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PubMed2002

Isolation and characterization of rabbit single chain antibodies to human Reg Ialpha protein.

To investigate the role of Reg Ialpha in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Ialpha. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Ialpha. Anti-Reg Ialpha scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Ialpha scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Ialpha in human IBD.

Amino Acid Sequence↗
PubMed2004

Coordinate regulation of PSP/reg and PAP isoforms as a family of secretory stress proteins in an animal model of chronic pancreatitis.

BACKGROUND: PSP/reg and PAP are secretory stress proteins (SSP) and may be part of a protective mechanism. They share structural homologies and form insoluble fibrils after tryptic activation. To further explore the regulation of these proteins, we investigated the male WBN/Kob rat, a model of pancreatic inflammatory and fibrotic disease similar to chronic pancreatitis. MATERIALS AND METHODS: Expression of PSP/reg and PAP I, II and III in the WBN/Kob rat pancreas was evaluated on the mRNA and protein level, by immunohistochemistry and by highly sensitive isoform specific ELISAs. RESULTS: The SSPs are constitutively secreted, PAP in nanomolar, PSP/reg in micromolar concentrations. Before conventional morphological changes are detectable in the WBN/Kob rat, focally increased expression of secretory stress protein is visible. SSP levels in pancreatic juice of WBN/Kob rats reach peak values 10- to 50-fold higher than in Wistar control rats. The highest expression was localized to acini with inflammatory infiltration. CONCLUSIONS: There is a tight spatial and temporal association between pre-inflammatory changes or inflammation and SSP-expression. These results support our concept that PSP/reg and PAP are coordinately regulated SSP.

Acute-Phase Proteins↗
PubMed2001

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV.

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.

Amino Acid Sequence↗
PubMed2002

No abnormalities of reg1 alpha and reg1 beta gene associated with diabetes mellitus.

In order to investigate whether there would be any association between abnormalities of either reg1 alpha or reg1 beta gene and diabetes mellitus in man, these two genes were analyzed in 42 patients with type 1 diabetes mellitus, 12 with fibrocalculous pancreatopathy, 37 with type 2 diabetes mellitus, and 22 normal controls, by PCR-SSCP analysis and nucleotide sequencing technique. Polymorphism in the reg1 alpha gene resulted in three mobility patterns in the PCR-SSCP analysis, due to nucleotide constituents at position -10 before exon 1 being either C/C, T/C or T/T. These three mobility patterns were observed in every group of subjects. The analysis of reg1 beta gene showed nucleotide substitutions in exon 4 in one patient, exon 5 in another patient with type 1 diabetes, and in exon 4 and intron 5 in one patient with fibrocalculous pancreatopathy. The nucleotide substitutions in exon 4 in the patient with type 1 diabetes and that with fibrocalculous pancreatopathy occurred at codons 103 and 84 while that in exon 5 in the patient with type 1 diabetes occurred at codon 141, changing the codons from CAT to CAC, GTG to GCG, and ACT to AAT and resulting in H103H silent, V84A and T141N missense mutations, respectively. In conclusion, using PCR-SSCP and nucleotide sequence analyses, we did not find any association between abnormalities of either reg1 alpha or reg1 beta gene with any type of diabetes studied.

Adult↗
PubMed1986

Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1.

Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1.

Animals↗
PubMed2000

Identification of a novel Reg family gene, Reg IIIdelta, and mapping of all three types of Reg family gene in a 75 kilobase mouse genomic region.

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of three subtypes (types I, II, III) based on the primary structures of the encoded proteins of the genes. We have isolated three types of mouse Reg family gene (Reg I, Reg II, Reg IIIalpha, Reg IIIbeta and Reg IIIgamma) [Unno et al. (1993) J. Biol. Chem. 268, 15974-15982; Narushima et al. (1997) Gene 185, 159-168]. In the present study, by Southern blot analysis of a mouse bacterial artificial chromosome clone containing the five Reg family genes in combination with PCR cloning of every interspace fragment between adjacent genes, the Reg family genes were mapped to a contiguous 75kb region of the mouse genome according to the following order: 5'-Reg IIIbeta-Reg IIIalpha-Reg II-Reg I-Reg IIIgamma-3'. In the process of ordering the genes, we sequenced the 6.8kb interspace fragment between Reg IIIbeta and Reg IIIalpha and encountered a novel type III Reg gene, Reg IIIdelta. This gene is divided into six exons spanning about 3kb, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg (regenerating gene product) proteins. Reg IIIdelta was expressed predominantly in exocrine pancreas, but not in normal islets, hyperplastic islets, intestine or colon, whereas both Reg I and Reg II were expressed in hyperplastic islets and Reg IIIalpha, Reg IIIbeta and Reg IIIgamma were expressed strongly in the intestinal tract. Possible roles of Reg IIIdelta and the widespread occurrence of the Reg IIIdelta gene in mammalian genomes are discussed.

Animals↗
PubMed2000

Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein.

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.

Acute-Phase Proteins↗
PubMedDate not supplied

Ectopic expression of reg protein: A marker of colorectal mucosa at risk for neoplasia.

Pancreatic regenerating gene (reg I) messenger RNA is overexpressed within the pancreas following injury and resection. Its level of expression corresponds to the level of cellular dedifferentiation. Human reg I has been localized to chromosome 2p12, and ectopic expression of its mRNA has been found within colorectal tumors. We postulated that colorectal production of reg I might either be a marker for the presence of cancer or define mucosa at risk for development of neoplasia. Using a monoclonal antibody to reg I, regenerating gene protein was histochemically mapped in 56 cases of documented colorectal adenocarcinoma. In sections of colon from normal control subjects no reg I protein was noted, whereas 58.9% of the specimens from cancer patients stained positive for reg I. Although a correlation was noted between reg I staining and Dukes' stage, there was no correlation with histologic grade or 5-year patient survival. In 39 of 55 cancer specimens the transition zone (interface) between the neoplasm and normal mucosa was visualized; 100% of the transition zones contained cells that stained strongly positive for reg I. We conclude that reg I protein is ectopically expressed in colorectal mucosa at the transition zone of colorectal cancer, and occasionally within the tumor itself. Although ectopic reg I expression in colorectal epithelia is not a marker for the presence of carcinoma, it may be a sensitive marker for mucosa at risk for development of neoplasia.

Adenocarcinoma↗
PubMedDate not supplied

Comprehensive proteomic analysis of human pancreatic juice.

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.

Agglutinins↗
PubMed2000

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Animals↗
PubMed1997

A Schwann cell mitogen accompanying regeneration of motor neurons.

Motor neurons are the only adult mammalian neurons of the central nervous system to regenerate following injury. This ability is dependent on the environment of the peripheral nerve and an intrinsic capacity of motor neurons for regrowth. We report here the identification, using a technique known as messenger RNA differential display, of an extracellular signalling molecule, previously described as the pancreatic secreted protein Reg-2, that is expressed solely in regenerating and developing rat motor and sensory neurons. Axon-stimulated Schwann cell proliferation is necessary for successful regeneration, and we show that Reg-2 is a potent Schwann cell mitogen in vitro. In vivo, Reg-2 protein is transported along regrowing axons and inhibition of Reg-2 signalling significantly retards the regeneration of Reg-2-containing axons. During development, Reg-2 production by motor and sensory neurons is regulated by contact with peripheral targets. Strong candidates for peripheral factors regulating Reg-2 production are cytokines of the LIF/CNTF family, because Reg-2 is not expressed in developing motor or sensory neurons of mice carrying a targeted disruption of the LIF receptor gene, a common component of the receptor complexes for all of the LIF/CNTF family.

Animals↗
PubMed2004

Transgenic overexpression of Reg protein caused gastric cell proliferation and differentiation along parietal cell and chief cell lineages.

Reg (regenerating gene product) was originally identified as a growth factor involved in pancreatic regeneration. During the healing course of gastric erosion, Reg expression is highly increased in the enterochromaffin-like (ECL) cells surrounding the ulcer crater, suggesting its role as a regulator of gastric mucosal regeneration. However, there has been no direct in vivo evidence of a growth-promoting role of Reg for the gastric mucosal cells. In the current study, Reg-transgenic mice were created and gastric mucosa were analysed for histological changes. Transgenic mice showed a marked increase in the thickness of the fundic mucosa. Anti-proliferating cell nuclear antigen (PCNA) staining of the fundic mucosa demonstrated the enlargement of the proliferating neck zone and the lower PCNA-negative zone. Histological analysis employing antibodies against cell-type markers revealed expansion of the chief cell and parietal cell populations and no change in the number of surface mucus-producing cells, ECL cells, or G cells. In conclusion, Reg has a growth-promoting effect on gastric progenitor cells and an activity to direct the differentiation of the cells into chief cell and parietal cell lineages. This was in contrast to other factors, all of which had been shown to drive differentiation towards mucus producing cells in vivo. In the injured gastric mucosa, Reg may play a unique and important part in the reconstruction of the properly organized mucosal architecture.

Animals↗
PubMed2006

Overexpression of regenerating islet-derived 1 alpha and 3 alpha genes in human primary liver tumors with beta-catenin mutations.

The Wnt/beta-catenin signaling pathway is activated in many human hepatocellular carcinomas (HCC). We tried to identify the genes involved in carcinogenesis and progression of HCC with beta-catenin mutations. We used PCR-based subtractive hybridization to compare gene expression between malignant and benign components of a human HCC occurring in pre-existing adenoma activated for beta-catenin. Two of the genes identified belong to the Regenerating gene (REG) family. They encode the Regenerating islet-derived 3 alpha (REG3A/HIP/PAP/REG-III) and 1 alpha (REG1A) proteins, both involved in liver and pancreatic regeneration and proliferation. Using siRNA directed against beta-catenin, we demonstrated that REG3A is a target of beta-catenin signaling in Huh7 hepatoma cells. The upregulation of REG3A and REG1A expression is significantly correlated to the beta-catenin status in 42 HCC and 28 hepatoblastomas characterized for their beta-catenin status. Thus, we report strong evidence that both genes are downstream targets of the Wnt pathway during liver tumorigenesis.

Adenoma↗
PubMed2006

Reg I-knockout mice reveal its role in regulation of cell growth that is required in generation and maintenance of the villous structure of small intestine.

Reg I (regenerating gene product I) is a growth factor that plays a central role in the generation and regeneration of the gastric mucosal architecture. On the other hand, mouse Reg I mRNA is expressed at the highest levels in the small intestine among the gastrointestinal tissues. In the current study, with the aim to clarify the role of Reg I protein in the small intestine, the temporal and spatial pattern of Reg I expression and the phenotype of Reg I-knockout mice in the tissue were examined. In the wild-type mice, immunohistochemistry localized Reg I protein expression in absorptive cells located in the lower half of the intestinal villi. Reg I expression was undetectable until embryonic day 13 (E13), when the fetal intestine still lacks villous structure; however, it dramatically increased at E17 along with the formation and maturation of the fetal intestinal villi. In the small intestine of the adult Reg I-knockout mice, less densely packed, round-shaped aberrant morphology of the absorptive cells was observed light microscopically, and electron microscopical examination revealed a strikingly loose connection of these cells to the basement membrane. Antiproliferating cell nuclear antigen staining and anti-Ki67 staining demonstrated the marked decrease in the number of proliferating cells in the small intestinal mucosa of the knockout mice. The cell migration speed visualized by one shot labeling of 5-bromodeoxyuridine was significantly slower in the knockout mice. These phenotypes of Reg I-knockout mice emerged, in accordance with the temporal pattern of Reg I expression described above, from E17. Reg I was considered to be a regulator of cell growth that is required to generate and maintain the villous structure of the small intestine.

Animals↗