ARTICLE |
Correspondence to: Catherine Figarella, Groupe de Recherche sur les Glandes Exocrines, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. E-mail: grge@medecine.univ-mrs.fr
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Summary |
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We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression. (J Histochem Cytochem 48:14011410, 2000)
Key Words: reg gene, reg protein, NOD mouse, localization, pancreas
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Introduction |
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Terazono et al., in 1988, isolated a clone from a cDNA library prepared from pancreatic islets isolated from 90% pancreatectomized nicotinamide-treated rats, a model of islet regeneration. The gene corresponding to the clone was named reg. This gene was found to be homologous to a previously described human exocrine pancreatic gene that encodes a 166 amino-acid protein (with an N-terminal signal peptide of 22 amino acids) whose proteolyzed form of 133 amino acids was designated "protein X"(
Since then, several reg and reg-related genes have been isolated from human, rat, and mouse and have been shown to constitute a multigene family, the Reg family (for review see
To resolve this issue, we determined in this present study the localization of the reg gene(s) in NOD mouse pancreatic tissues by an in situ hybridization (ISH) analysis with specific reg probes and an immunohistochemical evaluation of their corresponding protein(s).
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Materials and Methods |
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Animals and Treatment
NOD mice bred from a parental stock were provided by Dr. C. Boitard (Department of Immunology, INSERM U25, Necker Hospital, Paris, France). In our colony, the prevalence of diabetes at Day 210 was 43% in female and <1% in male mice. Insulitis, characterized by a lymphocytic infiltration, was observed from the fourth week of age and was present to a similar degree in both sexes. Glycosuria was detected using labsticks (Ketodiastix; Bayer Diagnostics, Puteaux, France). To select situations with various degrees of activity of the diabetogenic process we studied non-diabetic NOD female and male mice (age range 12245 days), diabetic NOD female, and cyclophosphamide-treated male NOD mice (aged 110 days). Cyclophosphamide (Endoxan; Lucien Laboratory, Colombes, France) in saline was injected SC at a dose of 300 mg/kg body weight 100 days after birth. Because in our colony the percentage of overt diabetes in NOD male mice was 16% after one injection and 83% after two injections of cyclophosphamide, the animals were sacrificed 10 days after the first injection while in a "prediabetic" period. In addition, female and male normal mice, age range 60135 days (IOPS-OF1 supplied by Iffa Credo; L'Arbresle, France), were studied as controls.
Tissue Sections
The pancreas was rapidly collected and fixed in 4% paraformaldehyde in 0.1 M PBS for 1248 hr at 4C. Tissues were treated in an automatic processor and embedded in paraffin. Paraffin sections of 34-mm thickness were cut for RNA ISH and immunohistochemistry and were mounted on SuperFrost Plus slides (Menzel-Glaser; Braunschweig, Germany) or Silane-prep slides (Sigma; L'Isle d'Abeau Chesnes, France).
Probes
The mouse regII probe was kindly provided by H. Okamoto (Sendai, Japan). It was full-length cDNA of 700 bp, subcloned into pBluescript vector at the XHoI site in the polycloning site. A 174-bp fragment of the mouse insulin cDNA was synthesized by RT-PCR as described by
Northern Blotting Analysis
Total mouse pancreatic RNA and synthesized regI and regII RNAs were prepared as described previously (
In Situ RNA Hybridization
In situ hybridizations were performed essentially as described previously by
Preparation of a Polyclonal Antibody Against Mouse reg Protein(s)
A polyclonal antibody was generated against mouse regI and regII protein using a peptide of 16 amino acids, VSLTSNTGYKKWKDDN(A), corresponding to position 137152 of regI and 145160 of regII. The protein sequences were deduced from the mouse cDNA sequences (
The 16-mer reg peptide was synthesized by the solid-phase technique (
The peptide of 1927 kD was coupled to the carrier molecule at a peptide:carrier ratio of 1:50. Peptide (1 µmole) in 0.05 M phosphate buffer, pH 6.1, was coupled to 20 nanomoles of the carrier KLH (keyhole limpet hemocyanin; Pierce, Rockford, IL), using EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide (Pierce) at a final concentration of 10 mg/ml. The mixture was stirred for 120 min at RT and the coupling reaction was stopped by addition of 0.1 mM sodium acetate, pH 4.0.
At Day 0, two New Zealand rabbits were given 200 µg of KLH-coupled peptide emulsified in complete Freund's adjuvant, 200 µg at Days 21 and 42, then 400 µg at Days 80, 130, and 280. Blood was collected in heparinized plastic tubes 10 days after each boost. A preimmune serum was collected before starting the immunization protocol for each rabbit. Sera were then prepared by centrifugation, aliquotted, and kept frozen until use. The immunoreactivity of the various bleedings was determined by immunoblotting as described below. Positive signal occured after the third boost and increased to reach a maximum in the following bleedings.
Characterization of the Antibody on Pancreatic Homogenates by Immunoblotting
Pancreatic homogenates were prepared from frozen mouse pancreas washed with a 0.15 M NaCl solution, then homogenized in a 0.15 M Tris buffer, pH 8.5, containing 1 mM benzamidine (10 ml per g of pancreas). After ultracentrifugation at 100,000 x g for 1 hr at 4C, supernatants containing pancreatic proteins were collected and stored by aliquots at -80C. Proteins present in supernatants were submitted to SDS-PAGE in a 15% polyacrylamide gel according to the method of
Purification of the Antibody for Immunocytochemistry
Mouse reg antibody was purified by adsorption to and elution from the protein immobilized on nitrocellulose according to
Immunohistochemical Staining
The avidinbiotinperoxidase complex method (Vectastain ABC kit; Vector, Burlingame, CA) was used for immunostaining. The paraffin sections were deparaffinized in xylene and hydrated through graded ethanols. They were immersed in preheated Dako target retrieval solution (Dako; Carpinteria, CA) in a water bath for 20 min to increase staining intensity with the primary antibodies and immersed in absolute methanol containing 0.5% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Sections were incubated with non-immune goat serum for 20 min to prevent nonspecific binding, then with the primary antiserum (diluted 1:500 in 150 mM PBS, pH 7.3, before purification or concentrated after purification) for 1 hr, and finally with biotinylated secondary antibody diluted. Sections were incubated for 30 min with the ABC reagent and then with the peroxidase substrate 3-amino-9-ethylcarbazol (AEC substrate kit; Vector). Slides were counterstained with Mayer's hemalum. Several controls were routinely performed : PBS or non-immune rabbit serum was used in place of the primary antiserum.
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Results |
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Specificity of the reg RNA Probes
Mouse RNA was analyzed by Northern hybridization using antisense and sense regII RNA probes after hydrolysis (reducing RNA probes from more than 700 nucleotides to an average of 200 nucleotides by controlled hydrolysis under alkaline conditions). As shown in Fig 1A, a single mRNA transcript of about 0.9 kb was detected in total mouse RNA with antisense regII RNA probes and no mRNA transcript was detected with sense regII RNA probes. Because of the homology that exists between regI and regII sequences, it was important to ensure the specificity of regII RNA probes after hydrolysis. We confirmed the cross-hybridization of the RNA probe (antisense) with synthetic RNAs regI and regII as shown in Fig 1B.
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Comparative reg and Insulin Gene Expression in NOD Mouse Pancreas Evaluated by ISH
As shown in Fig 2, expression of reg mRNA was detected at high levels in the exocrine portion of the pancreas of a 12-day-old female mouse (Fig 2A) but not in the endocrine portion where, as expected, expression of insulin mRNA was clearly visible (Fig 2B). Negative control was obtained with the reg sense probe (Fig 2C). Similar results were obtained in female and male 30-day-old mice, in which reg mRNA expression was detected in the exocrine compartment, whereas cells of the islets of Langerhans appeared negative for this transcript (data not shown). Comparative data for insulin and reg mRNA expression in two adjacent sections of the pancreas of a 30-day-old female mouse are shown in Fig 2D and Fig 2E. A strong hybridization signal was observed with the insulin antisense probe in the endocrine compartment (arrowheads) and in a few ductule cells (curved arrows) (Fig 2D), whereas expression of reg mRNA was detected at a high level in the exocrine portion of the pancreas but not in the endocrine portion (Fig 2E). As shown in Fig 3, a consistent exocrine localization of reg mRNA was observed in the presence of insulitis in the pancreas of a 95-day-old (Fig 3A) and a 140-day-old female NOD mouse (Fig 3B), as well as in the pancreas of a cyclophosphamide-treated male mouse (Fig 3C). Arrowheads in Fig 3A and Fig 3B point to the site at which a lymphocytic infiltration was visible in pancreatic islets. The reg signal remained specifically detected in the pancreatic acini of a diabetic female NOD mouse (Fig 3D).
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Similar results were obtained on pancreas sections of a control mouse (OF1 strain) (data not shown). Expression of reg mRNA was detected in the exocrine portion of the pancreas but not in the islets of Langerhans, whereas insulin mRNAwas specifically detected in the endocrine compartment and in a few duct cells, as observed in the NOD mouse pancreas.
Localization of reg Protein in NOD Mouse Pancreas by Immunocytochemistry
The specificity of mouse reg antibody was first determined by immunoblotting analysis performed on mouse pancreatic homogenates. Similar results were obtained with NOD or OF1 mouse pancreatic homogenates. As shown in Fig 4, the antibodies recognized a single band at 16.6 kD, consistent with the predicted molecular mass of the mouse reg protein. Immunocytochemical controls on NOD or OF1 mouse pancreas confirmed the specificity of the antibody. No immunoreactivity was observed (a) with preimmune serum, (b) after immunoabsorption of the antiserum with the corresponding free peptide before the immunocytochemical procedure, and (c) in appropriate controls in which the primary antibody was omitted.
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Immunohistochemical staining results presented in Fig 5 show that regardless of the age, sex, the severity of insulitis, and the presence of diabetes in NOD mouse, the results were identical. When pancreatic sections were treated with the purified antibody, acinar cells were intensely stained, whereas islet cells remained constantly negative. The presence of a mild (Fig 5B) or a severe (Fig 5E) insulitis did not modify the acinar staining. In a pancreatic section of a cyclophosphamide-treated male, the acinar staining appeared stronger in the cells surrounding the islets (Fig 5D) but the phenomenon was not constantly observed. Reg immunoreactivity was also observed in pancreatic ducts (Fig 5B, Fig 5D, and Fig 5G, arrowheads) demonstrating that, as expected, the reg protein was normally secreted. When the pancreatic sections were treated with the antibody against insulin, strong staining was observed only in islet cells and acinar cells were negative (Fig 5C).
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Discussion |
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The reg gene is normally expressed by the pancreatic acinar cells (
We first prepared sense and antisense riboprobes from the mouse regII cDNA and verified after hydrolysis the hybridization specificity of the antisense probe on total mouse pancreatic RNA and synthetic regI and regII RNAs. Using these riboprobes, we followed by ISH pancreatic reg mRNA expression in NOD mice at various stages of the diabetogenic process and compared it with insulin mRNA expression. The present data show that, regardless of the age, sex, or presence of insulitis, expression of reg mRNA was restricted to the exocrine portion of the pancreas, whereas, as expected, insulin mRNA expression was clearly visible in the islets of Langerhans. In some cases, insulin expression was observed in a few ductule cells, in agreement with our previous observations showing the presence of isolated endocrine cells in human fetal and adult tissue (
To complete our study, we performed immunocytochemical localization of the reg protein(s) in the NOD and the control mice. To identify the mouse protein(s), we raised antibodies to a synthetic peptide of 16 amino acids present in the two proteins and corresponding to the most favorable regions for potential antigenic sites. Immunoblotting analysis on mouse pancreatic homogenates with these polyclonal antibodies recognized a single band at 16.6 kD consistent with the predicted mass of the mouse regI protein. However, it is difficult to know if this single band also corresponds to the putative regII protein. Indeed, regI and regII share 76% homology and differ by the presence of a unique additional sequence of seven amino acids in the N-terminal region in regII (
It is important to note that reg mRNA and protein expression remains restricted to exocrine tissue, both in cyclophosphamide-treated males, which represent a prediabetic stage, and in overtly diabetic females. In these two situations, which correspond to a striking evolution in the organization of the lesion within the islet and in which reg mRNA levels were found to be very high, reg was never expressed in endocrine tissue. However, we previously observed that if, as expected, the diabetic NOD females did not express insulin mRNA, cyclophosphamide treated males exhibited a significantly higher level of insulin expression than that measured in non-treated males and in non-diabetic NOD females (
Do these data imply that reg cannot be considered as a growth factor involved in regeneration and/or growth of pancreatic islets, as hypothesized by Okamoto and his group? To address this issue, it appears necessary to distinguish between ß-cell regeneration and/or replication and proliferation of differentiated ß-cells. From the data that we have gathered on NOD mice, the possibility that reg may have a direct proliferative effect on preexisting ß-cells seems unlikely. On the other hand, an effect on ß-cell replication was observed by
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Acknowledgments |
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DS is supported by a fellowship of AJD (Aide aux Jeunes Diabétiques), France.
We are grateful to H. Okamoto (Japan) for providing us with the mouse regII cDNA probe and to B.Vialettes (Laboratoire de Diabétologie; Marseille, France) for allowing free access to the NOD mice.
Received for publication December 22, 1999; accepted April 26, 2000.
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Literature Cited |
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