European Society of Pediatric Endocrinology (ESPE).
Duality of interest
There is no duality of interest to report.