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Physiol. Genomics 24: 133-143, 2006; doi:10.1152/physiolgenomics.00156.2005
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Received 30 June 2005; accepted in final form 18 November 2005.
Physiological Genomics 24:133-143 (2006)
1094-8341/05 $8.00 © 2006 American Physiological Society

Identification of transcriptional targets during pancreatic growth after partial pancreatectomy and exendin-4 treatment

Diva D. De León1,2, Cyrus Farzad1, Michael F. Crutchlow1, John Brestelli3, John Tobias4, Klaus H. Kaestner3 and Doris A. Stoffers1

1 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine and the Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania School of Medicine, Philadelphia
2 Division of Endocrinology, Department of Pediatrics, The Children's Hospital of Philadelphia
3 Department of Genetics and the Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania School of Medicine, Philadelphia
4 Penn Bioinformatics Core, University of Pennsylvania, Philadelphia, Pennsylvania


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
After partial pancreatectomy (Ppx), substantial regeneration of the endocrine and exocrine pancreatic compartments has been shown in adult rodents. Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor agonist that augments endocrine ß-cell mass by stimulating neogenesis, proliferation, and cell survival. After Ppx, treatment with Ex-4 ameliorates hyperglycemia by stimulating ß-cell mass recovery. We utilized a cDNA microarray approach to identify genes differentially regulated during pancreatic regeneration after Ppx and/or Ex-4 administration. The pancreatic remnant after Ppx showed a large number of differentially regulated genes. In contrast, Ex-4 treatment resulted in a smaller number of differentially regulated genes. Of note, a common subset of genes regulated by Ex-4 and after Ppx was identified, including three members of the mitogenic Reg gene family, Reg2, -3{gamma}, and -3ß, as well as fragilis, a gene that maintains pluripotency during germ cell specification, and Serpin b1a, a member of an intracellular protease inhibitor family involved in cell survival. These observations were confirmed by real-time PCR. We determined that Reg3ß protein is also induced in the acinar pancreas after Ppx, suggesting a novel role for this factor in pancreatic growth or response to injury. Finally, comparison of transcription factor-binding sites present in the proximal promoters of these genes identified potential common transcription factors that may regulate these genes. Chromatin immunoprecipitation analyses confirmed Reg3{gamma} as a novel transcriptional target of Foxa2 (HNF3ß). Our data suggest molecular pathways that may regulate pancreatic growth and offer a unique set of candidate genes to target in the development of therapies aimed at improving pancreatic growth and function.

diabetes; ß-cell; pancreas; exenatide; glucagon-like peptide-1; islet; regeneration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
IN ADULT RODENTS, a regenerative capacity of the pancreas after partial pancreatectomy (Ppx) has been demonstrated. Thus, in rats, after a 90% Ppx, substantial well-defined regeneration of both exocrine and endocrine pancreas occurs. This process is the result of a well-orchestrated sequence of proliferative events that begin in the common pancreatic duct and continue in the smaller ductal structures, acini, and finally in the islets of Langerhans (5). This regenerative capacity of the pancreas, including both the exocrine and endocrine compartments, has important implications for a variety of disorders, including diabetes, pancreatitis, and pancreatic cancer. Understanding the molecular mechanisms involved in pancreatic regeneration is thus key to the development of effective preventive and therapeutic interventions relevant to these disorders.

Glucagon-like peptide-1 (GLP-1) is a potent glucose-dependent insulinotropic hormone secreted by the intestinal L cells in response to the ingestion of nutrients. GLP-1 also has important actions on gastric motility, suppression of plasma glucagon levels, promotion of satiety, and stimulation of glucose disposal in peripheral tissues (17). More recently, it has been appreciated that GLP-1 receptor agonists influence ß-cell growth in addition to ß-cell function. In the rat Ppx model, administration of the long-acting GLP-1 receptor agonist exendin-4 (Ex-4) stimulates the regeneration of ß-cell mass by increasing ß-cell neogenesis and proliferation, resulting in long-term attenuation of postpancreatectomy hyperglycemia (45). Similar results have been observed in the mouse subjected to a 70% Ppx (Stoffers DA and De León DD, unpublished observations). The observation of enhanced ß-cell recovery or maintenance in the face of genetic or metabolic insults has now been replicated in a growing number of rat and mouse models of diabetes in which GLP-1 receptor activation leads to increased rates of ß-cell replication, neogenesis (as indicated by several surrogate markers), and/or ß-cell survival (4, 7, 9, 23, 29, 37, 45).

The molecular mechanisms that regulate pancreatic regeneration and specifically ß-cell mass in the adult organism are still not fully understood. A significant level of regulation likely involves activation of transcription factor expression or function, leading to increased or decreased transcription of target genes that regulate the key processes of replication, neogenesis, and apoptosis of pancreatic epithelial cells. For example, the homeodomain transcription factor PDX-1 is essential for the early development of all pancreatic lineages and appears to be required for GLP-1 regulation of ß-cell growth, function, and survival (22). The endoderm transcription factor Foxa2 (HNF3ß) is a key regulator of PDX-1 gene transcription and expression (12, 21, 33, 44), and the ability of GLP-1 to activate PDX-1 expression may be mediated in part by Foxa2 (47). GLP-1 receptor activation also leads to the accumulation of intracellular cAMP, resulting in activation of cAMP-responsive element-binding protein (CREB) and promotion of ß-cell survival via induction of insulin receptor substrate-2 (IRS-2) (15). In the acinar cell carcinoma-derived cell line AR42J, GLP-1-induced insulin-positive differentiation appears to depend on BMP and transforming growth factor-ß (TGF-ß) signaling (46). The downstream genes regulated by these factors are largely unknown.

Here, we utilize cDNA microarray expression profiling with PancChip version 4.0 (PancChip4.0) that contains >10,000 pancreas-enriched elements to identify novel transcriptional targets involved in pancreatic regeneration and in particular targets of GLP-1 receptor signaling that could be involved in the expansion of pancreatic islet mass. We examined gene expression changes in total pancreas that occur during pancreatic regeneration after Ppx and in response to Ex-4 in BALB/c mice. We found both novel and known genes involved in cell growth and survival to be regulated by Ppx and Ex-4 and determined that one of these genes is a novel target of Foxa2. These data suggest molecular pathways that may regulate pancreatic growth and offer a unique set of potential candidate genes for the development of therapies aimed to improve pancreatic growth and function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
Experimental animals and surgical model.
Eight- to nine-week-old male BALB/c mice (Charles River Laboratories) were housed under standard conditions and allowed free access to standard mouse chow and water. These studies were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Ex-4 (Bachem, King of Prussia, PA) was administered intraperitoneally (ip; 1 nmol/kg body wt) immediately after surgery and every 24 h. Vehicle-treated animals received vehicle (1% BSA, 0.9% NaCl).

Mice were anesthetized (Nembutal Sodium, 50 mg/kg ip), and the entire splenic portion of the pancreas was surgically removed through a left lateral incision of the abdomen, resulting in an ~50% pancreatectomy, confirmed by weighing the removed and remnant portions during a pilot study. Sham operation involved anesthesia and abdominal incision while leaving the pancreas intact.

RNA isolation and cDNA preparation/labeling.
The pancreas remnant was harvested and RNA isolated using Trizol (Invitrogen, Carlsbad, CA) followed by DNase treatment (43). RNA integrity was confirmed by formaldehyde-agarose gel electrophoresis. Fifty-one RNA samples representing 13 groups were prepared: sham vehicle (SV), sham Ex-4 (SE), pancreatectomy vehicle (PV), and pancreatectomy Ex-4 (PE) at 12, 24, and 48 h after surgery and unoperated control pancreas. Each group contained at least four biological replicates except for PV, which contained three. Fluorescent labeling of samples was carried out as previously described (16).

Microarray.
Construction of the PancChip4.0 microarray and a source description of its >10,000 nonredundant elements have been previously reported (16). Samples were hybridized using a "reference design" in which two coupling reactions were set up for each pair of cDNAs. Each test sample was labeled with cyanine-3 (Cy3) and then paired with a control sample labeled with Cy5. Labeling was not alternated to control for variability between the dyes. Hybridization of the labeled samples to the PancChip4.0 and subsequent scanning and image analysis using Genepix Pro 3.0 were identical to those previously reported (16). The raw data results have been deposited into ArrayExpress (accession no. E-CBIL-5). The normalized data are available as Supplemental Data (available at the Physiological Genomics web site). 1

Data analysis.
Scanned data were thresholded using negative control spots. GenePix data for each array were imported into GeneSpring (version 6.2; Silicon Genetics, Redwood City, CA) and normalized using the intensity-dependent Lowess algorithm. Sample relationships were examined using principal components analysis, which revealed no strong technical effects that would encumber the subsequent analysis. To identify expression changes between experimental conditions, normalized data were exported to Significance Analysis of Microarrrays (SAM, version 1.2, Stanford University) (40), and a series of unpaired two-class comparisons was performed. For each comparison, the resulting ranked gene list and its associated q-values were saved in GeneSpring, where the lists were visualized and the Venn diagram tool was used to compare the lists.

To identify transcription factor-binding sites in the promoters of Ex-4- and Ppx-regulated genes, promoter sequences for Reg2, Reg3ß, Reg3{gamma}, Serpin b1a, and fragilis (–4800 to 0 relative to transcriptional start) were analyzed using Promoter Integration in Microarray Analysis (PRIMA) (8) to match all the transcription factor weight matrices (TFWM) in TRANSFAC version 8.0 (Biobase Biological Databases) to the promoters, using the sequences on the Mouse PromoterChip (10) as background. PRIMA aligned all TFWM against the background sequences and picked a score for each TFWM that produced a low false-positive rate (10%). All TFWM were then aligned against the five promoter sequences of interest, and a P value was calculated for each TFWM. The P value represents the probability of finding the number of found binding sites with a score greater than cutoff, using a hypergeometric distribution.

Quantitative real-time PCR validation.
Total RNA was converted to cDNA (First-Strand cDNA Synthesis Kit; Amersham Biosciences, Piscataway, NJ). Primers were designed for genes of interest using cDNA sequences deposited in the National Library of Medicine's database. Primer sequences are available on request. Quantitative real-time PCR (QPCR) was performed using a MyIQ Single-Color Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA). Logarithmic amplification and quantitative accuracy of all primer pairs were confirmed using serial dilutions of template cDNA. Product purity was assessed using melting-curve analysis. All reactions were performed in triplicate. For each sample, the threshold cycle (CT) for each target gene was compared with the CT for the internal control (ß-actin). ß-Actin mRNA levels were not changed under any of the conditions examined. Average difference values for the various experimental conditions (SV, SE, PV, PE) were compared. Fold effects were determined assuming each cycle represented a twofold difference in initial transcript quantity. Statistical significance was determined in log2-transformed data using one-way ANOVA and Fishers paired least significant difference test (Partek Pro version 6.1; Partek, St. Louis, MO).

Formaldehyde cross-linking and chromatic immunoprecipitation.
Pancreatic tissue was minced in cold PBS, passed through a 21-gauge needle, and cross-linked in 1% formaldehyde-PBS for 15 min. Chromatin isolation and immunoprecipitation were performed as previously described (10) on four independent chromatin preparations. Two hundred micrograms of input chromatin were used per immunoprecipitation. After preclearing, the supernatant was incubated overnight with Foxa2 antiserum (Ref. 3; Santa Cruz Biotechnology, Santa Cruz, CA) or control IgG. The average percentage bound for IgG was 0.47 and 3.17% for Foxa2. QPCR was performed to determine enrichment of promoter regions of interest. Enrichment of the region of interest was calculated using the following formula: 2[(CTIgG–CTInput)–(CTFoxa2–CTInput)]. Primer sequences are available on request.

Generation of polyclonal antisera directed against mouse Reg proteins.
Rabbit peptide-specific polyclonal antisera recognizing mouse Reg1 and -2 proteins were generated using a common 13-amino acid antigen (CKESGTTASNVWT) corresponding to amino acids 88–99 of Reg1 and 96–107 of Reg2. Reg3ß polyclonal antiserum was generated against a unique 12-amino acid antigen (RNPSTALDRAFC) corresponding to amino acids 135–146 of Reg3ß. Peptides were synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits (Rockland Immunochemicals, Gilbertsville, PA).

Western blot analysis.
Total pancreatic protein lysates (50 µg) were resolved on 12% NuPage Bis-Tris gels (Invitrogen Life Technologies) and transferred to nitrocellulose membranes, which were blocked and incubated in primary antisera (1:1,000). Mouse anti-ß-actin was used as loading control (Sigma, Milwaukee, WI), and secondary antisera [goat anti-rabbit IgG (H+L)-horseradish peroxidase (HRP) conjugated, goat anti-mouse IgG (H+L)-HRP conjugated; BioRad Laboratories] were used at 1:10,000. Signal was detected by chemiluminescence.

Immunohistochemistry.
Pancreas was fixed overnight at 4°C in 4% paraformaldehyde and embedded in paraffin. Sections (6 µm) through the maximal footprint region were stained with specific Reg1/2 and Reg3ß antisera (1:2,500) followed by secondary biotinylated goat anti-rabbit antibody (1:200; Vector, Burlingame, CA). Color development was performed using a Peroxidase Substrate DAB Kit (Vector Laboratories, Burlingame, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
Expression profiling of the pancreatic growth response.
We used a cDNA microarray approach to investigate gene expression during pancreatic regeneration after Ppx and in response to Ex-4. Male BALB/c 8- to 9-wk-old mice were divided into five groups: control, sham + vehicle (SV); sham + Ex-4 (SE); Ppx + vehicle (PV); and Ppx + Ex-4 (PE). Mice underwent 50% Ppx or sham operation and received a dose of Ex-4 (1 nmol/kg) or vehicle immediately after surgery and then every 24 h. Total pancreatic RNA was isolated at 12, 24, and 48 h after surgery (Fig. 1). Experimental and control reference cDNAs were fluorescently labeled and hybridized to PancChip4.0 containing 13,824 elements expressed in mouse pancreas. To identify expression changes between experimental conditions, we performed a series of unpaired two-class comparisons by SAM. Gene expression was compared between Ex-4- and vehicle-treated sham animals (SE vs. SV), vehicle-treated Ppx and sham animals (PV vs. SV), and Ex-4- and vehicle-treated Ppx animals (PE vs. PV) at 12, 24, and 48 h after surgery. Gene lists were created in the SAM program using a false discovery rate (FDR) of 29–33%. For each comparison, we saved the resulting ranked gene list and its associated q-values in GeneSpring, where we visualized the lists with respect to the expression data and used the Venn diagram tool to compare lists resulting from the various comparisons.



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  		Fig. 1. Experimental paradigm: 8- to 9-wk-old male BALB/c mice underwent partial pancreatectomy (Ppx) or sham operation and an ip injection of exendin-4 (Ex-4; 1 nmol/kg) or vehicle (1% BSA-0.9% NaCl) every 24 h. Total pancreas RNA was isolated at 12, 24, and 48 h.

 
Not surprisingly, the pancreatic remnant after Ppx showed a large number of differentially regulated genes (Table 1). Ninety-one genes were differentially regulated at 24 h and 37 genes at 48 h. Ppx regulated the expression of genes involved in growth, cell survival, mitochondrial and nuclear functions, signal transduction, and protein synthesis and degradation, which was the largest category. Twenty-three novel transcripts were differentially regulated. In the PE vs. PV comparison, no genes emerged at a reasonable FDR, except at 48 h when three genes were upregulated: amylase 2, Mlf2, and a novel transcript (data not shown).


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  		Table 1. Genes differentially regulated by pancreatectomy (PV vs. SV)

 
In contrast to Ppx, Ex-4 treatment resulted in a smaller number of differentially regulated genes. At 12 h, 3 genes were downregulated, while at 24 h, 13 genes were upregulated. These included known genes involved in cell growth and survival and signal transduction (Table 2). Of note, seven (54%) of the genes upregulated by Ex-4 were also upregulated by Ppx. The genes regulated during both pancreatic regeneration and in response to Ex-4 treatment include genes involved in growth (Reg2, Reg3ß, and Reg3{gamma}), cell survival (Serpin b1a), and fragilis, a gene involved in germ cell specification. We confirmed the regulation of these five genes by QPCR, whereas for two of the genes, hemopoietic cell phosphatase and AU-binding enoyl CoA hydratase, we were unable to amplify transcripts from pancreatic tissue (Fig. 2). The confirmation of five of seven genes in this category is consistent with the FDR of 30% applied to the microarray data analysis.


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  		Table 2. Genes differentially regulated by Ex-4 in sham mice (SE vs. SV)

 


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  		Fig. 2. Temporal expression pattern of genes regulated by both Ex-4 and Ppx. Normalized microarray intensity data were imported from GeneSpring and used to calculate fold change relative to the common, unoperated, untreated control samples for each condition and time point. Mean results ± SE are presented for Reg2 (P = 0.005, SE vs. SV, 24 h; P = 0.004, PV vs. SV, 24 h; P = 0.02, PV vs. SV, 48 h) (A), Reg3ß (P = 0.008, SE vs. SV, 24 h; P = 0.00006, PV vs. SV, 24 h; P = 0.006, PV vs. SV, 48 h; P = 0.04, PE vs. PV, 24 h) (B), Reg3{gamma} (P = 0.0004, SE vs. SV, 24 h; P = 0.05, PV vs. SV, 12 h; P = 0.002, PV vs. SV, 24 h; P = 0.02, PV vs. SV, 48 h) (C), Serpin b1a (P = 0.003, SV vs. SE, 24 h; P = 0.0005, PV vs. SV, 24 h) (D), and fragilis (P = 0.001, SV vs. SE, 24 h; P = 0.02, PV vs. SV, 24 h) (E). SV, sham + vehicle; SE, sham + Ex-4; PV, Ppx + vehicle; and PE, Ppx + Ex-4 at 12, 24, and 48 h (n = 3–5 biological replicates in all treatment groups and common control).

 
Regulation of the Reg gene family by Ex-4 and Ppx.
Reg2, Reg3ß, and Reg3{gamma} belong to the mitogenic Reg gene family that is encoded in a 75-kb region in the mouse genome and is grouped into four subtypes: type I (human RegI{alpha} and -Iß, rat RegI, and mouse Reg1); type II (mouse Reg2); type III [human pancreatitis-associated protein (PAP/HIP); human RegIII; rat PAP/Peptide23; rat RegIII/PAPII; rat PAPIII; mouse Reg3{alpha}, -3ß, -3{delta}, and -3{gamma}; and hamster islet neogenesis-associated protein (INGAP)]; and type IV (human RegIV) (1, 28). The founding member of this gene family, Reg1, was originally isolated from pancreatic secretions and from the islets of regenerating pancreas (39). Whereas Reg1 and -2 are expressed in exocrine tissue and hyperplastic islets and may be involved in pancreatic islet regeneration (31, 41), Reg3ß and Reg3{gamma} have been reported to be expressed strongly in the intestinal tract and only weakly in pancreas (27). Members of the mouse RegIII family were recently found to be upregulated in association with exocrine pancreatic proliferation after reduction of PDX-1 expression in adult mice (14).

In our study, Reg1 and -2 were upregulated during pancreatic regeneration, consistent with previous reports (31, 41). Interestingly, we found that members of the RegIII group, Reg3ß and Reg3{gamma}, were also strongly upregulated by Ppx (by 4.6- and 2.4-fold, respectively). Ex-4 also increased the expression of Reg2, Reg3ß, and Reg3{gamma} in sham animals by 2.3-, 2.0-, and 1.5-fold, respectively. The expression of these genes, particularly Reg2 and Reg3ß, was increased significantly in sham animals treated with vehicle compared with controls, suggesting an effect of anesthesia, surgical stress, or vehicle injection on the pancreatic expression of these genes. This apparent stress effect varied according to the specific Reg isoform and was early and time limited. In contrast, stimulatory response of Ex-4 and Ppx was greater and persisted for at least 24–48 h, suggesting a specific role for the Reg genes in the regenerative response (Fig. 2, A–C).

The regulation of Reg gene expression by Ppx and Ex-4 was validated by QPCR (Fig. 3, A–C). In parallel experiments, we examined Reg protein expression after Ppx. Isoform-specific antisera to detect Reg1/2 and Reg3ß were generated and used to confirm the upregulation of Reg1/2 and Reg3ß in protein lysates prepared from total pancreas at 24 h after Ppx (Fig. 4A). Immunohistochemical analysis of Reg3ß protein expression after Ppx showed that this upregulation is significant and localized to acinar tissue (Figs. 4, B and C), thereby suggesting a previously unrecognized role for Reg3ß in the response to Ppx.



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  		Fig. 3. Validation of pancreatic mRNA expression levels by quantitative real-time PCR (QPCR). Mean ± SE fold change expression relative to SV in mice treated with Ex-4 (SE) and after Ppx (PV) at 24 h for Reg2 [1-way ANOVA, P = 0.04, and Fishers paired least significant difference test (PLSD), P = 0.12, SE vs. SV; P = 0.01, PV vs. SV] (A), Reg3ß (1-way ANOVA, P = 0.03, and Fishers PLSD, P = 0.09, SE vs. SV; P = 0.01, PV vs. SV) (B), Reg3{gamma} (1-way ANOVA, P = 0.003, and Fishers PLSD, P = 0.04, SE vs. SV; P = 0.001, PV vs. SV) (C), Serpin b1a (1-way ANOVA, P = 0.005, and Fishers PLSD, P = 0.007, SE vs. SV; P = 0.002, PV vs. SV) (D), and fragilis (1-way ANOVA, P = 0.12, and Fishers PLSD, P = 0.19, SE vs. SV; P = 0.05, PV vs. SV) (E); n = 3–4 in all treatment groups.

 


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  		Fig. 4. Reg3ß is induced in the pancreatic remnant after Ppx. A: Reg1/2 and -3ß expression by Western blot analysis in SV and PV pancreas. Four biological replicates for each condition are presented. ß-Actin was used as the loading control. B and C: Reg3ß induction in the exocrine pancreas of PV mice (C) compared with SV mice (B), as shown by immunohistochemistry. Images were captured at x10 magnification.

 
Regulation of Serpin b1a and fragilis by Ex-4 and Ppx.
Expression of Serpin b1a, a member of an intracellular proteinase inhibitor family that is postulated to protect cells from uncontrolled proteolysis (35), was increased by Ex-4 treatment as demonstrated by microarray (1.38-fold, SE vs. SV) and by QPCR (2.41 ± 0.38-fold, SE vs. SV). Ppx increased the expression of Serpin b1a, by microarray (2.48-fold) and QPCR (3.25 ± 0.57-fold, PV vs. SV) (Figs. 2D and 3D). The expression of fragilis, an interferon-responsive gene involved in germ cell specification (32), was also stimulated by both Ex-4 and Ppx at 24 h (1.46-fold, SE vs. SV; 1.51-fold, PV vs. SV) (Fig. 2E). This observation was confirmed by QPCR (1.48 ± 0.07-fold, SE vs. SV; 2.26 ± 0.77-fold, PV vs. SV) (Fig. 3E).

Common transcription factor-binding sites in the promoter regions of genes regulated by Ex-4 and Ppx: identification of a novel transcriptional target of Foxa2.
To identify potential common transcriptional networks underlying the observed regulation of the five genes induced by Ex-4 and Ppx, we compared the transcription factor-binding sites present in the proximal promoters of these genes. The top weight matrices and P values for sites present in at least four of the five promoters are listed in Table 3. Putative binding sites for 10 transcription factors were identified, including transcription factors known to regulate pancreas development and function, such as HNF3ß (Foxa2), HNF6, and Nkx6.1 (reviewed in Ref. 13). Also included were SOX-5 and SOX-9, factors that are expressed during pancreatic development but whose specific role is yet to be determined (24). HMG-IY, a delayed early response gene proposed to be an important link in the sequence of nuclear events leading to proliferation (11) and more recently identified as a potential regulator of G protein-coupled cell proliferation in human pancreas (6), is also on this list. Of note, PDX-1 and CREB were not identified as common transcriptional activators of these proximal promoters. This may be a consequence of the specific early time points analyzed (12–48 h) and the restricted promoter region considered for this analysis.


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  		Table 3. Transcription factor weight matrices

 
Given the well-established role of Foxa2 in pancreas development and function (20, 38) as well as its ability to bind and transactivate PDX-1 (2, 21, 34, 44), we used chromatin immunoprecipitation (ChIP) to examine in vivo binding of Foxa2 to evolutionarily conserved potential Foxa2-binding sites in the promoter regions of Reg2 (–1386 relative to transcriptional start), Reg3{gamma} (–2730), Serpin b1a (–2957), and fragilis (–2694). ChIP assay for Foxa2 occupancy was carried out on four independent chromatin preparations, and QPCR was performed to quantify the degree of target sequence enrichment. Foxa2 antiserum provided a 4 ± 0.58-fold enrichment of Reg3{gamma} (Fig. 5, A and B). No reproducible enrichment of the Serpin b1a, Reg2, and fragilis enhancers was observed.



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  		Fig. 5. Foxa2 occupies the Reg3{gamma} promoter in vivo. Chromatin prepared from total mouse pancreas (n = 4) was immunoprecipitated with anti-Foxa2 or nonimmune IgG as indicated. Input chromatin and precipitated material were amplified with primers specific for Reg3{gamma}. A: resolution of PCR products by agarose gel electrophoresis. B: QPCR analysis. Enrichment of Reg3{gamma} relative to IgG was corrected for input. *95% confidence interval (CI) = 1.52–6.54.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 GRANTS
 REFERENCES
 
In an effort to identify genes involved in adult pancreatic growth, we utilized a cDNA microarray approach to examine the temporal pattern of gene expression in sham-operated or Ppx mice treated with vehicle or the ß-cell trophic peptide Ex-4 during the first 48 h after intervention. The PancChip4.0 that contains 13,824 distinct elements was used to identify novel transcriptional targets involved in cell growth and survival pathways as well as other targets involved in gene transcription, cytoskeletal structure and cell adhesion, signal transduction, and cell metabolism. A substantial number of novel genes were also identified.

The use of total pancreatic RNA in this microarray study allowed us to preserve the in vivo patterns of gene expression and minimize RNA degradation. Recent reports indicate that gene expression profiles are dramatically altered by tissue fractionation and culture (26). A significant caveat, however, is that the heterogeneity of the pancreas may result in dilution of signal from genes highly regulated in a subpopulation of cells by their unregulated expression in other cell types, which would minimize the apparent degree of regulation in total pancreatic RNA. This is likely to be a major reason for the low levels of gene induction observed for many of the genes identified in this microarray study. Future studies using in situ hybridization and laser capture microdissection will be required to precisely define the expression patterns of these genes within the exocrine and endocrine compartments of the pancreas.

Notably, more than one-half of the genes upregulated by Ex-4 in sham mice were also induced in the vehicle-treated Ppx remnant, suggesting a potential role for these genes in pancreatic growth. The most notable subset of these commonly regulated genes comprises members of the Reg gene family and includes members of the RegIII subfamily that were not previously known to play a role in the pancreas. Reg1, the founding member of this gene family, increases ß-cell replication (42). Similarly, Reg proteins appear to play regulatory roles in the proliferation of gastric epithelial cells and of hepatocytes after partial hepatectomy (18, 36). RegIII and Reg2 are anti-apoptotic in AR42J acinar cells and in neurons, respectively (25, 30). In addition to implicating Reg factors in the trophic effects of Ex-4, our results provide the first indication for a role of the RegIII family, specifically Reg3ß and -3{gamma}, in pancreatic regeneration.

Serpin b1a, a member of the serine proteinase inhibitor family, was induced by both Ex-4 and Ppx. Although members of this group of proteinase inhibitors are secreted into the circulation, the clade B serpins are intracellular proteins that protect cells from exogenous and endogenous proteinase-mediated injury (35). Upregulation of Serpin b1a after Ppx may be part of the cytoprotective response to injury. Interestingly, Ex-4 also regulates the expression of this gene, suggesting that the anti-apoptotic effects of Ex-4 may involve regulation of Serpin b1a.

Ex-4 and Ppx also regulated the expression of fragilis, an interferon-inducible transmembrane protein. Previous studies have shown fragilis expression predominantly in nascent primordial germ cells and gonadal germ cells as well as in pluripotent ES and embryonic germ cells; thus it has been proposed that fragilis may have a role in the propagation of the pluripotent state (19, 32). Cellular localization of fragilis expression in the pancreas will be required to determine its predominant site of expression and to determine whether its expression is compatible with localization to a potential progenitor population.

In an effort to identify common transcriptional networks involved in the regulation of these genes by Ex-4 and Ppx, we performed a computational analysis of their promoter regions. We identified putative binding sites for transcription factors known to have a critical role in ß-cell development and function, such as Nkx6.1, HNF3ß (Foxa2), and HNF6. Others such as SOX-5 and SOX-9 are expressed early during pancreatic development, but their precise roles are not yet established. Chromatin immunoprecipitation analysis verified Reg3{gamma} as a novel transcriptional target of Foxa2. Of note, these assays were conducted using chromatin prepared from quiescent adult pancreas. Future experiments will determine whether the occupancy of this promoter by Foxa2 is differentially regulated during pancreatic regeneration.

In summary, we have identified a set of genes that are differentially regulated by the in vivo administration of the ß-cell trophic factor Ex-4 and in association with pancreatic regeneration after Ppx. The genes commonly regulated by Ex-4 and Ppx may be particularly interesting candidates for a regulatory role in pancreatic growth. Indeed, the identification of the mitogenic Reg genes as transcriptional targets of Ex-4 suggests that the trophic effects of Ex-4 may be mediated in part by the upregulation of Reg gene expression via autocrine or paracrine effects. Furthermore, we have shown that members of the mouse RegIII family are upregulated, suggesting a previously unreported role for these genes in pancreatic growth. Finally, we have identified that Reg3{gamma} is a novel transcriptional target of Foxa2. This study provides a unique set of potential candidate genes to investigate the development of therapies aimed to improve pancreatic growth and function.


   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
GRANTS
 REFERENCES
 
D. A. Stoffers is a member of scientific advisory boards for Amylin Pharmaceuticals and Glaxo Smith Kline.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
REFERENCES
 
These studies were supported by an American Diabetes Association Career Development Award and National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-062965 (D. A. Stoffers) and U01-DK-56947 (K. H. Kaestner) and an individual National Research Service Award F32-DK-60273 and K12 Award K12-DK-063682-02 (D. D. De León). We acknowledge support from the Morphology Core of the University of Pennsylvania Center for Molecular Studies in Digestive and Liver Disease (P30-DK-50306) and the Functional Genomics Core of the Penn Institute for Diabetes, Obesity and Metabolism.


   ACKNOWLEDGMENTS
 
We thank Mira Sachdeva for advice and help with ChIP and Phillip Phuc Le for help with the analysis of the promoter regions of the genes of interest.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. A. Stoffers, Div. of Endocrinology, Diabetes and Metabolism, Dept. of Medicine, Clinical Research Bldg. 611 B, Univ. of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, PA 19104 (e-mail: stoffers{at}mail.med.upenn.edu)

10.1152/physiolgenomics.00156.2005.

1 The Supplemental Material for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/00156.2005/DC1. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 

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