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Endocrinology Vol. 142, No. 3 1033-1040
Copyright © 2001 by The Endocrine Society

ARTICLES

Hedgehog Signaling Regulation of Homeodomain Protein Islet Duodenum Homeobox-1 Expression in Pancreatic {beta}-Cells1

Melissa K. Thomas, Jee H. Lee, Naina Rastalsky and Joel F. Habener2

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, Wellman Building 320, Boston, Massachusetts 02114. E-mail: jhabener{at}partners.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin gene expression in pancreatic {beta}-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 {beta}-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic {beta}-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic {beta}-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ISLET DUODENUM homeobox-1 (IDX-1) is a master transcriptional regulator of both pancreas development and the differentiated {beta}-cell phenotype (1). Initially cloned in multiple laboratories, IDX-1 (2) is also known as IPF-1 (3), STF-1 (4), IUF-1 (5), GSF (6), and PDX-1 (7). Normal IDX-1 function is essential for the development of the pancreas. Homozygous disruption of IDX-1 results in pancreatic agenesis in mice and in humans (7, 8, 9).

In the pancreatic {beta}-cell, IDX-1 is of central importance in the sensing and response of the insulin promoter to varying levels of extracellular glucose (5, 10, 11). IDX-1 function is regulated by glucose-dependent phosphorylation (5, 12), nuclear translocation (13, 14), DNA-binding activity (5, 10), and transactivation potency (15, 16). The rat insulin I gene is regulated by glucose-responsive minienhancers consisting of binding sites for E2A proteins (E boxes) (17, 18) and AT-rich regions (A boxes) (19) that bind homeoproteins. Within the rat insulin I promoter, E boxes are designated Far and Nir, and A boxes are designated Flat and P1. Similar conserved sequences are found within all of the characterized promoters of mammalian insulin genes (20). A boxes contribute to {beta}-cell selectivity (11, 19, 21) and glucose-responsivity (6, 10, 11) of the insulin promoters. Homeoproteins, such as IDX-1, Lmx1, and Isl-1, bind to the A boxes, such as Flat or P1, and act in synergy with E2A heterodimers on adjacent E boxes to activate transcription of the insulin gene (19, 22, 23).

In humans, heterozygous mutations in IDX-1 predispose to maturity onset diabetes of the young (MODY4) and Type 2 (adult-onset) diabetes mellitus (24, 25, 26). Mice heterozygous for a disruption of the IDX-1 gene have impaired glucose tolerance (27, 28), and mice derived from a cre-lox model of pancreas-specific IDX-1 inactivation develop diabetes mellitus (27). Disruption of IDX-1 function may alter the development of the pancreas, the regulation of insulin production, or the total numbers of islet {beta}-cells.

Pancreatic {beta}-cell dysfunction, caused by altered IDX-1 levels, can occur in the absence of mutations in IDX-1. In {beta}-cell models of glucotoxicity, sustained exposure to hyperglycemia down-regulates IDX-1 expression concordant with decreased insulin gene transcription (29, 30, 31). Transfection of exogenous IDX-1 into glucotoxic insulinoma cells partially restores the activity of the insulin promoter (32). IDX-1 expression in insulin-producing cells is reduced also by palmitic acid or glucocorticoid exposure (33, 34). Strategies to increase levels of IDX-1 expression are restricted by the currently limited number of identified regulators of the IDX-1 promoter (34, 35, 36, 37).

Hedgehog (Hh) proteins are important developmental morphogens in Drosophila and in mice (38). Three vertebrate Hh proteins [sonic Hh (Shh), Indian Hh (Ihh), and desert Hh (Dhh)] all signal through the Hh signaling pathway by binding to the receptor signaling protein patched (Ptc) to activate the transmembrane protein smoothened (Smo), resulting in downstream transcriptional activation of target genes (38, 39, 40, 41). In early mouse pancreas development, the foregut endoderm from which the pancreas develops does not express Shh (42). However, reducing Hh signaling results in abnormal pancreas development, as indicated by the increased incidence of annular pancreas in mouse models in which Shh or Ihh genes are disrupted (43). After the pancreas has developed, Hh signaling seems to function in a different context. The Hh signaling proteins Ptc and Smo are preferentially expressed in the endocrine pancreas, and both Ihh and Dhh are expressed in subpopulations of insulin-producing pancreatic {beta}-cells (44). The expression of Ptc in pancreatic islets indicates that Hh signaling is active in the endocrine pancreas because the Ptc gene is a highly responsive transcriptional target for Hh signals (40, 45, 46, 47). In clonal pancreatic {beta}-cells, Hh signaling increases activation of the rat insulin I promoter (44).

An interplay between Hh signals and IDX-1 expression has been observed in some experimental systems. In the developing chick endoderm, in the presence of activin signaling, increasing Shh administration reduces IDX-1 expression, and an Shh-blocking antiserum induces IDX-1 expression (48). At later stages of development, inhibition of Hh signaling with the specific inhibitor cyclopamine results in heterotopic insulin expression only in segments of the developing gastrointestinal tract in which IDX-1 is expressed (49). In a transgenic mouse model in which Shh is misexpressed under the regulation of the IDX-1 promoter, the pancreas develops with insulin- and glucagon-expressing cells in a disorganized pattern, with the organ surrounded by smooth muscle (42). Interestingly, in mice in which the Ihh gene is disrupted, embryonic intestinal development is markedly abnormal, with reduced intestinal villi, decreased proliferation of cells between villi, and diminished numbers of cholecystokinin-producing cells. In this experimental model, IDX-1-expressing cells normally present in the developing duodenum (2, 7, 50) are not seen (43), suggesting either a failure of development of this subpopulation of neuroendocrine cells or a direct or indirect regulation of IDX-1 expression by Hh signaling.

In this report, we extend our studies of the Hh-mediated regulation of insulin gene transcription to identify IDX-1 as a direct target of Hh signaling converging on the insulin promoter. We demonstrate that IDX-1 gene expression is directly regulated by Hh signaling in clonal pancreatic {beta}-cells. By regulating the activation of genes, such as IDX-1 and insulin, that are essential for normal glucose metabolism and pancreatic {beta}-cell function, we propose that the Hh signaling pathway may provide new opportunities for the development of therapeutic strategies to enhance insulin production in patients with diabetes mellitus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
The -4.6 IDX-pGL3 plasmid was constructed by inserting the previously described -4.6-kb mouse IDX-1 promoter fragment (50) into a blunted HindIII restriction site of the pGL3-Basic Vector (Promega Corp. Life Sciences, Madison, WI). The pShh and insulin promoter-reporter plasmids were described previously (44, 51). FarFlat-CAT was a gift from J. L. Moss.

Cell culture and transfections
INS-1 cells were provided by C. Wollheim and were cultured in 11.1 mM glucose as described (44, 52). Transfections were conducted with Lipofectamine (Life Technologies, Inc., Gaithersburg, MD), and luciferase and CAT assays were performed as previously described (44, 53). Protein concentrations of cellular extracts were determined with the Bio-Rad Laboratories, Inc. Protein Assay (Bio-Rad Laboratories, Inc. Hercules, CA). P values were determined with Student’s t tests (Excel, Microsoft Corp., Redmond, WA).

Stock solutions of 10 mM cyclopamine (gift from W. Gaffield, United States Department of Agriculture) were prepared in 95% ethanol and diluted to the final concentrations indicated. Appropriately diluted ethanol vehicle solutions were used for control samples. For transfections, cells were treated with 0, 1, 10, or 20 µM cyclopamine in 0.19% ethanol, 90 min before transfection in serum-free medium, at the time of transfection by addition to the transfection cocktail, and after 5 additional hours of incubation when the transfection cocktail was replaced with INS-1 culture medium.

Nuclear extract preparation
Nuclear extracts were prepared by the method of Schreiber (54). For ectopic Hh expression, cells were transfected with 4 µg pShh or pED empty expression vector, and extracts were harvested 24 h after transfections. In experiments in which endogenous Hh signaling was inhibited, cells were treated with 20 µM cyclopamine in 0.19% ethanol or 0.19% ethanol vehicle twice daily, and extracts were harvested after 24 h of treatment. Protein concentrations of extracts were measured by the Bio-Rad Laboratories, Inc. Protein Assay.

Western blots
Cellular extracts were separated by SDS-PAGE, electroblotted (Immobilon-P membranes, Millipore Corp., Bedford, MA), incubated with rabbit polyclonal anti-IDX-1 antiserum (1:10,000 dilution), and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) (9, 53). As a control for protein loading, blots were washed and incubated with rabbit polyclonal anti-Creb antiserum (55) and visualized by enhanced chemiluminescence.

DNA-binding assays
DNA-binding assays were conducted, as described previously (2), with a [32P]-radiolabeled double-stranded oligonucleotide FarFlat probe encompassing nucleotides -247 to -198 of the rat insulin I promoter. To identify IDX-1-containing protein complexes, nuclear extracts were preincubated for 20 min at room temperature with rabbit polyclonal anti-IDX-1 antiserum or nonimmune rabbit antiserum (56).

Northern RNA blots
Total cellular RNA was prepared with Tri-Reagent (Sigma, St. Louis, MO). INS-1 cells were treated twice daily with 20 µM cyclopamine in 0.19% ethanol or 0.19% ethanol vehicle, and RNA was harvested after 24 h of treatment. Northern blots were prepared (44) and hybridized with a 1.3-kB NotI/NcoI fragment of rat IDX-1 complementary DNA (cDNA) derived from a pBJ5-IDX-1 expression vector (2) or with a 630-nucleotide cDNA fragment of rat {beta}-actin cDNA (44) as a control for gel loading. Autoradiograms were scanned by computing densitometry (Molecular Dynamics, Inc., Sunnyvale, CA), and data were quantitated using ImageQuant software (Molecular Dynamics, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Hh-responsive regions within the rat insulin I promoter
To investigate possible mechanisms for the activation of the rat insulin I promoter by Hh signaling, we used the previously developed experimental model system of ectopic Hh expression in which clonal INS-1 pancreatic {beta}-cells are transiently transfected with an expression plasmid for Shh (pShh) (44). In this system Shh is expressed, processed, and secreted into the culture medium, where it is available to interact with Ptc receptors on INS-1 cells. By transiently transfecting INS-1 cells with rat insulin I promoter-luciferase reporter 5'-deletion constructs in the presence of pShh or the empty expression vector, we determined the relative transcriptional activation of promoter segments in the presence or absence of ectopic Hh expression. Activation of Hh signaling in INS-1 cells increased the transcriptional activation of the -410 rat insulin I promoter construct, relative to control cells, as reported previously (44). By comparing the percent activation by ectopic Hh expression for each of the deletion constructs tested, we identified two Hh-responsive regions located between nucleotides -343 and -183 and between nucleotides -118 and -81 (Fig. 1Go). Notably, these Hh-responsive regions encompassed the glucose-responsive enhancers E2/A3/4 (Far/Flat) and E1/A1 (Nir/P1), regions of the rat insulin I promoter in which homeoproteins, such as IDX-1, bind to A boxes and contribute to synergistic promoter activation by interacting with heterodimers of the basic helix-loop-helix transcription factors, such as E2A and {beta}-2/neuroD, that bind to adjacent E boxes (17, 18, 57).



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  		Figure 1. Mapping of Hh-responsive regions within the rat insulin I promoter. INS-1 cells were transfected in duplicate with 2 µg each of 5'-deletion rat insulin I promoter-reporter constructs (51 ) and 4 µg pShh or the empty pED expression vector. Percent Hh activation (±SEM; n = 2) for each construct was compared with that of the -410 rat insulin I promoter-reporter construct (set at 100%). The upper schematic diagram of the rat insulin I promoter depicts positions of selected glucose-responsive elements (GSE) relative to the transcription start site (RIP-I: -410 bp of rat insulin I promoter; E2 (Far), A3/4 (Flat), E1 (Nir), A1(P1), glucose-responsive enhancer elements within the rat insulin I promoter).

 
Glucose-responsive elements within the rat insulin I promoter are regulated by Hh signaling
To determine whether Hh-responsiveness of the rat insulin I promoter could be mediated by glucose-responsive elements, we used a multimerized FarFlat enhancer-reporter construct, FarFlat-CAT (gift of J. L. Moss) in transient transfections of INS-1 cells. Ectopic Shh expression increased the activation of the FarFlat-CAT reporter, relative to the empty expression vector, by approximately 3-fold (Fig. 2AGo). However, the levels of endogenous Hh production and signaling in INS-1 cells determine and limit the available range for additional promoter activation by exogenous Hh. Therefore, we treated the INS-1 cells with cyclopamine, a highly specific inhibitor of Hh signaling (58, 59, 60). Cyclopamine markedly inhibited the activity of the FarFlat-CAT reporter (Fig. 2BGo). The extent of inhibition of FarFlat activity by cyclopamine (50-fold) is much greater than the inhibition of the -410 rat insulin I promoter (5-fold) (44), a strong indication that the glucose-responsive element FarFlat is a target of Hh signaling in the insulin gene. The marked sensitivity of the FarFlat-CAT reporter activity to inhibition by cyclopamine indicates that substantial endogenous Hh signaling is present in INS-1 cells. On the basis of our transfection studies with the FarFlat-CAT reporter construct, we concluded that Hh-responsiveness could be conferred to the rat insulin I promoter by proteins activating either E-(Far) or A-(Flat) box glucose-response elements.



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  		Figure 2. A, Activation of the glucose-responsive enhancer FarFlat is increased by Hh signaling. INS-1 cells were transfected in duplicate with 1 µg of the multimerized enhancer-reporter construct FarFlat-CAT and 4 µg pShh (+) or the empty pED expression vector (-). Fold activation ± SEM, normalized to the activation of FarFlat-CAT in the absence of ectopic Hh expression, is shown (n = 5; **, P < 0.05). B, Inhibition of Hh signaling with cyclopamine decreases activation of the glucose-responsive enhancer FarFlat. INS-1 cells were transfected, in duplicate, with 1 µg of the multimerized enhancer-reporter construct FarFlat-CAT and 4 µg of the empty pED expression vector and incubated with 20 µM cyclopamine in 0.19% ethanol (+) or 0.19% ethanol vehicle (-). Percent activation ± SEM, normalized to the activation of FarFlat-CAT in the absence of cyclopamine treatment, is shown (n = 3; ***, P < 0.001).

 
Hh signaling regulates DNA-binding of protein complexes that contain IDX-1
In DNA-binding studies with a [32P]-radiolabeled FarFlat oligonucleotide probe, nuclear extracts derived from INS-1 cells transfected with pShh exhibited increased DNA-binding, relative to nuclear extracts from cells transfected with the empty expression vector (Fig. 3Go, A and C). The addition of rabbit polyclonal antiserum directed against the pancreatic homeoprotein IDX-1 attenuated Hh-responsive protein-binding and supershifted the radiolabeled DNA probe. These findings identify the presence of IDX-1 in protein complexes activated by Hh signaling. In a complementary series of DNA-binding assays on the [32P]-radiolabeled FarFlat probe, the protein-binding activity in extracts derived from cyclopamine-treated INS-1 cells was diminished, relative to controls (Fig. 3BGo). The cyclopamine-sensitive protein complexes also contained IDX-1, as shown by the attenuation and supershift of protein binding in the presence of anti-IDX-1 serum.



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  		Figure 3. A, Hh-responsive protein complexes bind to FarFlat sequences and contain IDX-1. Nuclear extracts (NEx) were prepared from INS-1 cells transfected with pShh (Hh +) or the empty expression vector (Hh -), then preincubated with normal rabbit serum ({alpha}-IDX-1 -) or rabbit polyclonal anti-IDX-1 antiserum ({alpha}-IDX-1 +), incubated with a [32P]-radiolabeled oligonucleotide FarFlat probe, and separated by extended electrophoresis on native acrylamide gels (in which the free probe was run off of the gels). A portion of a corresponding autoradiograph from a representative experiment is shown. Two Hh-responsive complexes are indicated with arrows and were not observed in the same experiment with probe incubated in the absence of INS-1 extracts (lane at left). B, Cyclopamine-sensitive protein complexes bind to FarFlat sequences and contain IDX-1. NEx were prepared from INS-1 cells incubated with 20 µM cyclopamine in 0.19% ethanol (Cyc +) or 0.19% ethanol vehicle (Cyc -), then preincubated with normal rabbit serum ({alpha}-IDX-1 -) or rabbit polyclonal anti-IDX-1 antiserum ({alpha}-IDX-1 +), incubated with a [32P]-radiolabeled oligonucleotide FarFlat probe, and separated by extended electrophoresis on native acrylamide gels. A portion of a corresponding autoradiograph from a representative experiment is shown. Two cyclopamine-sensitive complexes are indicated with arrows and were not observed in the same experiment with probe incubated in the absence of INS-1 extracts (lane at left). C, Hh-responsive protein complexes bind to FarFlat sequences (full gel view). As in Fig. 3AGo, NEx were prepared from INS-1 cells transfected with pShh (Hh +) or the empty expression vector (Hh -), then preincubated with normal rabbit serum ({alpha}-IDX-1 -) or rabbit polyclonal anti-IDX-1 antiserum ({alpha}-IDX-1 +), and incubated with a [32P]-radiolabeled oligonucleotide FarFlat probe. Protein complexes were separated by a shorter gel electrophoresis on native acrylamide gels in which the radiolabeled probe was retained on the gel (probe). A corresponding autoradiograph from a representative experiment is shown. Two Hh-responsive complexes are indicated with arrows and were not observed in the same experiment with probe incubated in the absence of INS-1 extracts (lane at left). A nonspecific complex is designated (ns).

 
IDX-1 expression levels are regulated by Hh signaling
To determine whether changes in IDX-1 levels might account for the observed differences in DNA-binding activities on the FarFlat probe, we analyzed nuclear extracts from INS-1 cells transfected with pShh or the empty expression plasmid, by Western blotting with rabbit polyclonal anti-IDX-1 serum. We observed increased nuclear IDX-1 levels in INS-1 cells transfected with pShh, relative to control cells (Fig. 4AGo). This increase in IDX-1 protein could result from regulation of IDX-1 at the level of gene expression, messenger RNA (mRNA) stability, protein stability, or nuclear translocation. A substantial component of the regulation of IDX-1 levels by Hh signaling occurs at the level of mRNA expression (Fig. 4Go, B and C). After 24 h of cyclopamine administration, the IDX-1/actin mRNA ratio was reduced by approximately 40%, relative to vehicle-treated control cells.



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  		Figure 4. Hh signaling regulates IDX-1 expression. A, Activation of Hh signaling increases nuclear IDX-1 protein levels in INS-1 cells (upper panel). Nuclear extracts were prepared from INS-1 cells transfected with pShh (Hh +) or the empty pED expression vector (Hh -). Extract proteins (10 µg per lane) were separated by SDS-PAGE before Western blotting with rabbit polyclonal anti-IDX-1 antiserum. The IDX-1 protein migrates as a doublet near the 43-kDa marker. As a protein loading control, the Western blot was washed and reblotted with rabbit polyclonal anti-Creb antiserum (lower panel). B, Inhibition of Hh signaling with cyclopamine decreases IDX-1 mRNA levels. INS-1 cells were incubated with 20 µM cyclopamine in 0.19% ethanol (+) or 0.19% ethanol vehicle (-) for 24 h before preparation of total cellular RNA. Northern blots of INS-1 RNA were probed for IDX-1 and {beta}-actin (actin) as a gel loading control. Corresponding autoradiographs are shown. C. Northern blots were analyzed by scanning densitometry to calculate IDX-1 mRNA/actin mRNA ratios. Data shown are the average of two experiments in which the IDX-1/actin mRNA ratios for cyclopamine-treated cells ranged from 43–78% of those from vehicle-treated control cells.

 
Hh signaling activates the IDX-1 promoter
To directly assess the effects of Hh signaling on the IDX-1 promoter, we used a -4.6-kb IDX-1 mouse promoter fragment known to encode all of the regulatory elements necessary to impart the expression pattern of endogenous mouse IDX-1. The -4.6-kb promoter was shown earlier to mimic the temporal and spatial expression of IDX-1 during pancreas development and, ultimately, in differentiated pancreatic {beta}-cells (50). INS-1 cells were transiently transfected with a -4.6-kB IDX-1 promoter-luciferase reporter construct and pShh or the empty expression control plasmid. Ectopic Hh expression in INS-1 cells activated the IDX-1 promoter in a dose-dependent manner, indicating that activation of Hh signaling increases IDX-1 gene expression (Fig. 5AGo). A similar increase was observed in IDX-1 promoter activation in response to ectopic Hh expression in transfections of the clonal {beta}-cell line MIN6 (data not shown).



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  		Figure 5. A, Activation of Hh signaling increases IDX-1 promoter activation. INS-1 cells were transfected in duplicate with 2 µg of a -4.6-kb mouse IDX-1 promoter-reporter construct or the empty reporter construct (pGL3) and 0–4 µg of pShh with 4–0 µg of the empty pED expression vector, as indicated. Relative luciferase (LUC) activities for the IDX-1 promoter and the empty reporter vector (±SEM) from a representative transfection, normalized to protein concentrations, are shown. B, Inhibition of Hh signaling with cyclopamine decreases IDX-1 promoter activation. INS-1 cells were transfected in duplicate with 2 µg of a -4.6-kb mouse IDX-1 promoter-reporter construct and 4 µg of the empty pED expression vector. Cells were treated with 0, 1, 10, or 20 µM cyclopamine in 0.19% ethanol, as indicated. IDX-1 promoter activation was normalized to that of cells not treated with cyclopamine (100%). Luciferase activities were normalized to protein concentrations of cellular extracts. Percent activations ± SEM are shown (n = 3; ***, P < 0.01).

 
INS-1 cells were then transiently transfected with the -4.6-kB IDX-1 promoter-reporter construct without added Hh. In a pattern analogous to that observed for the rat insulin I promoter, treatment of transfected INS-1 cells with cyclopamine inhibited the basal IDX-1 promoter activity dose-dependently, by over 80% at the highest dose tested (Fig. 5BGo). Therefore a substantial component of IDX-1 promoter activity in INS-1 cells, in the absence of ectopic expression of Hh, is regulated by endogenously-generated Hh signaling in the INS-1 cells. The activation of the IDX-1 promoter by exogenous activation of Hh signaling, and the repression of IDX-1 promoter activity and mRNA levels by the Hh signaling inhibitor cyclopamine, identify the IDX-1 gene as a target for positive regulation by Hh signaling in pancreatic {beta}-cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies, we have identified the IDX-1 gene as a target for Hh signals in pancreatic {beta}-cells. IDX-1 levels directly impact pancreatic {beta}-cell function and glucose homeostasis. Heterozygous disruption of IDX-1 gene expression in mouse models results in impaired glucose tolerance (27, 28), likely because of impaired insulin gene expression and diminished pancreatic {beta}-cell mass. Heterozygous mutations of IDX-1 in humans confer a predisposition to the development of diabetes mellitus (24, 25, 26), highlighting the clinical importance of signal transduction pathways that regulate IDX-1 levels.

The finding that inhibition of Hh signaling reduced both IDX-1 mRNA levels and promoter activation indicates that a substantial component of basal IDX-1 expression in clonal pancreatic {beta}-cells is dependent on active Hh signals. The Hh proteins Ihh and Dhh are expressed in INS-1 cells and pancreatic islets (44). Because all vertebrate Hhs signal via the same receptor signaling proteins and activate the same downstream target genes in other experimental systems (38, 39, 40, 41), Ihh and Dhh likely provide the endogenous Hh signals in {beta}-cells that converge on the IDX-1 promoter. The identification of the IDX-1 promoter as a regulatory target of Hh signals suggests that the loss of embryonic duodenal IDX-1 expression in the Ihh knockout mouse model (43) likely was the result of impaired Hh signaling in developed cells rather than the result of a failure of intestinal neuroendocrine cell development. The observation that IDX-1 is expressed in the pancreas of Ihh knockout mice (43) may be explained by the expression of additional pancreatic Hh proteins, such as Dhh, that maintain Hh signaling in pancreatic islets and promote IDX-1 expression.

The Hh-responsive regulation of IDX-1 levels provides a mechanism for Hh signaling, to indirectly regulate glucose-responsive elements and the activity of the insulin promoter (Fig. 6Go). Our studies do not exclude the possibility that other protein regulators of insulin gene expression may also be targets of Hh signals. Activation or inhibition of Hh signals in INS-1 cells was sufficient to regulate nuclear IDX-1 levels within a critical range that modulated the binding of protein complexes to the glucose-responsive enhancer FarFlat. In our studies of the rat insulin I promoter, in which we identified FarFlat as a candidate Hh-response element, we also noted a decrement in Hh-responsiveness by disrupting the synergistic connection between the glucose-response element Nir (E box) and P1 (A box). This result is consistent with decreased IDX-1 protein levels available to bind A box sequences. It is also possible that additional Hh-responsive elements are present within the insulin promoter. We observed a decrement in IDX-1 mRNA levels after 24 h of treatment with cyclopamine; whereas, in a similar system, significant decreases in insulin mRNA levels were not observed until 72 h of cyclopamine treatment (44). This temporal gap in the effects of inhibition of Hh signaling on gene expression levels in clonal pancreatic {beta}-cells may primarily reflect differences in mRNA half-lives, but it is also consistent with a primary inhibition of IDX-1 gene expression followed by a secondary reduction in insulin gene expression.



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  		Figure 6. Proposed model of insulin promoter activation by Hh signaling. Schematic diagrams of the IDX-1 and insulin gene promoters are shown. In response to Hh signaling, IDX-1 expression is increased. We propose that Hh-responsive elements (HhRE) within the IDX-1 promoter directly mediate this process. The increase in IDX-1 expression levels results in an indirect activation of insulin gene expression in response to Hh signals, by increasing DNA-binding of IDX-1 to A boxes (i.e. Flat or P1) within the insulin promoter. This increase in DNA-binding allows IDX-1 to promote synergistic activation of insulin gene expression in conjunction with basic helix-loop-helix (bHLH) transcription factors (including E12, E47, and {beta}-2/neuroD) that bind to adjacent E boxes (i.e. Far and Nir). Inhibition of Hh signaling with the specific inhibitor cyclopamine decreases IDX-1 gene expression and DNA-binding activity to the insulin promoter, resulting in decreased insulin gene expression. POL II designates DNA polymerase II and associated basal transcription machinery at the transcription start sites of the IDX-1 and insulin genes in the schematic diagram.

 
The regulatory mechanisms by which Hh signaling alters IDX-1 expression levels likely differ in distinct cellular contexts. Although studies in embryonic chick endoderm cultures suggested that IDX-1 expression is negatively regulated by Hh signals (48), analysis of IDX-1 expression in the developing duodenum of Ihh knockout mice implied the converse (43). This apparent paradox likely reflects the complexity of Hh signaling pathways, as well as the importance of the combinatorial code of converging morphogen signals on IDX-1 expression. Furthermore, differentiated pancreatic {beta}-cells would be expected to use Hh signals to mediate cellular processes distinct from those required in developmental systems.

Multiple levels of regulation determine whether Hh signals result in the activation or repression of gene expression. For example, the Hh receptor Ptc is capable of either transmission or sequestration of Hh signals (61). Both activator and repressor forms of the transcriptional regulators in the Ci/Gli transcription factor family result from Hh signaling (62, 63). Hh proteins also have the capacity to travel substantial distances within tissues (64, 65, 66). Recent evidence, indicating that Smo transmembrane proteins are translocated to the cell surface in response to Hh signals (67), raises the possibility that unidentified signals associated with the cell membrane or emanating from extracellular sources provide additional levels of regulation.

The identification of the Hh signaling pathway in the regulation of IDX-1 expression in pancreatic {beta}-cells may provide opportunities for the development of novel therapeutic strategies for diabetes mellitus. Maintaining IDX-1 levels in a physiologic range is essential for pancreatic development, insulin gene transcription, preservation of pancreatic {beta}-cell mass, and normal glucose homeostasis. Hh signals likely converge on several of these IDX-1 functions, possibly including the differentiation or proliferation of new pancreatic {beta}-cells. Further elucidation of mechanisms by which Hh signals regulate IDX-1 and insulin gene expression should provide new insights regarding {beta}-cell functions, with important therapeutic implications for patients with diabetes mellitus.


   Acknowledgments
 
The authors thank W. Gaffield, A. McMahon, C. Miller, W. Moritz, J. L. Moss, and J. Seufert for generously providing reagents.


   Footnotes
 
1 We acknowledge support from NIH Grants DK-55365 and DK-30457 (to J.F.H.) and DK-02476 (to M.K.T.). Back

2 Investigator with the Howard Hughes Medical Institute. Back

Received October 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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