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Endocrinology Vol. 147, No. 12 5752-5759
Copyright © 2006 by The Endocrine Society

Trefoil Factors Are Expressed in Human and Rat Endocrine Pancreas: Differential Regulation by Growth Hormone

Malene Jackerott1, Ying C. Lee1, Kjeld Møllgård, Hans Kofod, Janne Jensen, Sonja Rohleder, Nicole Neubauer, Louise W. Gaarn, Jeanette Lykke, Rikke Dodge, Louise T. Dalgaard, Birgitte Søstrup, Dennis B. Jensen, Lars Thim, Ebba Nexø, Peter Thams, Hanne Cathrine Bisgaard and Jens H. Nielsen

Departments of Medical Biochemistry and Genetics (M.J., Y.C.L., H.K., J.J., S.R., N.N., L.W.G., J.L., R.D., L.T.D., B.S., D.B.J., P.T., H.C.B., J.H.N.) and Medical Anatomy (K.M.), University of Copenhagen, DK-2200 Copenhagen, Denmark; Department of Protein Chemistry (L.T.), Novo Nordisk A/S, DK-2880 Bagsværd, Denmark; and Department of Clinical Biochemistry (E.N.), Aarhus University Hospital, DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: Jens Høiriis Nielsen, Department of Medical Biochemistry and Genetics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. E-mail: jhn{at}imbg.ku.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human pancreas and islets by ELISA. Exposure of neonatal rat islets or insulinoma cells to GH, a known ß-cell growth factor, resulted in markedly increased TFF3 but decreased TFF1 mRNA levels. The effect of GH on TFF3 expression was confirmed by Western blot. Culture of neonatal rat islets in the presence of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC MORPHOGENESIS IS a complex process involving multiple factors, in which intracellular signaling molecules, hormonal influences, growth factors, extracellular matrix components, and integrins participate in a coordinated manner (1, 2, 3, 4, 5, 6). It is increasingly evident that the establishment of cell-to-cell contact and the three-dimensional organization is an integral part for the end-stage differentiation of the pancreatic islets. The factors responsible ensure the maintenance of cell surface integrity and preserve and limit their potential proliferative abilities (5, 6). Among the molecules involved in preserving cell-to-cell continuity and repair are the trefoil factor (TFF) family of peptides (7, 8, 9, 10, 11, 12) that are predominantly found in the gastrointestinal tract and other mucosal tissues. TFFs are small protease-resistant proteins consisting of three members, TFF1 (also termed as pS2) and TFF2 (known formerly as spasmolytic polypeptide) and TFF3 (also known as intestinal trefoil factor). They are recognized to be decisive in cell migration and spreading and cell renewal and have a role in inflammatory response in the gastrointestinal tract (12). Although TFF2 was first isolated from porcine pancreas (13), the localization and function of TFFs in the endocrine pancreas have so far not been investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human material and animals
Adult human paraffin-embedded, formaldehyde-fixed pancreatic tissue, frozen pancreatic tissue and islets and viable islets were obtained from the multicenter European Union-supported program on ß-cell transplantation in diabetes (directed by Professor D. Pipeleers, Brussels, Belgium), which has been approved by central and local ethical committees. The human islet preparations for the present study were treated as previously described (14). Series of paraffin sections from 9- to 24-wk gestation human fetuses fixed in Lillie’s fixative were available from previous studies (15, 16). Newborn Wistar rats (aged 3–5 d) were obtained from Taconics Europe (Ry, Denmark). The animal handling and experimentation were conducted in accordance with institutional guidelines and approved by the Animal Experiments Inspectorate in Denmark.

TFF peptides and antibodies
Recombinant human TFF1, TFF2, and TFF3 and rat TFF3 were obtained from Novo Nordisk A/S (Bagsværd, Denmark) and prepared as previously described (17, 18, 19). Recombinant human TFF3 was biologically active in protection of the gastrointestinal mucosa (7). Antibodies to human TFF1 (2239A), TFF2 (2240A), and TFF3 (2241A) were raised in rabbits, as previously described (20, 21). A rabbit antibody (NN4) raised to synthetic human dimer TFF3 peptide fragment (49–59) conjugated to keyhole limpet hemocyanin was found to have high affinity for mouse TFF3 in Western blot (Thim, L., unpublished data). This antibody was used to stain rat pancreatic sections and INS-1E cell extracts on Western blots.

Immunocytochemistry
Immunocytochemical stainings of human pancreas were performed on 5-µm sections of paraffin-embedded, formaldehyde-fixed tissue that was deparaffinized and incubated in methanol containing 0.03% H2O2 to inhibit endogenous peroxidase activity. After antigen retrieval by treatment with 0.2 mg/ml pronase for 10 min at 37 C, the sections were blocked with 10% normal horse serum. For peroxidase/diaminobenzidine stainings, the sections were incubated with rabbit antiserum against human TFF1 (diluted 1:1000; 2239A), TFF2 (diluted 1:1000; 2240A), or TFF3 (diluted 1:2000; 2241A) at 4 C overnight. The primary antibodies were detected by successive incubations with biotin-labeled secondary antibodies against rabbit IgG and peroxidase-conjugated streptavidin followed by development in diaminobenzidine-H2O2 mixture. The sections were counterstained using Mayer’s hematoxylin, dehydrated, and mounted in Eukitt medium. The specificity of the immunostainings was verified by performing similar immunostainings replacing the primary antiserum with TFF antiserum preabsorbed against 20 µg/ml of the corresponding human TFF peptide. For double immunofluorescence of adult human pancreas, the sections were incubated with human TFF3 antiserum (diluted 1:4000; 2241A) mixed with guinea pig antiserum to insulin (diluted 1:1000) (Dako, Glostrup, Denmark) or mouse monoclonal antibodies to glucagon (0.5 µg/ml; Glu001) (Novo Nordisk) or somatostatin (5 µg/ml; Som018) (Novo Nordisk). Subsequently, biotin-labeled antirabbit IgG mixed with fluorescein isothiocyanate (FITC)-labeled anti-guinea pig IgG or FITC-labeled antimouse IgG was applied. After addition of peroxidase-conjugated streptavidin, the TFF3 signal was amplified by peroxidase-catalyzed deposition of biotin-conjugated tyramide using the TSA biotin system (PerkinElmer, Boston, MA) and visualized by application of Texas red-conjugated streptavidin. For TFF3 and pancreatic polypeptide (PP) double stainings, TFF3 immunofluorescence was performed as described above followed by application of rabbit antiserum to human PP (kindly donated by Dr. L.-I. Larsson, Copenhagen, Denmark) and FITC-labeled antirabbit IgG. By using peroxidase-catalyzed deposition of tyramide, double staining with two primary antisera derived in the same species can be performed (22).

Deparaffinized sections of newborn rat pancreas were incubated in methanol containing 0.03% H2O2, and antigen retrieval was performed by treatment for 2 x 5 min in the microwave oven. After blockage in 10% normal horse serum, the sections were exposed to rabbit antiserum with a high affinity for mouse TFF3 (NN4) mixed with guinea pig antiserum to insulin. The primary antibodies were detected as specified above. The specificity of the staining was verified by preabsorption of the antibody with 20 µg/ml of the rat TFF peptide. For double immunofluorescence of human fetuses, dewaxed sections were incubated with 10% normal goat serum followed by exposure to human TFF3 antiserum (diluted 1:2000; 2241A) mixed with guinea pig antiserum to insulin (diluted 1:3000) or mouse monoclonal antibodies to glucagon (0.083 µg/ml) or somatostatin (0.167 µg/ml). The primary antibodies were visualized by rhodamine-coupled antirabbit IgG, FITC-labeled antiguinea pig, and FITC-labeled antimouse IgG. All secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Specimens were examined by both conventional transillumination and fluorescent epiillumination using selective FITC and Texas Red filters and photographed using a DC250 camera (Leica, Wetzlar, Germany).

ELISA
Human pancreatic and islet samples were homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, 1 mM orthovanadate, 1 µg/ml aprotinin, and 1 µg/ml leupeptin in PBS) followed by centrifugation at 10,000 x g for 10 min. The total protein concentrations in the supernatants were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The concentration of TFF3 peptide in the extracts was measured by ELISA using the rabbit antihuman TFF3 antibody (2241A), as previously described (20), and expressed in relation to total protein concentration.

Islet preparation and culture
Freshly dissected neonatal rat pancreas was subjected to collagenase digestion and purified on a Percoll gradient. Subsequently islets were handpicked under stereomicroscope with a constriction pipette and the pooled newborn islets were cultured in RPMI 1640 medium containing L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% newborn calf serum for 5 d as previously described (23).

Islet cell migration
For culture of whole islets, pooled pancreatic islets were subdivided into separate dishes containing equal number of islets cultured in RPMI 1640 containing 0.5% human serum, plus or minus human GH (hGH) (500 ng/ml) (Novo Nordisk A/S) or human TFF3 (10–7 M). The cell migration was quantified as the area covered by cells after 3 wk in culture.

Cell proliferation
Neonatal rat islet cells were dispersed by trypsin-EDTA treatment and cultured for a week in RPMI 1640 containing 0.5% human serum and hGH (500 ng/ml). After 24 h starvation in medium containing 0.5% human serum, the cells were cultured for 24 h in presence or absence of hGH (500 ng/ml) and/or TFF3 (100 nM). Bromodeoxyuridine (BrdU) (10 mM) was added to the medium 90 min before fixation of the cells in 4% paraformaldehyde. The islet cells were double immunostained for insulin and BrdU as described previously (24). The mitotic activity of insulin cells was determined by counting the number of insulin-positive and insulin and BrdU double-positive cells under the microscope.

INS-1 and INS-1E cells
INS-1 and INS-1E cells (kindly provided by Dr. C. B. Wollheim, University of Geneva, Geneva, Switzerland) were cultured at 37 C in a humidified atmosphere containing 5% CO2 in RPMI 1640 containing 11 mM glucose and Glutamax I (Invitrogen, Taastrup, Denmark) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and 50 µM ß-mercaptoethanol. One day before addition of various stimuli, the FCS was reduced to 0.5% in the culture medium.

Western blotting
INS-1E cells cultured in the presence of absence of 500 ng/ml hGH for 24 h were lysed using RIPA buffer, centrifuged at 10,000 x g for 10 min, and protein concentrations were determined in supernatants. One milligram of protein extracts were incubated with 4 µg of rabbit anti-TFF3 (NN4) antibody overnight at 4 C. Subsequently the antibody-bound protein was precipitated after incubation for 1 h at 4 C with Dynabeads M-280 sheep antirabbit IgG (Dynal Biotech, Oslo, Norway). After wash of the precipitates in PBS containing 0.1% BSA, the antigens are eluted by incubation for 5 min at 95 C in RIPA buffer containing 0.11 M ß-mercaptoethanol and 1x NuPage LDS sample buffer (Invitrogen). The proteins were separated by electrophoresis on 12% NuPage Novex Bis-Tris gel in MES running buffer (Invitrogen) and transferred to nitrocellulose membranes by electroblotting. After incubation of the membrane in blocking buffer (Tris-buffered saline containing 5% nonfat dry milk, 5% FCS, and 0.1%Tween 20), the membrane was exposed overnight at 4 C to rabbit anti-TFF3 (NN4) antibody diluted 1:200 in blocking buffer. The membrane was subsequently washed in Tris-buffered saline containing 0.1% Tween 20, incubated with peroxidase-conjugated swine antirabbit IgG (Dako), and proteins detected by chemiluminescence using ECL plus Western blotting detection reagent (GE Healthcare, Chalfont St. Giles, UK).

Apoptosis assay
The apoptotic activity of INS-1E cells was determined by stimulating the cells for 24 h with IL-1ß (40 pg/ml), interferon (IFN)-{gamma} (50 U/ml), and TNF{alpha} (0.5 ng/ml) in the presence of absence of hGH (500 ng/ml) or TFF3 (100 nM) as described previously (25). Apoptosis was measured using the cell death detection ELISA Plus (Roche, Hvidovre, Denmark).

Insulin secretion
INS-1E cells were cultured for 2 d in 12-well plates in RPMI 1640 containing 5.5 mM glucose. Three hours before the experiments, the media were changed to RPMI 1640 containing 3 mM glucose. The cells were subsequently stimulated for 2 h with 3 or 20 mM glucose in the presence or absence of 7.6 µM TFF3 dissolved in Krebs-Ringer medium supplemented with 10 mM HEPES, 5 mM NaHCO3, 2.54 mM CaCl2, and 0.2% BSA. The insulin content in the medium was determined by RIA using guinea pig antiinsulin serum, monoiodinated human insulin as tracer, rat insulin as standard, and ethanol to separate antibody-bound insulin from free insulin.

Quantitative real-time PCR
Total RNA from human islets and from rat islets and INS-1 cells cultured in the presence of absence of hGH (500 ng/ml) for 4 or 24 h was isolated using the RNeasy kit (QIAGEN, Hilden, Germany). RNA samples (1.25 µg) were reversed transcribed with oligo dT using the Access reverse transcription system (Promega, Madison, WI). Quantitative real-time PCR was carried out using the LightCycler (Roche) with Quantitect SYBR Green PCR mix (QIAGEN) according to the recommended protocol. The real-time PCR runs were all performed using the same reaction conditions for denaturation, amplification, and extension [initial denaturation for 15 min at 95 C; three-cycling step for 35–45 cycles: 15 sec denaturation at 94 C, 20 sec annealing at 55 C, 20 sec extension at 72 C]. Gel electrophoresis and/or melting curve analysis were used to confirm the fidelity of the LightCycler PCR.

For human islet PCR, TFF primer sequences were identical with those as described (26). For rat islet and INS1 cell PCR, the following primers were used: TFF 1 primers, 5'-CAAGGT GACCTGTGTCCTC-3' (sense) and 5'-CTTGCTGGTTCTCAATGACC-3' (antisense) (218 bp product); TFF 2 primers, 5'-TCTTGGTAGTGGTCCTTGTCTTG-3' (sense) and 5'-GAAGATCAGGTTGGAAAAGCAG-3' (antisense) (317 bp product); TFF 3 primers, 5'-ATGGAGACCAGAGCCTTCT-3' (sense) and 5'-GGATGCTGGAGTCAAAACAG-3' (antisense) (193 bp product); phosphoglycerate kinase 1 (PGK) primers, 5'-CTGGAAAACCTCCGCTTTCA-3' (sense) and 5'-TGGCAGATTCACAC-3' (antisense) (191 bp product); ribosomal protein L13 (RPL13) primers, 5'-CCACCCTATGACAAGAAAAAGC-3' (sense) and 5'-ACATTCTTTTCTGCCTGTTTCC-3' (antisense) (227 bp product). The RPL13 and PGK primer pairs were acting as the reference controls for the relative real-time PCR of human islets and rat islets and INS-1 cells, respectively. To create the calibration standards for the real-time PCR, PCR fragments generated with the above-mentioned primer pairs using either human or rat islet cDNA as template were cloned into pCR 2.1 using the TOPO cloning kit (Invitrogen), and the identity of the PCR products was confirmed by sequencing. These respective individual PCR fragment-containing plasmids were linearized using HindIII and serial diluted by a factor of 10 to create a series of diluted PCR fragment-bearing plasmid solutions. They were used as the external calibration standards for the quantification of the respective TFFs in the real-time PCR runs.

Statistical analyses
Data are presented as mean ± SE. Statistical significances were evaluated by one-way ANOVA and Newman-Keuls multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of TFFs in adult human pancreas
To explore the presence of TFFs in the pancreas, immunocytochemical staining of human pancreatic sections using antihuman TFF1, TFF2, and TFF3 was performed (Fig. 1Go). Strong reactivity for TFF3 was found in islets and some pancreatic duct cells, whereas only a faint staining for TFF1 and no TFF2 staining was observed in islets (Fig. 1Go). No TFF1 or TFF2 immunoreaction was detected in pancreatic duct cells. By preabsorption of the TFF antisera with the corresponding TFF antigen, the TFF1 and TFF3 stainings were eliminated (Fig. 1Go). To identify the TFF3-positive cells, double immunofluorescence with antibodies to insulin, glucagon, somatostatin, and PP was performed. TFF3 was present in most insulin cells (Fig. 2Go, A–C), some glucagon (Fig. 2Go, D–F), and PP cells (Fig 2Go, J–L) but not somatostatin cells (Fig. 2Go, G–I). The localization of TFF1–3 was similar in both pancreatic samples analyzed.


Figure 1
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  		FIG. 1. Immunoperoxidase staining for TFF peptides in human pancreatic islets and ducts. Sections through pancreatic tissue were immunostained using antibodies against TFF1 (A), TFF2 (C), or TFF3 (E). As negative controls, similar immunostainings were performed using TFF1 antiserum preabsorbed with TFF1 peptide (B), TFF2 antiserum preabsorbed with TFF2 peptide (D), or TFF3 antiserum preabsorbed with TFF3 peptide (F). Section through human pancreatic duct stained by immunoperoxidase using TFF3 antiserum (G) or TFF3 antiserum preabsorbed with TFF3 peptide (H). Immunoreactive TFF3 was localized to some duct cells and a small cluster of cells close to the duct. Bar, 30 µm.

 

Figure 2
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  		FIG. 2. Section through human pancreatic islet stained by double immunofluorescence for TFF3 and insulin (A–C), TFF3 and glucagon (D–F), TFF3 and somatostatin (G–I), and TFF3 and PP (J–L). TFF3 was visualized by red fluorescence (A, D, G, and J), whereas islet hormones were detected by green fluorescence (B, E, H, and K). C, F, I, and L are merged images of the corresponding red and green images. Note that TFF3 immunoreactivity was detected in most insulin cells as well as some glucagon and PP cells (arrows) but not somatostatin cells. Bar, 30 µm.

 
Expression of TFF in isolated human pancreatic islets
To determine whether the TFF immunoreactivity coheres with the levels of TFF transcripts in human islets, quantitative RT-PCR analysis of TFF mRNA expression in isolated human islets was performed. It was found that there was significant expression of TFF3 mRNA in all four human islet samples examined, whereas undetectable to minimal expression of TFF2 and TFF1 was found (Fig. 3Go, A and B). Although TFF2 transcripts appeared to be expressed in human and rat islets as analyzed by RT-PCR, the levels of TFF2 mRNA present in islets was close to the detection limit of the assay, and because no immunoreactive TFF2 in human pancreatic islets was observed, the TFF2 expression was not further assessed. To verify that TFF3 protein is expressed in the islets, extracts of human pancreas and isolated islets were analyzed by a highly sensitive ELISA developed for human TFF3 (Fig. 3CGo). Low levels of TFF3 were detected in the pancreatic extracts, whereas higher levels were found in the isolated islets in accordance with the immunocytochemical results (Figs. 1Go and 3CGo).


Figure 3
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  		FIG. 3. Quantification of TFF3 mRNA and protein levels in human islets. A, Gel analysis of PCR products derived from TFFs and RPL13 cDNA of islets from four human donors. B, The TFF3 expression levels of the four individual islet samples were indicated in relation to RPL13 levels measured by real-time PCR. C, TFF3 protein levels in extracts from three individual human pancreas or islet preparations measured by ELISA.

 
Expression of TFF3 in fetal human and newborn rat pancreas
To evaluate the expression of TFF3 during human pancreatic development, sections of 14-wk-old human fetuses were stained with antibodies to TFF3 together with antibodies to either insulin, glucagon or somatostatin (Fig. 4Go, A–I). TFF3 was detected in insulin (Fig. 4Go, A–C) and glucagon (Fig. 4Go, D–F) but not somatostatin-positive cells (Fig. 4Go, G–I). Also, in newborn rat pancreas, TFF3 was localized to most insulin cells in addition to several noninsulin cells in the periphery of islets (Fig. 4Go, J–L).


Figure 4
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  		FIG. 4. Sections of a 14-wk human fetal pancreas (A–I) or newborn rat pancreas (J–L) stained by double immunofluorescence for TFF3 and insulin (A–C and J–L), TFF3 and glucagon (D–F), and TFF3 and somatostatin (G–I). TFF3 immunoreactivity was labeled by red fluorescence (A, D, G, and J), whereas insulin (B and K), glucagon (E), and somatostatin (H) immunopositive cells were visualized by green fluorescence. C, F, I, and L are merged images of the corresponding red and green images. Note in A–C and J–L that TFF3 localizes to most insulin cells (exemplified by arrows), whereas other cells are immunopositive for only TFF3 (arrowheads). In D–F, all glucagon cells also are TFF3 positive (exemplified by arrows), and some TFF3 cells are negative for glucagon (arrowheads). In contrast, TFF3 and somatostatin are localized to different cell populations (G–I). Bar, 30 µm.

 
Effects of GH on TFF expression in rat islets and insulinoma cells
Because GH and prolactin are known to stimulate ß-cell replication and insulin gene expression (24), we assessed the effect of GH on TFF mRNA expression using isolated neonatal rat islets and INS-1 cells acting as a ß-cell model system. To a large degree, INS-1 cells mimic pancreatic ß-cells, particularly their insulin responses to physiological doses of glucose (27). The quantitative analyses showed that the basal expression levels of TFF1 and TFF3 mRNAs are comparable in the control pancreatic neonatal islets; however, their responses to GH treatments follow a contrasting pattern. There was a marked decrease of TFF1 expression at 4 h in GH-treated rat pancreatic islets (4-fold as compared with control islets), whereas a significant increase of TFF3 expression (2-fold as compared with the control) was seen with rat islets treated with GH for 24 h (Fig. 5Go, A and B). Both TFF1 and TFF3 are expressed in the INS-1 cells (Fig. 5Go, C and D). The effect of GH on TFF1 and TFF3 expression patterns followed closely with what we observed for the isolated neonatal islets. For the GH-treated INS-1 cells, GH caused a significant attenuation of TFF1 expression, more so for those cells having been exposed to GH for 24 h than those for 4 h. On the other hand, similar to the rat islet data, a significant increase of TFF3 expression was seen when INS-1 cells had been treated with GH for 24 h. To determine whether the GH effect on TFF3 mRNA expression was reflected at the protein level, Western blot was performed on protein extracts from INS-1E cells cultured for 24 h with and without GH (Fig. 5EGo). The results confirmed that GH stimulated the expression of TFF3 protein.


Figure 5
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  		FIG. 5. Quantification of TFF expression in rat islets and INS-1 cells. A–D, Measurement by real-time PCR of TFF3 (A and C) and TFF1 (B and D) mRNA expression in rat pancreatic islets (A and B) and INS-1E cells (C and D) stimulated with hGH (500 ng/ml) for 0, 4, or 24 h. The INS-1E cell experiments were carried out on four separate occasions, and each cDNA sample was run in duplicates by real-time PCR, and data were normalized against PGK expression. GH treatment caused a significant increased TFF3 expression at 24 h and a significant decreased TFF1 expression at 24 h. Statistical analysis was assessed using one-way ANOVA, followed by Newman-Keuls multiple comparison test, *, P < 0.05. E, Analysis of TFF3 protein levels in INS-1E cells stimulated with or without hGH (500 ng/ml) for 24 h. INS-1E protein extracts were immunoprecipitated, separated by electrophoresis, and immunoblotted using the anti-TFF3 (NN4) antibody. Right lane shows 2 µg rat TFF3 as control. cont, Control.

 
Effects of TFF3 on islet cell migration and proliferation
Because hGH has been shown to cause attachment and spreading of newborn pancreatic islets that normally do not attach and spread under the present culture conditions (24), we compared the effects of TFF3 and hGH on islet attachment and migration. It was found that TFF3 added to the culture medium was able to stimulate islet cell attachment and migration on a solid surface (Fig. 6Go, A and C). However, the effect was significantly lower than that of hGH (Fig. 6Go, B and D). To see whether TFF3 also could stimulate proliferation of neonatal rat ß-cells, the effect on BrdU incorporation was evaluated in the presence and absence of hGH. No significant effect of TFF3 was observed (Fig. 6EGo).


Figure 6
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  		FIG. 6. Effect of TFF3 on islet spreading and proliferation. A–D, Neonatal rat islets cultured for 3 wk in the absence (A) or presence (B) of hGH (500 ng/ml) or TFF3 (10–7 M) (C). Islet area in relation to control islets (cont) is shown as means ± SE in D. ***, P < 0.001. E, Dispersed islet cells from newborn rats were cultured for 24 h in the presence or absence of hGH (500 ng/ml) and/or TFF3 (100 nM) and in the presence of BrdU followed by double immunostaining for BrdU and insulin. The percentage of BrdU-positive insulin cells was determined by counting the cells under the microscope. More than 300 insulin cells were counted in each preparation (n = 4–6). **, P < 0.01; ***, P < 0.001 vs. control.

 
Effects of TFF3 on apoptosis and insulin release in INS-1E cells
To determine whether TFF3 is able to prevent cytokine-induced apoptosis, INS-1E cells were exposed to a mixture of the cytokines IL-1ß, TNF{alpha}, and IFN{gamma} (mix) in the absence or presence of GH or TFF3. TFF3 did not affect the cytokine-induced apoptosis, whereas GH had a significant protective effect as previously shown (Fig. 7AGo) (25). To analyze the effect of TFF3 on glucose-stimulated insulin secretion, INS-1E cells were incubated for 2 h at 3 and 20 mM glucose with and without TFF3. TFF3 had no effect on basal or glucose-stimulated insulin release (Fig. 7BGo).


Figure 7
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  		FIG. 7. Effect of TFF3 on apoptosis and insulin secretion in INS-1E cells. A, INS-1E cells were treated with a mixture (mix) of IL-1ß (40 pg/ml), IFN{gamma} (50 U/ml), and TNF{alpha} (0.5 ng/ml) in the presence or absence of hGH (500 ng/ml) or TFF3 (100 nM) for 24 h. Apoptosis was determined by measurement of nucleosomal release by ELISA (n = 3). *, P < 0.05 vs. control (cont). B, Insulin secretion from INS-1E cells cultured for 60 min in 3 or 20 mM glucose in the presence or absence of TFF3 (7.6 µM) in 12-well plates. Results are means ± SE of four experiments performed in triplicates. *, P < 0.05 vs. 3 mM glucose.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Considering the abundance of TFFs in mucosal epithelia and the localization of TFF2 in the exocrine compartment of the porcine pancreas, the almost exclusive expression of TFF3 in the human endocrine pancreas was unexpected. The immunocytochemical colocalization of TFF3 with most insulin and some glucagon-expressing cells in the adult human pancreas correlates with the mRNA expression in isolated human islets. There was also expression of TFF3 in some pancreatic duct cells. Interestingly, in one recent report, using microarray cDNA analysis to profile gene expressions of intraductal papillary-mucinous tumors of the pancreas, the data revealed that the most highly expressed genes are the TFFs among the approximately 5000 human genes analyzed, and weak immunoreactive TFF3 was observed in ductal mucinous metaplasia and endocrine islets (28). The expression of TFF3 in the endocrine pancreas may have been neglected because the islets only constitutes 1–2% of the pancreas and because the level of expression is much lower than in the goblet cells of the intestine, which makes it difficult to detect TFF3 in the rat pancreas with antibodies to human TFF3 that readily detect TFF3 in the rat goblet cells. As measured by ELISA, the level of TFF3 in human islets is approximately 100 pmol/g protein, which is about one hundredth of the amount found in extracts from human intestines (our unpublished results). Recent reports on the presence of TFFs outside the mucosal epithelia comprise the central nervous system and oxytocin-containing cells in the pituitary (29, 30, 31, 32), which is consistent with the possibility that TFFs have different yet overlapping functions within and outside the gastrointestinal tract.

Similar to the expression of TFF3 in the embryonic gastrointestinal tract (33, 34), TFF3 was localized to the endocrine pancreas of fetal human and newborn rats. Because no information about abnormalities in pancreas development in the TFF3 mutant mouse has been reported (10), it remains to be studied whether TFF3 is dispensable for pancreatic development or whether compensatory mechanisms are activated. It may be speculated that the most prominent function of the TFFs is protection and restitution in response to cell damage as is evident from numerous studies of the gastrointestinal tract (11, 12). Thus, despite no obvious phenotype under normal conditions, the TFF3 mutant mouse is extremely vulnerable to the toxic effect of dextran sulfate (10). Gene expression profiling of regenerating rat pancreas after 90% pancreatectomy was found to be associated with changes in TFF expression in the pancreatic ducts, indicating a role in islet regeneration (35). These results are in agreement with the expression of TFF3 in some ducts in the adult human pancreas (Fig. 1Go). In preliminary studies of islet neogenesis in collagenase-treated fetal rat pancreas, we found up-regulation of TFF3 along with a number of other genes in parallel with ß-cell formation, although the cellular localization remains to be determined (36). In the neonatal rat pancreas, the TFF3 staining showed a marked heterogeneity in intensity, suggesting that TFF3 is confined to a subpopulation of ß-cells (Fig. 4Go, J–L) similar to another GH regulated protein, {delta}-like protein/preadipocyte factor-1 that has been implicated in ß-cell differentiation and proliferation without being a growth factor per se (37).

Because we and others previously found that GH and prolactin stimulate both ß-cell proliferation and insulin gene expression in rat islets in vitro and in vivo (24, 38, 39), it is of interest that hGH that activates both GH and prolactin receptors in rodents up-regulates the expression of TFF3 in both rat islets and INS-1 cells, suggesting that TFF3 may be involved in some of the actions of hGH in ß-cells. One possible function is the promotion of cell attachment and migration as shown in the present study. Although the effect was significant, it was considerably lower than that of GH (Fig. 6DGo). The molecular mechanisms are currently being investigated. No effect of exogenous TFF3 on ß-cell proliferation was observed (Fig. 6EGo). These observations are in line with the recent finding that mice overexpressing GH showed an enhanced mucosal repair and promotes intestinal mucosal regeneration, and this improvement of intestinal mucosal repair by GH was related to a preferential increased TFF3 expression (40) but not with those of TGF-ß1–3, epidermal growth factor, or TGF-{alpha} that were associated with increased crypt cell proliferation. hGH does indeed protect INS-1E cells against the apoptotic effect of the proinflammatory cytokines IL-1ß, IFN{gamma}, and TNF{alpha} (25). However, the present results show that exogenous TFF3 does not mimic the protective effect of GH cytokine-induced apoptosis (Fig. 7AGo). In addition TFF3 had no effect on glucose-induced insulin release in INS-1E cells (Fig. 7BGo). Very recently it has been reported that overexpression of TFF3 in a RIN cell line and rat islets using recombinant adenovirus resulted in increased proliferation and that knockdown by small interfering RNA resulted in attenuated proliferation (41). These results may indicate that endogenous TFF3 may have a different effect than exogenously added TFF3. This intriguing possibility has recently been supported by a study of mammary carcinoma in which a marked up-regulation of TFF3 expression was found with autocrine expressed hGH but not by exogenous added hGH (42). Overexpression of TFF3 resulted in increased anchorage-independent growth of the cells, whereas suppression by small interfering RNA resulted in attenuation of growth (42).

In contrast to TFF3 expression, hGH attenuated the expression of TFF1 in rat islets and INS-1E cells consistent with the proposed antiproliferative function of TFF1 (9). Interestingly, reciprocal regulation of TFF1 and TFF3 was reported in mice deficient in cytokine receptor subunit gp130 signaling via signal transducers and activators of transcription 1/3 that reduced TFF3 but not TFF1 production (43). In contrast to ß-cells, autocrine hGH in mammary carcinoma cells was reported to increase the expression of TFF1 that is known to be highly expressed in mammary cancer (42).

In summary, the present results demonstrate a distinct expression pattern of TFF3 in the endocrine pancreas in humans and rats. The finding that hGH causes reciprocal effects on TFF3 and TFF1 expressions in the pancreatic islets and ß-cells suggest that certain GH actions in islet function may be mediated through the actions of TFFs. Exogenous TFF3 has been found to promote islet cell attachment and migration but show no effect on proliferation, apoptosis, or insulin secretion. It remains possible that regulation of the endogenous TFF levels in the ß-cells modulate cellular functions in an autocrine manner.


   Acknowledgments
 
The excellent technical assistance by I. Tønnesen, H. Hadberg, P. S. Thomsen, H. Nguyen, I. M. Jensen, and K. Ottosen is greatly appreciated. Preparation of islets and RNA was performed by Hanne Richter-Olesen and Tina Kisbye (Hagedorn Research Institute) and Tina M. Olsen (Novo Nordisk A/S). Isolated human islets and adult human pancreatic tissue were obtained from the European Union and Juvenile Diabetes Research Foundation supported ß Cell Transplant program (directed by Daniel Pipeleers, Free University Brussels, Brussels, Belgium). INS-1 and INS-1E cells were kindly provided by Claes Wollheim (University of Geneva, Geneva, Switzerland).


   Footnotes
 
This work was supported by Juvenile Diabetes Research Foundation (Grant 1-2004-107), European Union integrated project BetaCell Therapy, Danish Medical Research Council (to Danish Stem Cell Research Center), Danish Diabetes Association, Aase and Ejnar Danielsens Fund, Novo Nordisk Foundation, and the Vera and Carl Johan Michaelsen Foundation.

Disclosure statement: M.J., Y.C.L., K.M., H.K., J.J., S.R., N.N., L.W.G., J.L., R.D., L.T.D., B.S., D.B.J., E.N., P.T., and H.C.B. have nothing to declare. L.T. is employed by and has equity interests in Novo Nordisk A/S. J.H.N. consults for and has equity interests in Novo Nordisk A/S.

First Published Online September 14, 2006

1 M.J. and Y.C.L. contributed equally to this work. Back

Abbreviations: BrdU, Bromodeoxyuridine; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; hGH, human GH; IFN, interferon; PGK, phosphoglycerate kinase; PP, pancreatic polypeptide; RIPA, radioimmunoprecipitation assay; RPL13, ribosomal protein L13; TFF, trefoil factor.

Received May 5, 2006.

Accepted for publication September 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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