Maintaining pancreatic β-cell mass and function is essential for normal insulin production and glucose homeostasis. Regenerating islet-derived 2 (Reg2, Reg II, human ortholog Reg1B) gene is normally expressed in pancreatic acinar cells and is significantly induced in response to diabetes, pancreatitis, and high-fat diet (HFD) and during pancreatic regeneration. To evaluate the role of endogenous Reg2 production in normal β-cell function, we characterized Reg2 gene-deficient (Reg2-/-) mice under normal conditions and when subjected to several pathological challenges. At a young age, Reg2 gene deficiency caused no obvious change in normal islet morphology or glucose tolerance. There was no change in the severity of streptozotocin-induced diabetes or caerulein-induced acute pancreatitis in the Reg2-/- mice, indicating that the increased Reg2 expression under those conditions was not essential to protect the islet or acinar cells. However, 13- to 14-month-old Reg2-/- mice developed glucose intolerance associated with significantly decreased islet β-cell ratio and serum insulin level. Similarly, after young mice were fed an HFD for 19 weeks, diminished islet mass expansion and serum insulin level were observed in Reg2-/- vs wild-type mice. This was associated with a decline in the rate of individual β-cell proliferation measured by Ki67 labeling. In both conditions, the β-cells were smaller in gene-deficient vs wild-type mice. Our results indicate that normal expression of Reg2 gene is required for appropriate compensations in pancreatic islet proliferation and expansion in response to obesity and aging.

The onset of severe acute pancreatitis (SAP) is clinically harmful as it may rapidly progress from a local pancreatic inflammation into proemial systemic inflammatory reactions. Patients with SAP have a high mortality, with most cases of death resulting from complications involving the failure of organs other than the pancreas. The distinctive feature of SAP is that once it starts, it may aggrevate the clinical condition of the patient continuously, so that the levels of injury to the other organs surpass the severity of the pancreatic lesion, even causing multiple organ failure and, ultimately, death. In clinical practice, the main complications in terms of organ dysfunctions are shock, acute respiratory failure, acute renal failure, among others. The acute renal injury caused by SAP is not only able to aggravate the state of pancreatitis, but it also develops into renal failure and elevates patients' mortality. Studies have found that the injury due to massive inflammatory mediators, microcirculation changes and apoptosis, among others, may play important roles in the pathogenic mechanism of acute renal injury.

The cystic fibrosis (CF) mouse pancreas has constitutively elevated expression of the Reg/PAP cell stress genes (60-fold greater Reg3alpha, and 10-fold greater PAP/Reg3beta and Reg3gamma). These genes are suggested to be involved in protection or recovery from pancreatic injury.

To test this idea the supramaximal caerulein model was used to induce acute pancreatitis in wild type and CF mice. Serum amylase, pancreatic water content (as a measure of edema), pancreatic myeloperoxidase activity, and Reg/PAP expression were quantified.

In both wild type and CF mice caerulein induced similar elevations in serum amylase (maximal at 12 h), pancreatic edema (maximal at 7 h), and pancreatic myeloperoxidase activity (MPO, a marker of neutrophil infiltration; maximal at 7 h). By immunohistochemistry, Reg3alpha was strongly expressed in the untreated CF pancreas but not in wild type. During pancreatitis, Reg3alpha was intensely expressed in foci of inflamed tissue in both wild type and CF.

These data demonstrate that the severity of caerulein-induced pancreatitis is not ameliorated in the CF mouse even though the Reg/PAP stress genes are already highly upregulated. While Reg/PAP may be protective they may also have a negative effect during pancreatitis due to their anti-apoptotic activity, which has been shown to increase the severity of pancreatitis.

A mouse model using repetitive acinar cell injury caused by supraphysiologic doses of cerulein to induce the characteristic fibrosis and loss of acinar cell mass found in human chronic pancreatitis was employed to identify early changes in gene expression. A gene array was used to detect changes in 18,000 expressed sequence tags; changes in specific transcripts were confirmed by RNase protection assays. These methods identified SPINK3, the mouse homologue of human and rat protease inhibitor genes, as being highly expressed in the pancreas and induced after pancreatic injury. Because SPINK3 may be an important serine protease inhibitor, its up-regulation may reflect an important endogenous cytoprotective mechanism in preventing further injury. The up-regulation of SPINK3 was specific; the mouse homologue of the zymogen-processing protein ZG-16p was also highly expressed in the pancreas but sharply down-regulated early in the course of injury. These findings suggest that the pancreatic acinar cell may respond to injury with a program of self-preservation and loss of normal function.

Regenerating gene (Reg or REG) family, within the superfamily of C-type lectin, is mainly involved in the liver, pancreatic, gastric and intestinal cell proliferation or differentiation. Considerable attention has focused on Reg family and its structurally related molecules. Over the last 15 years, 17 members of the Reg family have been cloned and sequenced. They have been considered as members of a conserved protein family sharing structural and some functional properties being involved in injury, inflammation, diabetes and carcinogenesis. We previously identified Reg IV as a strong candidate for a gene that was highly expressed in colorectal adenoma when compared to normal mucosa based on suppression subtractive hybridization (SSH), reverse Northern blot, semi-quantitative reverse transcriptase PCR (RT-PCR) and Northern blot. In situ hybridization results further support that overexpression of Reg IV may be an early event in colorectal carcinogenesis. We suggest that detection of Reg IV overexpression might be useful in the early diagnosis of carcinomatous transformation of adenoma. This review summarizes the roles of Reg family in diseases in the literature as well as our recent results of Reg IV in colorectal cancer. The biological properties of Reg family and its possible roles in human diseases are discussed. We particularly focus on the roles of Reg family as sensitive reactants of tissue injury, prognostic indicators of tumor survival and early biomarkers of carcinogenesis. In addition to our current understanding of Reg gene functions, we postulate that there might be relationships between Reg family and microsatellite instability, apoptosis and cancer with a poor prognosis. Investigation of the correlation between tumor Reg expression and survival rate, and analysis of the Reg gene status in human malignancies, are required to elucidate the biologic consequences of Reg gene expression, the implications for Reg gene regulation of cell growth, tumorigenesis, and the progression of cancer. It needs to be further attested whether Reg gene family is applicable in early detection of cancer and whether Reg and Reg-related molecules can offer novel molecular targets for anticancer therapeutics. This has implications with regard to prognosis, such as in monitoring cancer initiation, progression and recurrence, as well as the design of chemotherapeutic drugs.

The exocrine pancreas of the cystic fibrosis (CF) mouse (cftr(m1UNC)) is only mildly affected compared with the human disease, providing a useful model to study alterations in exocrine function. The CF mouse pancreas has approximately 50% of normal amylase levels and approximately 200% normal Muclin levels, the major sulfated glycoprotein of the pancreas. Protein biosynthetic rates and mRNA levels for amylase were not altered in CF compared with normal mice, and increases in Muclin biosynthesis and mRNA paralleled the increased protein content. Stimulated pancreatic amylase secretion in vitro and in vivo tended to be increased in CF mice but was not statistically significant compared with normal mice. We show for the first time that the CF mouse duodenum is abnormally acidic (normal intestinal pH = 6.47 +/- 0.05; CF intestinal pH = 6.15 +/- 0.07) and hypothesize that this may result in increased signaling to the exocrine pancreas. There were significant increases in CF intestinal mRNA levels for secretin (310% of normal, P < 0.001) and vasoactive intestinal peptide (148% of normal, P < 0.05). Furthermore, CF pancreatic cAMP levels were 147% of normal (P < 0.01). These data suggest that the CF pancreas may be chronically stimulated by cAMP-mediated signals, which in turn may exacerbate protein plugging in the acinar/ductal lumen, believed to be the primary cause of destruction of the pancreas in CF.

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.

We originally isolated the HIP/PAP gene in a differential screen of a human hepatocellular carcinoma cDNA library. This gene is expressed at high levels in 25% of primary liver cancers but not in nontumorous liver. HIP/PAP belongs to the family of C-type lectins and acts as an adhesion molecule for hepatocytes. In normal adult human tissues, HIP/PAP expression is found in pancreas (exocrine and endocrine cells) and small intestine (Paneth and neuroendocrine cells). In order to gain insight into the possible role of HIP/PAP in vivo, we have investigated the pattern of HIP/PAP expression in the developing postimplantation mouse embryo by in situ hybridization. Detailed analysis of developing mouse embryos revealed that HIP/PAP gene exhibits a restricted expression pattern during development. Thus, HIP/PAP transcripts are first observed within the nervous system from day 14.5 onwards in trigeminal ganglia, dorsal root ganglia, and spinal cord where it appears to be an early specific marker of a subpopulation of motor neurons. At laster stages, HIP/PAP transcripts were detected in intestine and pancreas at day 16.5 but not in embryonic liver. This highly restricted expression pattern suggests that HIP/PAP might participate in neuronal as well as intestinal and pancreatic cell development.

Free radicals are involved in the pathogenesis of acute pancreatitis, during which pancreatitis-associated protein (PAP)-I is overexpressed. We explored whether PAP-I expression could be induced by oxidative stress and whether it could affect apoptosis.

AR4-2J cells were exposed to H2O2 or menadione, and PAP-I messenger RNA (mRNA) expression was analyzed by Northern blotting.

Maximal expression was observed with 0.1 mmol/L H2O2 or with 0.05 mmol/L menadione. Induction was detectable after 12 hours, reached a climax at 18 hours, and then decreased. Pretreatment of the cells with pyrrolidine dithiocarbamate completely abolished PAP-I mRNA induction, suggesting involvement of NFkappaB in the signaling pathway. These findings were confirmed in transient transfection assays using a plasmid containing the PAP-I promoter linked to the chloramphenicol acetyltransferase reporter gene. Then the relationship between PAP-I induction and protection against cell damage during oxidative stress was considered. Constitutive PAP-I expression in AR4-2J cells after transfection with PAP-I complementary DNA conferred significant resistance to apoptosis induced by low doses of H2O2 but not to necrosis induced by high doses of H2O2.

These results suggest that during oxidative stress, PAP-I might be part of a mechanism of pancreatic cell protection against apoptosis.