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Endocrinology, doi:10.1210/en.2007-0865
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Endocrinology Vol. 150, No. 1 87-97
Copyright © 2009 by The Endocrine Society

The Homeodomain-Interacting Protein Kinase 2 Regulates Insulin Promoter Factor-1/Pancreatic Duodenal Homeobox-1 Transcriptional Activity

Marie-Josée Boucher, Mélanie Simoneau and Helena Edlund

Umeå Center for Molecular Medicine (M.-J.B., H.E.), Umeå University, Umeå 901 87, Sweden; Service de Gastroentérologie/Département de Médecine (M.J.-B.), Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4; and Département d’Anatomie et Biologie Cellulaire (M.S.), Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Cananda J1H 5N4

Address all correspondence and requests for reprints to: Marie-Josée Boucher, Service de Gastroentérologie/Départment de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Cananda J1H 5N4. E-mail: marie-josee.boucher{at}usherbrooke.ca; or Helena Edlund, Umeå Center for Molecular Medicine, University of Umeå, S-901 87 Umeå, Sweden. E-mail: helena.edlund{at}ucmm.umu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homeodomain transcription factor insulin promoter factor (IPF)-1/pancreatic duodenal homeobox (PDX)-1 plays a crucial role in both pancreas development and maintenance of β-cell function. Targeted disruption of the Ipf1/Pdx1 gene in β-cells of mice leads to overt diabetes and reduced Ipf1/Pdx1 gene expression results in decreased insulin expression and secretion. In humans, mutations in the IPF1 gene have been linked to diabetes. Hence, the identification of molecular mechanisms regulating the transcriptional activity of this key transcription factor is of great interest. Herein we analyzed homeodomain-interacting protein kinase (Hipk) 2 expression in the embryonic and adult pancreas by in situ hybridization and RT-PCR. Moreover, we functionally characterized the role of HIPK2 in regulating IPF1/PDX1 transcriptional activity by performing transient transfection experiments and RNA interference. We show that Hipk2 is expressed in the developing pancreatic epithelium from embryonic d 12–15 but that the expression becomes preferentially confined to pancreatic endocrine cells at later developmental stages. Moreover, we show that HIPK2 positively influences IPF1/PDX1 transcriptional activity and that the kinase activity of HIPK2 is required for this effect. We also demonstrate that HIPK2 directly phosphorylates the C-terminal portion of IPF1/PDX1. Taken together, our data provide evidence for a new mechanism by which IPF1/PDX1 transcriptional activity, and thus possibly pancreas development and/or β-cell function, is regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Insulin promoter factor (IPF)-1/pancreatic duodenal homeobox (PDX)-1 is a homeodomain transcription factor that is first expressed in the prospective pancreatic region of the developing foregut on embryonic day (e) 8.5 in mouse (1, 2). The key role for IPF1/PDX1 in pancreas development has been demonstrated in genetic studies in which targeted inactivation of the Ipf1/Pdx1 gene in mice (2, 3) and homozygosity for a nonsense mutation in the human IPF1 gene (4) resulted in pancreatic agenesis. Later in development and in adulthood, IPF1/PDX1 is highly expressed in β-cells in which it ensures β-cell function. Conditional inactivation of the Ipf1/Pdx1 gene selectively in β-cells leads to diabetes in mice (5), whereas heterozygosity for the null mutation, and hence reduced IPF1/PDX1 expression levels, affect insulin expression (5), insulin secretion (6), and predispose islets to apoptosis (7). In humans, heterozygosity for the nonsense mutations has been linked to maturity-onset diabetes of the young (8) and missense mutations have been linked to type 2 diabetes (9). Taken together, these findings clearly emphasize the key role for Ipf1/Pdx1 in β-cell function and maintenance of normoglycemia.

IPF1/PDX1 controls the expression of several genes that together ensure β-cell function. IPF1/PDX1 has been shown to directly bind to and regulate the promoter activity of various β-cell genes such as insulin (1), glut2 (10), glucokinase (11), and islet amyloid polypeptide (12, 13). However, like many other transcription factors, the regulation of IPF1/PDX1 target genes is also governed by interactions between IPF1/PDX1 protein and other transcription factors, coactivators, or corepressors. Hence, IPF1/PDX1 transcriptional activity has been shown to be strengthened by interactions with Pbx1 (pre-B-cell leukemia homeobox 1)(14, 15), p300 (16, 17, 18), and bridge-1 (19), whereas interaction with PCIF1 inhibits IPF1/PDX1 transcriptional activity (20, 21). We and others have recently shown that IPF1/PDX1 can be posttranslationally modified by phosphorylation (22, 23), and other reports have suggested that glycosylation (24) and sumoylation (25) events can also affect IPF1/PDX1 function. Thus, IPF1/PDX1 transcriptional activity, and hence the level of IPF1/PDX1 target gene expression, will depend on interactions with other transcription factors and cofactors as well as on posttranslational modifications.

The family of homeodomain-interacting protein kinases (HIPK1, HIPK2, HIPK3) was originally identified as binding partners of the homeodomain protein neurokinin (NK)-3 (26). NK-3 belongs to the NK-2 family of protein, which includes a large number of homeodomain transcription factors that have important functions during embryonic development and organogenesis (27). Several independent studies have shown that the HIPK family, and in particular the most studied member HIPK2, interacts with, phosphorylates, and modulates the function of other homeodomain proteins as well as other transcription factors, including p53, carboxy-terminal binding protein 1, and myeloblastosis oncogene (28, 29, 30, 31, 32), providing evidence for an important role for HIPKs in the regulation of transcription.

The current study was undertaken to define molecular mechanisms that modulate IPF1/PDX1 transcriptional activity. Here we show that Hipk2 expression overlaps with IPF1/PDX1 expression in the developing and adult pancreas. Moreover, we demonstrate that HIPK2 positively modulates IPF1/PDX1 transcriptional activity. In addition, we show that HIPK2 is able to directly phosphorylate the C-terminal portion of IPF1/PDX1. Hence, our data provide evidence of a new mechanism by which IPF1/PDX1 transcriptional activity is regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Islet isolation and cell culture
Mouse islets were isolated by collagenase digestion of the pancreas (33). The mouse {alpha}TC1/16, βTC3, and MIN6 cell lines and the human embryonic kidney 293 (HEK293 and HEK293T) cells were cultured as previously described (22). The rat insulinoma INS1E cell line was a kind gift of C. B. Wollheim and P. Maechler (Geneva, Switzerland) and was cultured as originally published (34).

In situ hybridization, immunohistochemistry, and immunofluorescence
In situ hybridization and immunohistochemistry were performed either successively on the same section or on consecutive sections of isolated and fixated embryonic and adult tissue essentially as described elsewhere (35). To circumvent the problem of cross-reactivity with Hipk2 relatives, Hipk1 and Hipk3, the digoxigenin-labeled RNA probe used for this study was a 671-bp fragment overlapping the 3' coding region and 3' untranslated region of hipk2.

For indirect immunofluorescence studies, HEK293 cells were grown on sterile glass coverslips and cotransfected with Flag-HIPK2- and His-IPF1/PDX1-expressing plasmids. Cells were then washed twice with ice-cold PBS and fixed in methanol/acetone (30:70) for 15 min at –20 C. After being dried, cells were blocked for 30 min with 10% fetal bovine serum (room temperature). Cells were then immunostained 2 h at room temperature with the primary antibody and washed in PBS before incubation for 1 h with appropriate secondary antibody.

The following antibodies were used: mouse anti-Flag M2 (Sigma, Stockholm, Sweden), rabbit anti-IPF1/PDX1 (1, 22), Alexa-488-coupled goat antimouse (Molecular Probes, Stockholm, Sweden), and CY3-coupled goat antirabbit (Jackson Laboratories, Sulzfeld, Germany). Chromosomal DNA was revealed by staining with 4',6-diamidino-2-phenylindole (Sigma).

Transient transfections and luciferase assays
Experiments were performed essentially as described previously (22). Briefly, cells were transfected either with Lipofectamine or Lipofectamine 2000 according to the manufacturer’s recommendations. The pcDNA3.1-Ipf1/Pdx1 wild-type (WT) (His tagged), S61A, S61D, S66A, S66D, and S61/66A expression vectors and the insulin reporter construct, RIP WT-luciferase were as published (22). The mutant rat insulin promoter (RIP) promoter, which transcriptional activity is about 2.5-fold lower of that of the wt promoter (1), carries a mutation in the IPF1/PDX1 binding site (the AA residues in the TAATGGG P1 motif were changed to CC) to which IPF1/PDX1 fails to bind (1), and was cloned upstream of the luciferase gene (RIP MUT-luciferase). The IPF1/PDX1-dependent reporter construct (5XP1-luciferase) used for luciferase assays contains five copies of the IPF1/PDX1 minimal binding site (P1) linked to the β-globin TATA box (1) and cloned upstream of the luciferase gene. Flag-tagged expression vectors for Hipk2 WT and the kinase-inactive Hipk2 (K221A) were a kind gift of L. Schmitz (Giessen, Germany) and S. Soddu (Rome, Italy) and have been described elsewhere (29, 30). The following gal4-IPF1/PDX1 constructs were a kind gift of M. D. Walker (Rehovot, Israel). The gal4-IPF1/PDX1[amino acids (aa) 1–91] and gal4-IPF1/PDX1(aa1–138) constructs displayed higher transactivation potential (300 and 1000 times, respectively) than the gal4-IPF1/PDX1(aa1–283) and gal4-IPF1/PDX1(aa1–215) constructs (data not shown and Glick, E., and M. Walker, personal communication) but the relative expression from each independent gal4-IPF1/PDX1 construct in absence of HIPK2 was normalized to 1. For the gal4-IPF1/PDX1 (aa168–283), the C-terminal IPF1/PDX1 (aa168–283) portion was cloned in frame downstream of the gal4 DNA binding domain in the pM2 vector. The p300-expressing vector and the pFR-luciferase reporter gene were a kind gift of C. Asselin (Sherbrooke, Canada) (36). The mouse cDNA coding for E2A (pCMV-SPORT6-E2A) was from an image clone (GI:17390605). The control small interfering RNA (siRNA), Hipk2 siRNA and p300 siRNA used were from Santa Cruz Biotechnology (Falkenberg, Sweden). Empty expression plasmid was used to maintain a constant total amount of DNA among wells. Cells were harvested 36–48 h after transfection and RNA extraction, for cDNA preparation and RT-PCR analyses, or protein cell extraction, for Western blot analyses, or for luciferase assays were carried out. For luciferase assays, the data are expressed as firefly activity normalized to the activity of Renilla luciferase (pRL-CMV, Promega).

RNA extraction and RT-PCR
Total RNAs were extracted using a NucleoSpin RNA II kit (BD Biosciences, Stockholm, Sweden) following the manufacturer’s instructions. Reverse transcription reactions were performed using the SuperScript first-strand synthesis kit (Invitrogen, Stockholm, Sweden), and then 50 ng of cDNA were used as templates for PCR. The primers used were the following: Hipk2 (5'-AGTTTTCTCCCCTCACAC-3' and 5'-TAGGTTATGTGGTCCACC-3'), Ipf1/Pdx1 (5'-AGAAGCTGGCCACTAGCCTCT-3' and 5'-CTGTGGGCAACAAGGGAGTT-3'), and gapdh (5'-ACGGCAAATTCAACGGCACAG-3' and 5'-GGTCATGAGCCCTTCC ACAAT-3'). Parameters for DNA amplification were 94 C for 30 sec, annealing temperature (55 C for Hipk2 and 67 C for Ipf1/Pdx1 and gapdh) for 30 sec, and 72 C for 1 min. DNA amplification products were analyzed by gel electrophoresis on an agarose gel stained with ethidium bromide.

Western blotting
The cells were washed twice with ice-cold PBS, lysed in high-salt lysis buffer [1% Nonidet P-40, 50 mM Tris (pH 7.5), 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 10 mM NaF, 10% glycerol, 0.2 mM orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 µg/ml aprotinin] and cleared of cellular debris by centrifugation (10,000 rpm, 10 min, 4 C). Protein concentrations were measured using bicinchoninic acid reagent (Pierce, Stockholm, Sweden) with BSA as standard. Equal amounts of proteins were separated by SDS-PAGE, and proteins were detected immunologically after electrotransfer onto nitrocellulose membranes. Membranes were blocked in PBS containing 5% nonfat dry milk and 0.05% Tween 20 for 1 h at 25 C. Membranes were then incubated with appropriate primary antibodies in blocking solution followed by incubation with horseradish peroxidase-conjugated secondary antibodies. After extensive washing in PBS-0.05% Tween 20, blots were visualized with chemiluminescence reagent. The following antibodies were used: mouse anti-Flag M2 (Sigma), mouse antiglutathione-S-transferase (GST), rabbit anti-p300, mouse anti-gal4 (Santa Cruz), mouse anti-His (QIAGEN, Solna, Sweden), rabbit anti-ERK1/2 (Cell Signaling, Stockholm, Sweden) and rabbit anti-panIPF1/PDX1 (22).

IPF1/PDX1-GST fusion protein purification
Mouse IPF1/PDX1 full-length (amino acids 1–283), mouse N-terminal (amino acids 1-168), and mouse C-terminal IPF1/PDX1 (amino acids 213–283) cDNAs were ligated downstream of the glutathione S-transferase sequence in pGEX plasmid. The recombinant plasmid was introduced into Escherichia coli BL21, and the fusion protein was produced by growing 1 liter of a bacterial culture to an OD of 0.4 (at 600 nm) and then treating the culture with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside for 3 h. Bacteria were recovered, resuspended in lysis buffer [PBS (pH 7.5), EDTA 10 mM, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptine, 0.7 µg/ml pepstatin, 0.5 µg/ml aprotinin], and sonicated. Triton X-100 was added to the lysate to a final concentration of 1%. The bacterial lysate was incubated on ice for 10 min and centrifuged at 10,000 rpm for 15 min. The supernatant was recovered and mixed with glutathione-Sepharose 4B beads, and mixture was rotated for 30 min at 4 C. The beads were then washed extensively first in lysis buffer containing 1% Triton X-100 and then in 100 mM HEPES (pH 7.6). The IPF1/PDX1-GST proteins were eluted with 100 mM HEPES containing 50 mM glutathione (pH 7.6).

HIPK2 kinase assays
Kinase assays were essentially done as described by the manufacturer. Briefly, IPF1/PDX1 was immunoprecipitated as previously described (22). Protein G-Sepharose complexes were washed three times with Triton buffer [1% Triton X-100, 50 mM Tris (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 0.2 mM orthovanadate, 0.1 mM phenylmethyl sulfonyl fluoride, 0.5 µg/ml leupeptine, 0.7 µg/ml pepstatin, 0.5 µg/ml aprotinin] and twice in kinase buffer [40 mM 3(N-morholino)propanesulfonic acid (pH 7.0), 1 mM EDTA]. The beads were equally split in two Eppendorf tubes and kinase assays were performed by adding or not 50 ng of an active HIPK2 (Upstate, Lake Placid, NY) in kinase buffer supplemented with 500 µM ATP and 2 µCi [{gamma}-32P]ATP. Alternatively, 100–200 ng IPF1/PDX1-GST protein was incubated with or without 50 ng of an active HIPK2 in kinase buffer supplemented with 500 µM ATP and 2 µCi [{gamma}-32P]ATP. Reaction was stopped after incubation at 30 C for 30 min by addition of Laemmli buffer [62.5 mM Tris-HCl (pH 6.8), 2.2% SDS, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5% β-mercaptoethanol, 0.005% bromophenol blue]. Radiolabeled IPF1/PDX1 was separated by SDS-PAGE and processed for autoradiography.

Data presentation and statistical analysis
All of the experiments were performed at least in triplicate, and results were analyzed by the Student’s t test and were considered significantly different at P < 0.05. Typical Western blots are shown. Densitometric analyses were carried out using the Scion Image 4.02 software package (Scion Corp., Frederick, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hipk2 is expressed in the developing and adult pancreas
To begin to explore a potential role for HIPK2 in the regulation of IPF1/PDX1 transcriptional activity, we first mapped Hipk2 expression in the pancreas by in situ hybridization. Hipk2 expression was not detectable in e10 dorsal and ventral pancreatic buds, although, as previously reported (37, 38), strong expression of Hipk2 was observed in the developing neural tissues at this stage (data not shown). From e12 to e15, Hipk2 was expressed throughout the pancreatic epithelium (Fig. 1AGo). At later stages of development (e17), Hipk2 was preferentially expressed in the differentiating endocrine cells (Fig. 1AGo). At both early and late stages of pancreatic development the expression of Hipk2 overlapped with that of IPF1/PDX1, providing evidence that Hipk2 is expressed in both pancreatic progenitor cells and differentiating β-cells.


Figure 1
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  		FIG. 1. Hipk2 expression overlaps with IPF1/PDX1 expression. A, In situ hybridization of e12, e13, e15, and e17 mouse pancreas using a digoxigenin-labeled Hipk2 cDNA probe (blue) and immunostaining of consecutive sections with antibodies against IPF1/PDX1 (green). Note that in the e12 and e17 overlays, Hipk2 expression is in red pseudocolor. Bar, 100 µM. B, RT-PCR analysis with Hipk2 primers using cDNA from pancreatic cell lines or islets as templates as indicated. C. HEK293 cells were transiently transfected with expression vectors encoding a Flag-tagged HIPK2 WT and a His-tagged IPF1/PDX1 WT. Forty-eight hours after transfection, indirect immunofluorescence studies were undertaken using Flag (in green) and His (in red) antibodies.

 
To elucidate whether Hipk2 expression was maintained in adult islet cells, we assessed Hipk2 expression by RT-PCR using cDNA prepared from isolated mouse islets. Hipk2 expression was detected in islets as well as in the insulinoma cells βTC3, MIN6, and INS-1E (Fig. 1BGo). In agreement with the previously described role for HIPK2 in modulation of paired box transcription factor (Pax)-6 transcriptional activity (28), Hipk2 expression was also observed in the pancreatic glucagonoma cell line {alpha}TC1/16 (Fig. 1BGo). Taken together, our expression analyses provide evidence that the expression of Hipk2 overlaps with that of IPF1/PDX1 from e12 onward in the developing and adult mouse pancreas.

Due to the lack of a commercially available antibody that can recognize the endogenous HIPK2, the subcellular localization of HIPK2 and IPF1/PDX1 proteins were determined after cotransfection of Flag-HIPK2- and His-IPF1/PDX1-tagged proteins in HEK293 cells. In agreement with previous findings (26, 29, 30), HIPK2 was mostly localized to nuclear dots, whereas IPF1/PDX1 was uniformly distributed throughout the entire nucleus (Fig. 1CGo). In contrast to other studies that have reported changes in subcellular localization of c-Ski (39) and p53 (29, 30) after HIPK2 coexpression, the subcellular localization of IPF1/PDX1 was identical, regardless of whether HIPK2 was overexpressed (Fig. 1CGo). Thus, HIPK2 and IPF1/PDX1 both localize to the nucleus.

HIPK2 influences IPF1/PDX1 transcriptional activity
Given that HIPK2 was originally identified as a kinase modulating the activity of homeodomain transcription factors (26), we investigated whether HIPK2 could also modulate IPF1/PDX1 transcriptional activity. Transfection of increasing amounts of wild-type Hipk2 (HIPK2 WT) in a non-β-cell line, HEK293, dose-dependently increased the IPF1/PDX1-induced activity of a reporter construct that contains five copies of the minimal binding site for IPF1/PDX1 (5XP1) (1), whereas a kinase-inactive form of Hipk2 (HIPK2 KA) had no effect (Fig. 2AGo). Thus, HIPK2 positively influences IPF1/PDX1 transcriptional activity, and the kinase activity of HIPK2 is required for this effect. Similarly, HIPK2, in a dose-dependent manner, significantly enhanced IPF1/PDX1 transactivation of the insulin promoter (Fig. 2BGo). In absence of IPF1/PDX1, HIPK2 had no effect on either the 5XP1-luciferase or the insulin promoter reporter gene activity (Fig. 2Go, C and D, and data not shown).


Figure 2
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  		FIG. 2. HIPK2 increases IPF1/PDX1 transcriptional activity. A and B, HEK293 cells were transfected with 0.1 µg of pcDNA3-Ipf1/Pdx1 WT, the indicated amount of Hipk2-encoding vector, 0.1 µg 5XP1-luciferase (A), or 0.1 µg RIP-luciferase (B), and 0.01 µg Renilla-luciferase. Total amount of DNA was maintained by addition of empty expression plasmid. *, Significantly different from cells transfected with Ipf1/Pdx1 alone P < 0.001. C, HEK293 cells were transfected with 0.1 µg of pcDNA3-Ipf1/Pdx1 WT, 0.3 µg of p300-encoding vector, 0.2 µg of Hipk2-encoding vector, 0.1 µg RIP-luciferase and 0.01 µg Renilla-luciferase. Equal total amount of DNA was maintained by addition of empty expression plasmid. *, Significantly different from cells transfected with Ipf1/Pdx1 alone P < 0.001; **, significantly different from cells transfected with Ipf1/Pdx1 + Hipk2, P < 0.001. D, HEK293 cells were transfected with 0.1 µg of pcDNA3-Ipf1/Pdx1 WT, 0.1 µg of E2A-encoding vector, 0.2 µg of Hipk2-encoding vector, 0.1 µg RIP-luciferase and 0.01 µg Renilla-luciferase. Equal total amount of DNA was maintained by addition of empty expression plasmid. *, Significantly different from cells transfected with Ipf1/Pdx1 alone P < 0.001; **, significantly different from cells transfected with Ipf1/Pdx1 + Hipk2 P < 0.001. E, HEK293T cells were transfected with a control (ctl) or a p300 siRNA. Protein lysates were prepared 36 h after transfection. Western blot analysis of p300 and ERK1/2 protein expression levels in control siRNA- and p300 siRNA-transfected cells are showed. F, HEK293T cells were transfected with a control (ctl) or a p300 siRNA together with 0.1 µg of pcDNA3-Ipf1/Pdx1 WT, 0.2 µg of Hipk2-encoding vector, 0.1 µg 5XP1-luciferase, and 0.01 µg Renilla-luciferase. Relative activity in pcDNA3 + control siRNA-transfected cells was set at 1. *, P < 0.001.

 
Because the insulin promoter is not solely regulated by IPF1/PDX1 but also influenced by coactivators and other transcription factors (40), we next investigated whether HIPK2 could cooperate with the coactivator p300 or the transcription factor E2A to modulate insulin promoter activity. In absence of IPF1/PDX1, cotransfection of p300 or E2A with HIPK2 WT or HIPK2 KA had no effect on the insulin promoter activity, (Fig. 2Go, C and D). Cotransfection of IPF1/PDX1 with either p300 (Fig. 2CGo) or E2A (Fig. 2DGo) significantly enhanced the IPF1/PDX1-induced insulin promoter activity by 1.84- and 2.93-fold, respectively. Transfection of HIPK2 WT increased the IPF1/PDX1-induced insulin promoter activity by nearly 3-fold. (Fig. 2Go, A–D), and transfection of HIPK2 WT together with p300 increased by more than 5-fold the IPF1/PDX1-induced insulin promoter activity (Fig. 2CGo). Similarly, cotransfection of E2A and HIPK2 WT resulted in a 6.7-fold increase in IPF1/PDX1-mediated transactivation of the insulin promoter (Fig. 2DGo). Taken together, these results suggest that HIPK2, in an additive manner, cooperates with coactivators and transcription factors to positively modulate the insulin promoter activity. The lack of increased transcriptional activity upon cotransfection of HIPK2, and either p300 (Fig. 2CGo) or E2A (Fig. 2DGo), but not IPF1/PDX1, emphasizes the key role for IPF1/PDX1 in HIPK2-mediated stimulation of insulin promoter activity.

To further evaluate whether HIPK2 WT stimulation of IPF1/PDX1 transcriptional activity was dependent on p300, IPF1/PDX1 ± HIPK2 WT was cotransfected with p300 siRNA or control siRNA. Transfection of the p300 siRNA decreased by more than 80% the p300 protein expression levels (Fig. 2EGo). As expected, p300 siRNA did not affect the basal activity of the 5XP1-luciferase reporter gene but reduced the IPF1/PDX1-induced 5XP1-luciferase reporter gene activity by approximately 40% (Fig. 2FGo). Nevertheless, cotransfection of HIPK2 WT to either control siRNA- or p300 siRNA-transfected cells was still able to significantly increase the IPF1/PDX1-induced 5XP1-luciferase reporter gene activity (Fig. 2FGo). These results suggest that although p300 contributes to the transactivation potential of IPF1/PDX1, reduced levels of p300 do not affect the HIPK2-mediated stimulatory effect on IPF1/PDX1 transcriptional activity.

HIPK2 independently affects IPF1/PDX1 transcriptional activity and protein levels
During the course of contransfection studies in HEK293 cells, we repeatedly observed an increase in IPF1/PDX1 protein levels upon cotransfection with HIPK2 WT. This observation prompted us to explore whether the enhanced IPF1/PDX1 transcriptional activity by HIPK2 in HEK293 cells merely reflected increased IPF1/PDX1 levels. Thus, we first cotransfected His-tagged Ipf1/Pdx1 with Flag-tagged Hipk2 and evaluated the effect on IPF1/PDX1 levels. As shown in Fig. 3AGo, HIPK2 WT dose-dependently increased IPF1/PDX1 protein levels, and this effect was dependent on the activity of the kinase domain of HIPK2. Of note, HIPK2 WT proportionally increased both the unphosphorylated (Fig. 3Go, lower band) and phosphorylated (Fig. 3Go, upper band) forms of IPF1/PDX1 (22).


Figure 3
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  		FIG. 3. In HEK293 cells, HIPK2-induced IPF1/PDX1 protein expression levels does not totally account for HIPK2-induced IPF1/PDX1 transcriptional activity. A, HEK293 cells were transiently transfected with equal amount of DNA from pcDNA3-derived vector encoding His-tagged Ipf1/Pdx1 WT (0.5 µg) and the indicated amount of Flag-tagged Hipk2-encoding vector or empty vector (–). Equal amounts of whole-cell lysates were separated by SDS-PAGE and submitted to Western blot analysis using the indicated antibodies. B, HEK293 cells were transfected with the indicated amount of Ipf1/Pdx1 WT- and Hipk2 WT-encoding vectors, 0.1 µg RIP-luciferase and 0.01 µg Renilla-luciferase. Equal amount of DNA was maintained by addition of empty expression plasmid. Relative activity in Ipf1/Pdx1 WT (0.1 µg)-expressing cells was set at 1. *, P < 0.02. C, HEK293 cells were transiently transfected with the indicated amount of His-tagged Ipf1/Pdx1 WT and Flag-tagged Hipk2 WT-encoding vector. Empty expression plasmid was used to maintain a constant total amount of DNA among wells. Equal amounts of whole-cell lysates were separated by SDS-PAGE and submitted to Western blot analysis using the indicated antibodies. A graphic representation of IPF1/PDX1 (HIS) expression is shown. Ns, Not significant.

 
To determine whether the HIPK2-mediated increase in IPF1/PDX1 levels alone could explain the observed increase in IPF1/PDX1 transcriptional activity, we next expressed proportionally increasing amounts of Ipf1/Pdx1 in absence of HIPK2 WT or constant amounts of Ipf1/Pdx1 with increasing amounts of HIPK2 in HEK293 cells and evaluated the effect on insulin reporter activity (Fig. 3BGo) and protein levels (Fig. 3CGo). As expected, IPF1/PDX1 levels increased with increasing amounts of transfected IPF1/PDX1 construct, but this increase only moderately influenced insulin reporter activity (Fig. 3Go, B and C). In contrast, a significant and dose-dependent enhancement of insulin reporter activity (Fig. 3BGo) was observed when cotransfecting increasing amounts of HIPK2 with constant amount of the IPF1/PDX1 construct (Fig. 3BGo). A Ipf1/Pdx1 to Hipk2 transfection ratio of 1:3 resulted in a relative level of IPF1/PDX1 protein comparable with that obtained with a 3-fold higher amount of IPF1/PDX1 construct alone (Fig. 3CGo). However, the transcriptional activity obtained at a 1:3 ratio of Ipf1/Pdx1 to Hipk2 was about 2.5-fold enhanced over that observed with 3-fold increase in the amount of transfected Ipf1/Pdx1 construct alone (Fig. 3BGo). These results provide evidence that the HIPK2-positive effect on IPF1/PDX1 in HEK293 cells is mediated in part via an increase in IPF1/PDX1 levels but that this alone cannot fully account for the HIPK2-mediated increase in IPF1/PDX1 transcriptional activity. Taken together, data provide evidence that HIPK2, in HEK293 cells, primarily stimulates IPF1/PDX1 transcriptional activity and secondary also IPF1/PDX1 protein levels.

Reduction of Hipk2 expression by siRNA in β-cells attenuates IPF1/PDX1-dependent transcription
To determine whether HIPK2 also affects IPF1/PDX1 transcriptional activity in a context in which HIPK2 and IPF1/PDX1 are endogenously expressed, we used RNA interference approaches to reduce Hipk2 expression in the pancreatic β-cell line MIN6. The Hipk2 siRNA specifically decreased Hipk2 mRNA levels in MIN6 cells by approximately 70% (Fig. 4AGo). Moreover, in HEK293 cells transfected with Flag-tagged HIPK2 WT the Hipk2 siRNA efficiently reduced HIPK2 protein levels (Fig. 4CGo). Thus, the Hipk2 siRNA, but not control siRNA, reduces Hipk2 expression.


Figure 4
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  		FIG. 4. Down-regulation of Hipk2 expression by siRNA in MIN6 cells reduces IPF1/PDX1-dependent transcriptional activity. A, RT-PCR analysis of Hipk2, Ipf1/Pdx1, and gapdh were performed using cDNA prepared from control siRNA- (1, 1') and Hipk2 siRNA-transfected (2, 2') MIN6 cells. Two independent experiments are represented. B, MIN6 cells were transfected with a control siRNA (ctl) or a HIPK2 siRNA. Protein lysates were prepared 48 h and 72 h after transfection. Western blot analysis of IPF1/PDX1 and ERK1/2 expressions in control siRNA- and Hipk2 siRNA-transfected MIN6 cells are showed. C, HEK293 cells were transfected with the human HIPK2 plasmid (1 and 2) or the mouse Hipk2 plasmid (3 and 4) together with a control siRNA (1 and 3) or a Hipk2 siRNA (2 and 4). Equal amounts of whole-cell lysates were separated by SDS-PAGE and subjected to Western blot analysis using the anti-Flag (HIPK2) and the anti-ERK1/2 (as a protein loading control) antibodies. D, MIN6 cells were transfected with a control (ctl) or a Hipk2 siRNA together with 0.1 µg RIP WT-luciferase or 0.1 µg RIP MUT-luciferase as indicated and 0.01 µg Renilla-luciferase. Thirty-six hours after transfection, luciferase activity was measured. Relative activity in control siRNA-transfected cells was set at 1. *, P < 0.001. NS, Not significant.

 
The siRNA mediated reduction of Hipk2 expression in the β-cell line MIN6 did not affect IPF1/PDX1 mRNA or protein levels 48 h after transfection (Fig. 4Go, A and B). At 72 h after transfection, we observed, however, a 40% reduction in IPF1/PDX1 protein levels (Fig. 4BGo).

Transfection of Hipk2 siRNA resulted in a clear reduction in the activity of the IPF1/PDX1-dependent reporter construct 5XP1-luciferase (data not shown) and reduced by approximately 50% the activity of the insulin promoter in MIN6 cells already 36 h after transfection (Fig. 4DGo). In contrast, the Hipk2 siRNA did not affect the transcriptional activity of an insulin promoter reporter construct in which the IPF1/PDX1 binding site had been mutated (Fig. 4DGo). Thus, the positive effect that HIPK2 exerts on insulin promoter activity is IPF1/PDX1 dependent. Taken together, these results provide evidence that HIPK2 positively modulates IPF1/PDX1 target gene expression in an IPF1/PDX1-dependent manner.

HIPK2 phosphorylates IPF1/PDX1
The positive effect of HIPK2 on IPF1/PDX1 transcriptional activity prompted us to explore whether IPF1/PDX1 could be directly phosphorylated by HIPK2. For this purpose we performed an in vitro kinase assay using an active HIPK2 and either the immunoprecipitated, endogenous IPF1/PDX1 protein or different IPF1/PDX1-GST fusion proteins as substrates.

In agreement with previous studies, HIPK2 was autophosphorylated in presence of ATP (41, 42), which also served as a positive control for the kinase assay (Fig. 5Go, A and B). Additionally, the immunoprecipitated, endogenous 45-kDa IPF1/PDX1, the 60-kDa IPF1/PDX1 full-length-GST fusion protein, and a 37-kDa IPF1/PDX1 C-terminal (aa213–283) GST-fusion protein, all incorporated [{gamma}-32P] in presence of HIPK2 (Fig. 5Go, A and B). Interestingly, no [{gamma}-32P] was incorporated in the N-terminal portion (aa1–168) of IPF1/PDX1. Moreover, no phosphorylation of IPF1/PDX1 was observed in absence of added HIPK2, and no phosphorylated band corresponding to the 26-kDa GST protein alone was observed (Fig. 5Go, A and B, and data not shown). Together, these data provide evidence that HIPK2 directly phosphorylates IPF1/PDX1 and that the last 70-aa C-terminal region alone contains aa that serve as HIPK2 substrates.


Figure 5
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  		FIG. 5. HIPK2 phosphorylates IPF1/PDX1. In vitro phosphorylation of immunoprecipitated IPF1/PDX1 (IP IPF1/PDX1) (A) and full-length (FL) IPF1/PDX1-gst, N-terminal (NT) IPF1/PDX1(aa1–168)-gst or C-terminal (CT) IPF1/PDX1(aa213–283)-gst (B) by HIPK2 was determined by kinase assays using active HIPK2. Autoradiograms (KA) show radiolabeled IPF1/PDX1 and HIPK2. Western blots (WB) are included as IPF1/PDX1 protein loading controls C, HEK293 cells transfection assay using 0.1 µg of vectors encoding either WT or mutant forms of Ipf1/Pdx1, 0.2 µg Hipk2 WT-encoding vector, and 0.1 µg RIP-luciferase and 0.01 µg Renilla-luciferase reporter constructs. Relative activity in Ipf1/Pdx1 WT-expressing cells was set at 1. D, HEK293T cells were transfected with 0.1 µg pFR-luciferase, 0.01 µg Renilla-luciferase, 0.1 µg of the indicated gal4-IPF1/PDX1 construct together or not (pcDNA3) with 0.2 µg of HIPK2-encoding vector. The relative transcriptional activity of each individual gal4-IPF1/PDX1 construct in absence of HIPK2 was set at 1. A schematic representation of the different gal4-IPF1/PDX1 constructs used is shown. E, HEK293 cells were transiently transfected with the indicated gal4-Ipf1/Pdx1 construct together (+) or not (–) with Flag-tagged Hipk2-encoding. Equal amounts of whole cell lysates were separated by SDS-PAGE and submitted to Western blot analysis using the indicated antibodies.

 
We recently showed that a minor portion of IPF1/PDX1 is phosphorylated on serine 61 and/or serine 66 in pancreatic β-cells and that these phosphorylation sites influence IPF1/PDX1 function (22). To investigate whether phosphorylation of serine 61 and/or serine 66 were involved in the effect of HIPK2, we next analyzed the previously described serine-to-alanine, which abolishes phosphorylation, and serine-to-aspartic acid, which mimics phosphorylation, IPF1/PDX1 mutants at positions 61 and 66 (22), respectively, in our HIPK2 cotransfection assay. Cotransfection of HIPK2 WT increased IPF1/PDX1-dependent transcriptional activity of the different IPF1/PDX1 mutants (Fig. 5CGo) to the same extent as that observed for the IPF1/PDX1 WT construct. These data demonstrate that the stimulatory effect of HIPK2 on IPF1/PDX1 transcriptional activity is independent of phosphorylation at serine 61 and serine 66.

The finding that the IPF1/PDX1 (aa213–283) C-terminal-GST fusion protein still served as a HIPK2 substrate (Fig. 5BGo), suggest that at least one of the last 70 aa of IPF1/PDX1 is sufficient for phosphorylation by HIPK2. To investigate whether the C-terminal region is also required for the positive effect of HIPK2 on IPF1/PDX1 transcriptional activity, we next determined the transcriptional activity of different gal4-IPF1/PDX1 fusion proteins. HIPK2 WT significantly enhanced both the gal4-IPF1/PDX1(aa1–283)- and the gal4-IPF1/PDX1(aa168–283)-induced transcription of a pFR-reporter construct that contains gal4-responsive elements (Fig. 5DGo). In contrast, HIPK2 WT did not stimulate the transcriptional activity of gal4-IPF1/PDX1 constructs containing IPF1/PDX1 aa1–91, aa1–138, or aa1–215 fused to the gal4 DNA binding domain (Fig. 5DGo). As previously noticed in HEK293 cells (Fig. 3AGo), we observed a slight increased (1.5-fold for aa1–293, aa1–91, aa1–215 and 2-fold for aa168–283) in gal4-IPF1/PDX1 protein expression levels when HIPK2 was coexpressed (Fig. 5EGo). We also previously demonstrated that this level of increase in IPF1/PDX1 protein expression only moderately influence its transactivation potential (Fig. 3Go, B and C). Moreover, gal4-IPF1/PDX1 aa1–91, aa1–215, and to a lesser extent aa1–138 also increased in expression after HIPK2 overexpression without increasing pFR-luciferase activity, suggesting that increased expression of IPF1/PDX1 (to these levels) is not sufficient to influence its transactivation potential. Taken together, these data provide evidence that the IPF1/PDX1 C-terminal aa168–283 are required for the positive effect of HIPK2 on IPF1/PDX1 transactivation potential.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite the clearly established role for IPF1/PDX1 in pancreas development and β-cell function, many important mechanistic questions with regard to its regulation remain unanswered. In this study, we have identified the serine/threonine kinase HIPK2 as a novel modulator of IPF1/PDX1 transcriptional activity.

Our data show that HIPK2 increases IPF1/PDX1-dependent transcription in HEK293 cells. In these cells, part of the positive effect of HIPK2 on IPF1/PDX1 function is likely to result from posttranscriptional events that lead to the observed increase in IPF1/PDX1 protein levels. HIPK2 has previously been shown to affect, albeit in a negative manner, the expression levels of other proteins (31, 32). In addition to a positive effect of HIPK2 on levels of IPF1/PDX1 protein, HIPK2 significantly enhanced IPF1/PDX1-dependent transcriptional activity beyond that observed with a comparable increase in expression of IPF1/PDX1 alone. Thus, in HEK293 cells, the HIPK2-mediated increase in IPF1/PDX1 protein levels cannot fully account for the HIPK2-induced IPF1/PDX1 transcriptional activity and HIPK2 is likely to directly influence IPF1/PDX1 transcriptional activity.

siRNA-mediated reduction of Hipk2 expression in MIN6 cells rapidly (36 h) resulted in a decrease of IPF1/PDX1 transcriptional activity, whereas IPF1/PDX1 protein levels were reduced only 72 h after hipk2 siRNA transfection. These results suggest that reduction of HIPK2 initially leads to reduced IPF1/PDX1 transcriptional activity without affecting protein levels, and upon prolonged reduction of HIPK2, IPF1/PDX1 protein levels also become affected. Collectively our data thus show that, independent of its effect on IPF1/PDX1 protein levels, HIPK2 positively modulates IPF1/PDX1 transcriptional activity.

HIPK2 possess a homeodomain-interacting domain that is important for enhancing the repressor activities of NK homeodomain transcription factors (26), leaving open the possibility that HIPK2 may affect IPF1/PDX1 transcriptional activity by physically interacting with IPF1/PDX1. Preliminary coimmunoprecipitation studies have unfortunately so far failed to reveal a clear interaction between HIPK2 and IPF1/PDX1. Whether this is reflective of a brief, transient interaction between HIPK2 and IPF1/PDX1 or/and due to a low amount of IPF1/PDX1 interacting with HIPK2 (see Fig. 1CGo) will require further analyses. Regardless, our data show that, like that reported for Pax6 (28), the kinase activity of HIPK2 is required for its stimulatory effect on IPF1/PDX1 function; the kinase-inactive form of HIPK2 had no effect on IPF1/PDX1 transcriptional activity. Inactive forms of kinases usually do not affect interaction with their targets and can even display stronger interaction, as it has been demonstrated for Pax6 and the kinase-dead HIPK2 (28). Consequently, a mere interaction of HIPK2 with IPF1/PDX1 does not seem to be the major mechanism by which HIPK2 affects IPF1/PDX1 transcriptional activity. Thus, the positive effect of HIPK2 on IPF1/PDX1 function is most likely attributed to the protein kinase activity of HIPK2.

In this study we showed that HIPK2 is able to directly phosphorylate both the native, immunoprecipitated IPF1/PDX1 protein and an IPF1/PDX1-GST fusion protein produced in bacteria. It is important to note that the IPF1/PDX1 produced in bacteria has the theoretically expected molecular mass of 31 kDa, whereas the IPF1/PDX1 expressed in mammalian cells has a molecular mass of approximately 45 kDa. Thus, it seems that the modifications occurring on IPF1/PDX1 in mammalian cells are dispensable for its phosphorylation by HIPK2. At present, we do not know on which amino acid HIPK2 phosphorylates IPF1/PDX1. HIPK2 was, however, still able to phosphorylate the C-terminal, aa213–283, IPF1/PDX1-GST fusion, suggesting that at least one amino acid within this region represents a HIPK2 phosphorylation site. Identification of phosphorylation sites on the few known HIPK2 targets suggest that HIPK2 may be a proline-directed serine/threonine kinase (28, 29, 30, 43). Analysis of the IPF1/PDX1 protein sequence has revealed four potential phosphorylation sites that meet this requirement: serine61-proline (Ser61), serine66-proline (Ser66), threonine214-proline (Thr214), and serine269-proline (Ser269). HIPK2 was, however, unable to phosphorylate a N-terminal (aa1–168) IPF1/PDX1-GST, thus containing the Ser61 and Ser66 potential phosphorylation sites. Moreover, HIPK2 was still able to enhance the transcriptional activity of the Ser61 and Ser66 mutants to the same extent as that observed with the wild-type Ipf1/Pdx1 construct. Ser61 and Ser66 phosphorylation are therefore unlikely to be target sites for phosphorylation by HIPK2. These results are in agreement with our previous data showing that these phosphorylation sites do not per se modulate IPF1/PDX1 transcriptional activity (22). Moreover, as opposed to the positive effect of HIPK2 on IPF1/PDX1 function, we have previously associated Ser61 and/or Ser66 phosphorylation with IPF1/PDX1 protein degradation (22). Further analyses will be require to elucidate whether the Thr214 or Ser269, located in the C-terminal region of IPF1/PDX1, or other sites are phosphorylated by HIPK2 and whether they are involved in the positive effect of this kinase.

In addition to the N-terminal transactivation domain, the IPF1/PDX1 C terminus has also been shown to be important for full IPF1/PDX1 transactivation potential (20, 21, 44). Our data support this observation because HIPK2 WT was able to increase the transactivation potential of the gal4-IPF1/PDX1 (aa 1–283) and gal4-IPF1/PDX1 (aa168–283) fusion protein but not that of the (aa 1–91); (aa1–138); or (aa1–215) gal4-IPF1/PDX1 fusion proteins. Thus, not only does the C-terminal 70 aa of IPF1/PDX1 still serve as a substrate for HIPK2 but at least the C-terminal part aa168–283 of IPF1/PDX1 is also required for HIPK2’s positive effect on IPF1/PDX1 transcriptional activity. Surprisingly, we observed a decrease in the gal4-IPF1/PDX1 aa1–91 transactivation potential on HIPK2 overexpression. Thus, although HIPK2 does not appear to phosphorylate the N-terminal portion (aa 1–168) of IPF1/PDX1, the possibility that HIPK2 negatively influences a coactivator that binds and regulates the gal4-IPF1/PDX1 aa 1–91 transcriptional activity cannot be excluded. Previous studies (44, 45) and our unpublished data provide evidence that the N- and C-terminal portions of IPF1/PDX1 possesses their independent transactivation potential. Hence, the overall effect of HIPK2 on the full-length IPF1/PDX1 transcriptional activity might be a balance between an indirect effect of HIPK2 on the N-terminal portion and a direct effect on the C-terminal portion of IPF1/PDX1. The expression of both Hipk2 and IPF1/PDX1 in fetal pancreatic epithelium, differentiating β-cells, and adult islets is suggestive of a role for HIPK2 in the regulation of IPF1/PDX1 transcriptional activity, which in turn is supportive of a functional role for HIPK2 in the developing and adult pancreas. Direct functional analyses of a role for hipk2 in pancreatic development and/or β-cell function in vivo will, however, have to await the generation of pancreatic and β-cell-specific targeted deletions of the hipk2 gene in mice.

Collectively, our data provide evidence that HIPK2 is a new regulator of IPF1/PDX1 transcriptional activity. By virtue of its effect on IPF1/PDX1 function, our data are consequently suggestive of a role for HIPK2 in pancreatic development and/or β-cell function. Thus, the identification of HIPK2 as a new positive modulator of IPF1/PDX1 function may have potential therapeutic relevance for the preservation and/or restoration of β-cell function and glucose homeostasis in patients with diabetes.


   Acknowledgments
 
We are grateful to Dr. L. M. Schmitz and Dr. S. Soddu for providing us with the human and mouse Flag-tagged HIPK2 plasmids, respectively; Dr. C. Asselin for the p300-expressing vector; Dr. C. B. Wollheim and P. Maechler for the INS-1E cell line; Drs. V. Poitout and D. J. Drucker for MIN6 cells used in Canada; and Dr. M. D. Walker for gal4-IPF1/PDX1 (aa1–283), (aa1–91), (aa1–138). and (aa1–215) constructs. We thank Dr. Kelly Loffler for helpful discussions and critical reading of the manuscript and members of the laboratory for technical suggestions and helpful discussions.


   Footnotes
 
This work was supported by grants from the Swedish Research Council and the European Union (Integrated Project EuroDia LSHM-CT-2006-518153 in the Framework Program 6 of the European Community) (to H.E.), the Natural Sciences and Engineering Research Council of Canada (to M.-J.B.), and a postdoctoral fellowship from the Fonds pour la Recherche en Santé du Québec (to M.-J.B.).

Current Address for M.-J.B.: Service de Gastroentérologie/Départment de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: marie-josee.boucher@usherbrooke.ca.

Disclosure Statement: M.-J.B. and M.S. have nothing to declare. H.E. is a cofounder, shareholder, and consultant of Betagenon AB, Sweden.

First Published Online September 4, 2008

Abbreviations: aa, Amino acids; e, embryonic day; GST, glutathione-S-transferase; HEK, human embryonic kidney; HIPK, homeodomain-interacting protein kinase; IPF, insulin promoter factor; NK, neurokinin; Pax, paired box transcription factor; PDX, pancreatic duodenal homeobox; RIP, rat insulin promoter; siRNA, small interfering RNA; WT, wild type.

Received June 27, 2007.

Accepted for publication August 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals