Human Reg Research Articles

Expression of human REG family genes in inflammatory bowel disease and their molecular mechanism.

The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members have been reported to be expressed in Crohn's disease (CD) and ulcerative colitis (UC) and to be involved as proliferative mucosal factors in IBD. However, expression of all the REG family genes in IBD is still unclear. Here, we analyzed expression of all the REG family genes (REGIα, REGIβ, REG III, HIP/PAP, and REG IV) in biopsy specimens of UC and CD by real-time RT-PCR. REG Iα, REG Iβ, and REG IV genes were overexpressed in CD samples. REG IV gene was also overexpressed in UC samples. We further analyzed the expression mechanisms of REG Iα, REG Iβ, and REG IV genes in LS-174T and HT-29 human colonic epithelial cells. The expression of REG Iα was significantly induced by IL-6 or IL-22, and REG Iβ was induced by IL-22. Deletion analyses revealed that three regions (- 220~- 211, - 179~- 156, and - 146~- 130) in REG Iα and the region (- 274~- 260) in REG Iβ promoter were responsible for the activation by IL-22/IL-6. The promoters contain consensus transcription factor binding sequences for MZF1, RTEF1/TEAD4, and STAT3 in REG Iα, and HLTF/FOXN2F in REG Iβ, respectively. The introduction of siRNA for MZF1, RTEF1/TEAD4, STAT3, and HLTF/FOXN2F abolished the transcription of REG Iα and REG Iβ. The gene activation mechanisms of REG Iα/REG Iβ may play a role in colon mucosal regeneration in IBD.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters.We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I−/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I−/- and wild-type mice.ATLANTIS was found in both wild-type and Reg I−/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I−/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I−/- mice did not differ from that of wild-type mice.These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Recombinant Human REG I<i>α</i> Aggregates <i>Staphylococcus aureus</i>—Exhibits a Lectin-Like Function

Staphylococcus aureus is pathogenic to humans with worldwide health care concern due to its ability to evade the immune system and develop resistance to multiple drugs. Reg family proteins are C-type lectins with antimicrobial properties. Bacterial aggregation through binding to microbial cell surface sugar and/or lipid moieties is key mechanism employed in the process. In the present study we have analysed the antimicrobial effect of human REG Iα on S. aureus. Aggregation of mid-log phase culture of S. aureus was observed in presence of purified recombinant REG Iα. Therefore REG Iα can be applied in eliminating S. aureus infections in humans.

Abstract 4941: Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types, with a 5-year survival rate <5%. Tumor specific biomarkers that can aid in early detection and treatment monitoring are urgently needed in order to improve the outcomes. Human Reg family proteins (Reg1A, Reg1B, Reg3A, Reg3G and Reg4) are a group of calcium-dependent (C-type) lectins that promote cell growth and respond to inflammatory stimulants. We examined their associations with pancreatic ductal adenocarcinoma (PDAC). In patients with PDAC, the serum levels of Reg1A and 1B were significantly elevated (p = 0.0003 and 0.0018), which correlated with TNM stages of the patients. By using immunohistochemistry (IHC), the expression of Reg1A, Reg1B and Reg4 was observed in the malignant glandular epithelium of pancreatic intraepithelial neoplasia (PanIN) and invasive PDAC. The intensity of Reg1A and Reg1B staining positively correlated with the histological grades of PanIN. The serum levels and tissue expression of Reg1A and Reg1B were negatively correlated with the differentiation grades of invasive PDAC. Additionally, patchy expression of Reg1A andReg3A/G was also observed in areas with acinar-to-ductal metaplasia (ADM) adjacent to cancer cells. Our data suggests that the changes in Reg proteins were closely associated with the carcinogenesis of PDAC. Reg1A and 1B proteins showed specificity for pancreatic ductal differentiation and could be used as clinical markers for early detection of PDAC, monitoring therapeutic response and predicting tumor recurrence after therapy and tumor metastasis. The expression of Reg family proteins in both ADM and PanIN cells supports the notion that ADM could be an alternative mechanism of PDAC carcinogenesis. Citation Format: Qing Li, Hao Wang, George Zogopoulos, Qin Shao, Jun-Li Liu, Zu-Hua Gao. Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4941.

Involvement of autoimmunity to REG, a regeneration factor, in patients with primary Sjögren's syndrome

The regenerating gene (Reg) was isolated originally as a gene specifically over-expressed in regenerating pancreatic islets and constitute a growth factor family. Reg gene product (Reg) is important in the pathophysiology of various human inflammatory diseases. Recently, the possible involvement of human REG in the regeneration of salivary ductal epithelial cells of patients with primary Sjögren's syndrome (SS) was reported. However, the expression of the REG family genes in minor salivary glands (MSG) and the occurrence of anti-REG Iα autoantibodies in SS patients were obscured. In this study, we examined the expression of REG family genes in the MSG of SS and screened anti-REG Iα autoantibodies in SS. The mRNA levels of REG family genes in MSG were quantified using real-time reverse transcription-polymerase chain reaction (RT-PCR) and REG Iα expression in the MSG was analysed by immunohistochemistry. The mRNA level of REG Iα in the MSG of SS patients was significantly higher than that of control. REG Iα protein was expressed highly in SS ductal epithelial cells. Anti-REG Iα autoantibodies in the sera were found in 11% of SS. All the MSG in the anti-REG Iα autoantibody-positive group showed REG Iα expression, whereas only 40% showed REG Iα expression in the anti-REG Iα autoantibody-negative group. The anti-REG Iα autoantibody-positive group showed significantly lower saliva secretion and a higher ratio of grade 4 (by Rubin-Holt) in sialography. These data suggest strongly that autoimmunity to REG Iα might play a role in the degeneration of MSG ductal epithelial cells in primary SS.

Reg IV Regulates Normal Intestinal and Colorectal Cancer Cell Susceptibility to Radiation-Induced Apoptosis

Regenerating (Reg) gene IV is predominantly expressed in gastrointestinal cells and highly up-regulated in many gastrointestinal malignancies, including colorectal cancer (CRC). Human CRC cells expressing higher levels of Reg IV gene and its protein product (Reg IV) are resistant to conventional therapies, including irradiation (IR). However, the underlying mechanism is not well defined. A murine model of IR-induced intestinal injury and in vitro and in vivo models of human CRC were used to determine the role of Reg IV in regulation of normal intestinal and colorectal cancer cell susceptibility to IR-induced apoptosis. Treatments of recombinant human Reg IV (rhR4) protein protected normal intestinal crypt cells from IR-induced apoptosis by increasing the expression of antiapoptotic genes Bcl-2, Bcl-XL, and survivin. However, overexpression of Reg IV in human CRC cells was associated with increased resistance to IR-induced apoptosis. Therefore, we used antagonism of Reg IV as a tool to increase CRC cell susceptibility to IR-induced cell death. Two complementary approaches using specific monoclonal antibodies and small interfering RNAs were tested in both in vitro and in vivo models of human CRC. Both approaches resulted in increased apoptosis and decreased cell proliferation, leading to decreased tumor growth and increased animal survival. Furthermore, these approaches increased CRC cell susceptibility to IR-induced apoptosis. These results implicate Reg IV as an important modulator of gastrointestinal cell susceptibility to IR; hence, it is a potential target for adjunctive treatments for human CRC and other gastrointestinal malignancies.

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.

Dysregulation of reg gene expression occurs early in gastrointestinal tumorigenesis and regulates anti-apoptotic genes

Expression of anti-apoptotic genes is frequently elevated in tumors, where they increase resistance to chemotherapeutic agents and predict poor patient outcomes. However, key cellular factors regulating anti-apoptotic genes in tumors remain unknown. Increased expression of the regenerating (Reg) genes is commonly observed in gastrointestinal (GI) malignancies including colorectal cancer (CRC). We therefore examined Reg gene expression and associated changes in anti-apoptotic genes in an animal model of GI tumorigenesis. Using real time RT-PCR, we measured expression of Reg genes in human colorectal adenocarcinoma specimens, colon adenocarcinoma cell lines and adenomas from multiple intestinal neoplasia (min) mice heterozygous for a germ-line mutation of the adenomatous polyposis coli (APC) gene. Expression of Reg genes is increased in human colorectal adenocarcinomas and in the intestine of APCmin/+ mice at 4 weeks of age, a time preceding the spontaneous second mutation in the APC gene. Individual Reg genes exhibited regional expression profiles across the GI tract in mice. Adenomas from 14-week old mice had significant increases in at least one member of the Reg gene family, most commonly Reg IV and an associated increase in expression of the anti-apoptotic gene, Bcl-2. Addition of exogenous recombinant human Reg IV to human colon adenocarcinoma cells significantly increased Bcl-2 and Bcl-xL expression and induced resistance to ionizing radiation. These results show that dysregulation of Reg genes occur early in tumorigenesis. Furthermore, increased expression of Reg genes, specifically Reg IV contribute to adenoma formation and lead to increased resistance to apoptotic cell death in CRC.

Reg IV Activates the Epidermal Growth Factor Receptor/Akt/AP-1 Signaling Pathway in Colon Adenocarcinomas

Reg IV, a secreted protein and member of the Reg multigene family, is up-regulated in malignancies of the human gastrointestinal tract, including colorectal carcinoma (CRC). However, in vitro signal transduction pathway(s) utilized by Reg IV are not yet known. To determine the signaling pathway(s) responsive to Reg IV, we examined the effects of purified recombinant human Reg IV (rhR4) on HCT116 and HT29 colon adenocarcinoma cells. Addition of rhR4 to cultures led to a dose-dependent increase in cell number similar to that observed after treatment with epidermal growth factor (EGF). In addition, rhR4 treatment resulted in rapid phosphorylation of EGF receptor at Tyr992 and Tyr1068 and Akt at Thr308 and Ser473. Using luciferase reporter gene assays, we demonstrated that Reg IV signaling through EGF receptor and Akt results in increased activator protein-1 (AP-1) transcription factor activity. Real-time reverse-transcription polymerase chain reaction and Western blot analyses revealed quantitative increases in c-Jun, JunB, JunD, and FosB expression associated with increased AP-1 activity. Electrophoretic mobility shift assay further revealed significant increases in AP-1 binding activity in rhR4-treated cells, with increased supershift in the presence of antibodies to JunB, JunD, and FosB. Furthermore, rhR4 treatments led to the increased expression of Bcl-2, Bcl-XL, survivin, and matrilysin, genes associated with a poor prognosis in advanced CRC. Reg IV is a potent activator of the EGF receptor/Akt/AP-1 signaling pathway in CRC. Disruption of Reg signaling may have utility as a therapeutic intervention for human gastrointestinal adenocarcinomas.

High expression of RELP (Reg IV) in neoplastic goblet cells of appendiceal mucinous cystadenoma and pseudomyxoma peritonei

The regenerating protein (Reg)-like protein (RELP, also known as Reg IV) is a recently characterized fourth member of the human Reg protein family. The Reg proteins are small, about 20-kD-sized, secretory proteins of C-lectin type. The previously known Reg proteins have been functionally implicated in regeneration, proliferation, and differentiation of the pancreas, liver, and gastrointestinal mucosa. To study the tissue expression of RELP, we raised a monoclonal antibody to RELP. By immunohistochemistry and in situ hybridization, we found a robust de novo expression of RELP in the neoplastic goblet cells of appendiceal mucinous cystadenomas and in the epithelial implants of pseudomyxoma peritonei (PMP). Our findings indicate that RELP serves as a marker for appendiceal mucinous cystadenomas and PMP, and that RELP may contribute to the pathogenesis of these disorders.

DISCOVERY OF A NEW REG3?? HAMSTER GENE HOMOLOGOUS TO THE HUMAN REG3 GENE

For over 60 years, insulin injections have been the standard treatment for diabetes mellitus even though they cannot prevent the devastating complications associated with the disease. Another approach to diabetes therapy would be to propagate new β-cells in the pancreas that could compensate for any insulin deficit. A family of regenerating genes (Reg) has been identified to be strongly expressed during islet neogenesis. We previously found a type 3 Reg gene in hamsters, Islet Neogenesis Associated Protein (INGAP) and have recently shown that administration of a portion of this protein can reverse hyperglycemia in rodents. Since then, a mouse INGAP related protein (INGAPrP/Reg 3δ) has been discovered, as has the human Reg 3 gene, which is claimed to be the counterpart to INGAPrP. Validating human Reg 3 as the INGAP/INGAPrP homolog is difficult since the Reg family is underrepresented in humans and hamsters compared to mice and rats. Our aim was to find new members of the Reg gene family in hamsters by using a tissue culture that was known to constitutively express INGAP. Isolated hamster acinar cells were embedded into a collagen matrix and cultured in DMEM/F12 leading to the formation of duct-like structures (DLS). To amplify Reg genes by reverse transcriptase polymerase chain reaction (RT-PCR), we designed degenerate primers based on the conserved domains of the Reg protein family. PCR products were visualized on agarose gel and appropriately sized products were gel eluted, sequenced and searched against the GenBank database. The RACE system was performed to further sequence the mRNA ends outside the degenerate primer boundaries. When PCR reactions were carried out using hamster DLS cDNA and the degenerate Reg primers, it resulted in the amplification of a novel type 3 Reg gene. The translated protein sequence was found to have the highest similarity to the rat Reg3γ protein at 80% and the mouse Reg 3γ protein at 79% indicating that it was a novel hamster Reg 3γ gene. Compared to the other hamster Reg proteins, it was found to be 55% similar to INGAP and 48% similar to Reg 1. When the human Reg 3 protein sequence was evaluated, it was discovered that human Reg 3 has 69% homology to hamster Reg 3γ compared with only 54% homology to INGAP and only 50% to its reported counterpart, INGAPrP. The discovery of hamster Reg3γ provides strong evidence that human Reg 3 is not the human INGAP/INGAPrP homolog. Elucidating all the Reg family members, including the human INGAP homolog, could provide us with an appropriate means for promoting islet growth in diabetic individuals.

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene ( Reg), first isolated from a regenerating islet cDNA library [J. Biol. Chem. 263 (1988) 2111], encodes a secretory protein with a growth stimulating effect on pancreatic β cells that ameliorates the diabetes of 90% depancreatized rats [Proc. Natl. Acad. Sci. USA 91 (1994) 3589] and non-obese diabetic mice [Diabetes 51(Suppl. 3) (2002) S478]. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5′-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far ( REG Iα, REG Iβ, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158–175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5′ HIP/PAP 3′-5′ RS 3′-3′ REG Iα 5′-5′ REG Iβ 3′-3′ REG III 5′. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the Mut S strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Production and characterization of Reg knockout mice: reduced proliferation of pancreatic beta-cells in Reg knockout mice.

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced beta-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [(3)H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control Reg(+/+) mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in beta-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [(3)H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic beta-cell mass. These results indicate that Reg plays an important role in beta-cell growth/regeneration.

Expression of the Regenerating Gene Family in Inflammatory Bowel Disease Mucosa: Reg Iα Upregulation, Processing, and Antiapoptotic Activity

BackgroundThe pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury related to an ongoing inflammatory process and mucosal reparative mechanisms. Proreparative mucosal factors may offer new therapeutic...

Human aspartyl (asparaginyl) beta-hydroxylase monoclonal antibodies: potential biomarkers for pancreatic carcinoma.

Pancreatic adenocarcinoma is among the top 10 leading causes of death due to cancer in the United States. The lack of reliable and sensitive biomarkers for this disease makes it difficult to render an early diagnosis. To evaluate carcinoma-associated monoclonal antibodies (MoAbs), including AF-20, SF-25, and FB-50, for their binding specificity to pancreatic adenocarcinoma relative to normal pancreatic tissue. In addition, binding of the Th9 MoAb to human Reg 1 protein was studied because of its potential role in cell growth. Adjacent histologic sections were immunostained with each of the MoAbs and graded on a scale of 0 to 4+, corresponding to the relative distribution and intensity of immunoreactivity within the tumor and normal adjacent tissue. Intense levels (grade 3 or 4) of FB50 immunoreactivity were detected in 19 of 19 tumors but not in normal adjacent pancreatic tissue. In addition, increased levels of FB50 immunoreactivity were detected in at least 75% of the tumor cells in 18 of the 19 cases. SF-25 immunoreactivity similarly distinguished pancreatic adenocarcinoma from normal pancreas in 14 of 19 cases. In contrast, AF20 immunoreactivity was detected in 6 of 19 pancreatic adenocarcinomas, and for the most part, the labeling was focal and of a low level. TH9 immunoreactivity was detected in 5 of 19 tumors but also in normal as well as inflamed adjacent pancreatic tissue. These results suggest that the FB50 and SF25 MoAbs represent excellent potential biomarkers of pancreatic adenocarcinoma and could be configured in an immunoassay for detecting pancreatic adenocarcinoma cells in biologic fluids.

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

To investigate the role of Reg Iα 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 Iα. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Iα. Anti-Reg Iα 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 Iα 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 Iα in human IBD.

Human REG I gene is up-regulated in intrahepatic cholangiocarcinoma and its precursor lesions.

The Reg I gene (regenerating gene) and its product (Reg protein) are a regenerating and/or proliferating factor(s) of pancreatic islet cells. The ectopic expression of REG Ialpha was shown in colorectal carcinomas, suggesting that REG Ialpha is related to their carcinogenesis. In this study, we examined the expression of REG I in intrahepatic cholangiocarcinoma (ICC) and its precursor lesion (biliary dysplasia). By polymerase chain reaction and in situ hybridization (ISH) studies using a total of 16 fresh liver specimens, REG Ialpha mRNA was demonstrated in 6 of 11 (55%) ICC cases, but in 0 of 5 (0%) normal livers. Immunohistochemistry for REG I protein was performed in 100 formalin-fixed, paraffin-embedded sections obtained from the 18 cases of ICC alone, 45 hepatolithiasis with ICC (n = 19) or biliary dysplasia (n = 26), 21 hepatolithiasis alone (all with hyperplasia), and 16 normal livers. In ICC, the expression of REG I protein was significantly dependent on the histologic differentiation; 12 of 13 (92%) cases in papillary and well-differentiated, 6 of 16 (38%) cases in moderately differentiated, and 0 of 8 (0%) cases in poorly differentiated types. Moreover, in the lesions of hyperplasia, low-grade dysplasia, and high-grade dysplasia in hepatolithiasis, REG I protein was expressed in 4 of 21 (19%), 7 of 12 (58%), and 13 of 14 (93%) cases, respectively. In normal liver, intrahepatic bile ducts were constantly negative for REG I protein. These findings suggest that neoexpression of REG I is a good marker for biliary mucosa at risk for development of ICC, and also that REG I plays a role in the early stages of biliary carcinogenesis, probably via a cell-proliferative effect.

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.