PAP1 signaling involves MAPK signal transduction.

Pancreatitis-associated protein 1 (PAP1) belongs to the Reg family of secretory proteins. Several important biological roles have been attributed to PAP1 but the signaling pathways activated by this protein remain only partially understood. Here, we describe the intracellular pathways triggered by PAP1 in a pancreatic acinar cell line. Taking advantage of the fact that PAP1 induces its own transcription, we performed ChIP assays to analyze the recruitment of transcriptional factors on its promoter. Our results show that PAP1 increased the transactivation activity of pap1 and the binding on its promoter of the nuclear factors C/EBPbeta, P-CREB, P-ELK1, EGR1, STAT3, and ETS2, which are downstream targets of MAPK signaling. p44/42, p38, and JNK MAPKs activity increased after PAP1 treatment. In addition, pharmacological inhibition of these kinases markedly inhibited the induction of pap1 mRNA. Taken together, these results indicated that the mechanism of PAP1 action involves the activation of the MAPK superfamily.

Toll-like receptor 2 is critical for induction of Reg3 beta expression and intestinal clearance of Yersinia pseudotuberculosis.

OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.

Ionic interactions between bovine chymotrypsinogen A and chondroitin sulfate A.B.C.. A possible model for molecular aggregation in zymogen granules.

The formation of large aggregates by ionic interactions between acidic glucosaminoglycans and cationic secretory proteins has been proposed as one of the critical steps in the concentration process in the condensing vacuoles of secretory cells. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A and chondroitin sulfate as a simplified model. Small amounts of chondroitin sulfate were found able to induce chymotrypsinogen precipitation. Like zymogen granules, the resulting aggregates were moderately sensitive to ionic strength and insensitive to osmolality. Moreover, their pH dependence was similar to that of isolated zymogen granules. When sulfated glucosaminoglycans isolated from the zymogen granules of the guinea pig pancreas were used instead of chondroitin sulfate, the same kind of interactions with chymotrypsinogen were obtained. Our data support the hypothesis that the strong ionic interactions between those sulfated glucosaminoglycans and cationic proteins could be responsible for the concentration process.

Genetic alterations in precancerous pancreatic lesions and their clinical implications.

Pancreatic adenocarcinoma, with an incidence/death ratio of 0.99, has the worst prognosis of all cancers. Risk factors associated with the sporadic form of pancreatic adenocarcinoma are unknown and less than 10% of patients receive curative treatment (surgery associated with radiation therapy or chemotherapy) with a low 5-year survival rate (10 to 20%). In more than 90% of patients, the tumor discovered at diagnosis is not resectable or has already metastasized. Thus, a better understanding of the etiology of pancreatic cancer is essential to identify new prognostic markers and new therapeutic targets. There is a wealth of data on the identification of genetic alterations associated with pancreatic cancer and their role in its development. This review will focus on the current knowledge of genetic alterations associated with two pancreatic lesions that can potentially evolve into pancreatic adenocarcinoma, Pancreatic Intraepithelial Neoplasia (PanIN) and Intraductal Papillary Mucinous Neoplasm (IPMN). These two lesions share a large panel of typical genetic alterations which are close to those found in pancreatic adenocarcinoma. A better understanding of these alterations may lead to therapeutic targets that could help prevent the progression of PanIN and IPMN to cancer.

Human pancreatitis-associated protein. Messenger RNA cloning and expression in pancreatic diseases.

A human pancreatic cDNA library was screened with the cDNA encoding rat "pancreatitis-associated protein" (PAP). The selected clone encoded a secretory protein structurally related to rat PAP. The protein had the same size as rat PAP and showed 71% amino acid identity, the six half-cystines being in identical positions. Domains of the proteins showing homologies with calcium-dependent lectins were also conserved. In addition, expression in pancreas of the genes encoding the human protein and rat PAP showed similar characteristics: both were expressed at very low levels in control tissue and overexpressed during the acute phase of pancreatitis, contrary to most secretory products. The human protein was therefore named human pancreatitis-associated protein (PAP-H). Antibodies raised to a synthetic peptide of PAP-H detected a single band with an M(r) compatible with PAP-H in Western blot analysis of proteins extracted from a pancreas presenting with acute pancreatitis. In that tissue, the protein could be immunolocalized to the apical regions of acinar cells. An immunoassay was also constructed to quantify the protein in serum. Elevated PAP-H levels were observed in patients with acute pancreatitis and in some patients with chronic pancreatitis. Values were close to background in healthy subjects and in patients with other abdominal diseases. These results confirm that PAP-H synthesis increases during inflammation and suggest a possible use of the protein as biological marker of acute pancreatitis.

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Dietary regulation of pancreatic protein synthesis. I. Rapid and specific modulation of enzyme synthesis by changes in dietary composition.

Dietary adaptation of pancreatic protein synthesis and of pancreatic enzyme concentration, was studied over the first 24 h of exposure to a new diet. Rats were adapted to a carbohydrate-rich (G) or to a protein-rich diet (P) and were switched to the opposite regime after a 15 h fast. The evolution of the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen and of the pancreatic concentration of amylase and chymotrypsinogen were followed. Fasting caused important modifications in the relative rate of synthesis of the three enzymes in rats adapted to a P diet. Adaptative changes in the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen were seen within 2 h after the beginning of refeeding. These changes were followed by corresponding adaptative modifications in pancreatic contents 4 h after the beginning of refeeding. After 24 h of refeeding, significant adaptative changes had occurred in both the relative rates of synthesis and in enzyme concentrations. Thus exocrine pancreatic protein synthesis can be modulated as early as 2 h after refeeding and this modulation is followed by adaptative changes in pancreatic enzyme content.

Dietary regulation of pancreatic protein synthesis. II. Kinetics of adaptation of protein synthesis and its effect on enzyme content.

The kinetics of the adaptative changes in the relative rates of synthesis and pancreatic concentrations of amylase, chymotrypsinogen and trypsinogen were studied over 10 days of adaptation to a carbohydrate-rich (G), or a protein-rich (P) diet. During adaptation to the P diet, 60% of the adaptative decrease of the amylase to chymotrypsinogen ratio of incorporation was complete within 24 h of feeding and plateau values were obtained after five days. Adaptation to the G diet was only 20% complete after 24 h and plateau values were obtained later than with the P diet. The evolution of the ratio of concentrations of amylase and chymotrypsinogen followed those of incorporation in the adaptation to both diets. These results support the determinant role of adaptative changes in the rates of synthesis of individual enzymes on the dietary adaptation of enzyme proportions in the pancreas. The differences in the kinetics of adaptation to the two diets suggest that different mechanisms are involved in the adaptative regulation of protein synthesis to a carbohydrate-rich diet or a protein-rich diet.

Different action of hormonal stimulation on the biosynthesis of three pancreatic enzymes.

The rates of biosynthesis of amylase, lipase and chymotrypsinogen were followed in rat pancreas in vivo after intravenous injection of the hormone cholecystokinin-pancreozymin (8 IDU/kg) associated with secretin (5 CU/kg). This pancreatic stimulation resulted in non-parallel variations of the rates of biosynthesis of the three studied enzymes, suggesting independent regulation of synthesis. The result of one stimulation was calculated in terms of quantities of enzymes synthesized by the pancreas in comparison to control. It was found that chymotrypsinogen, amylase and lipase productions were increased by 63, 26 and 10%, respectively, indicating that repeated cholecystokinin-pancreozymin plus secretin stimulations could induce "adaptation-like" modifications of the pancreatic enzyme content.

Cloning, sequencing and expression of the L5, L21, L27a, L28, S5, S9, S10 and S29 human ribosomal protein mRNAs.

During systematic analysis of the mRNAs expressed in a human colorectal carcinoma with the aim of evidencing new makers of the disease (Frigerio et al. (1995), in press), we isolated several clones corresponding to homologs of rat ribosomal protein mRNAs L5, L21, L27a, L28, S5, S9, S10 and S29. Because expression of several mRNAs encoding ribosomal proteins was found strongly altered during colorectal carcinogenesis, sequence of these transcripts, not previously described in human, was completed and their expression analyzed. Northern blot analysis of RNAs extracted from colorectal cancer and ajdacent normal tissue from 6 patients revealed in all of them perturbations of expression in cancer, compared to normal. No correlation could however be made between the level of expression and the severity of the disease. Yet, abnormal patterns with additional larger transcripts were observed in some patients for rpL5, rpL28 and rpS10.

Rapid PCR cloning and sequence determination of the rat lithostathine gene.

The rat lithostathine gene was isolated from a genomic library using a rapid screening procedure involving PCR amplification. It was characterized over 2.7 kbp of gene sequence and 2.43 kbp of 5'-flanking sequence. The 5'-end of the coding sequence was determined by primer extension of lithostathine mRNA. The lithosathine sequence spanned over six exons. The promoter region of the gene contained the TATAAA and CCAAT consensus sequences 30 and 107 bp upstream of the cap site, respectively. Furthermore, a tract of (TG)22 repeat, with potential Z-DNA conformation, was found at position-1081.

Two transcripts are generated from the pancreatitis associated protein II gene by alternative splicing in the 5' untranslated region.

We have previously reported the coding sequence of the rat PAP II mRNA. We show in this paper the existence in rat pancreas of two forms of PAP II mRNA with identical coding sequence but a different 5'-untranslated region. We demonstrate that this is the result of a differential splicing.

The pancreatitis associated protein III (PAP III), a new member of the PAP gene family.

A third member of the rat pancreatitis associated protein (PAP) gene family is described here. Its messenger RNA was cloned from an intestinal cDNA library and sequenced. The encoded protein, designated PAP III, shows 66% and 63% identity with the rat PAP I and II, respectively. The PAP III gene is constitutively expressed in the small intestine and in the pancreas with acute pancreatitis, but not in the healthy pancreas.

Expression of the stress-induced p8 mRNA is transiently activated after culture medium change.

We report here that the mere fact of changing culture medium for fresh medium induced in several cell lines the expression of stress-activated genes including protein kinases p38, JNK and ERK1/2 and the transcription factor C/EBPbeta. As a consequence, p8, a gene induced by stress in several tissues, was strongly up-regulated. Induction did not occur after change for cell-conditioned medium. Induction was however transient, with a peak at 60 min for p38, at 15-30 min for JNK and at 15 min for ERK1/2, at 2-3 hours for C/EBPbeta and at 4-6 hours for p8. Repression of the induction was due to the secretion of thermolabile molecule(s) that progressively conditioned the medium. As low as 25% of conditioned medium added to fresh culture medium was sufficient to abolish the stress response. Taken together, our data indicate that the renewal of culture medium induces a transient cellular stress that may be a source of artifacts in experiments performed shortly after a change of culture medium.

Cdx1 promotes cellular growth of epithelial intestinal cells through induction of the secretory protein PAP I.

Expression of the Cdx1 homeobox gene in epithelial intestinal cells promotes cellular growth and differentiation. Cdx1and the Pancreatitis Associated Protein I (PAP I) are concomitantly expressed in the epithelial cells of the lower part of the intestinal crypts. Because Cdx1 is a transcription factor and PAP I, in other tissues, is a proliferative factor, we looked for a relationship between these two proteins in the intestinal-derived IEC-6 cells. After stable transfection with a Cdx1 expression vector, they produce high levels of the PAP I transcript and protein indicating a functional link between the two genes. Demonstration of Cdx1 binding to the PAP I promoter region and suppression of PAP I induction after deletion of the corresponding sequence indicated that Cdx1 is a transcription factor controlling PAP I gene expression in intestinal cells. By infecting IEC-6 cells with adenoviruses expressing PAP I, we demonstrated that PAP I induces mitosis in these cells. On the other hand, inhibition of the PAP I expression in the IEC-6 Cdxl-expressing cells using an antisense strategy confirmed the requirement of this protein for the effect of Cdx1 on cell growth. Finally, addition of the immunopurified PAP I to the culture medium promotes cell growth of the IEC-6 cells in a dose-dependent manner. Maximal effect was obtained at 1 ng/ml. Taken together these results demonstrate that PAP I is a target of the Cdx1 homeobox gene in intestinal cells which participates in the regulation of intestinal cell growth via an autocrine and/or paracrine mechanism.

Pancreatitis associated protein I (PAP-I) alters adhesion and motility of human melanocytes and melanoma cells.

Pancreatitis associated protein I is a secretory stress protein first characterized in pancreas during pancreatitis but also expressed in several tissues including hepatic, gastric, and colon cancer. Its concentration in serum can be significant. The relationship of pancreatitis associated protein I to skin cancers was investigated in normal melanocytes, melanoma tumors, and melanoma cell lines. None of them expressed pancreatitis associated protein I, even after stress induction. Adenovirus-mediated pancreatitis associated protein I expression, however, reduced cell adhesion to laminin-1 and fibronectin with a loss of integrin participation. Pancreatitis associated protein I expression stimulated haptotactic and directed migrations of some melanoma cells, but only directed migration was activated in normal melanocytes. Importantly, directed migration and spreading on fibronectin of the responsive melanoma cells were also enhanced when purified rat pancreatitis associated protein I was added to the culture medium of noninfected cells. This indicates that effects in infected cells were elicited by pancreatitis associated protein I after its secretion. Exogenous pancreatitis associated protein I can therefore modify the adhesion and motility of normal and transformed melanocytes, suggesting a potential interaction with melanoma invasivity.

A limited genomic region contains the human REG and REG-related genes.

A glycoprotein expressed in exocrine pancreas (where it has been called lithostathine) and endocrine pancreas (where it has been called the regeneration protein) is encoded by a gene (REG) which maps to 2p12. A REG-related sequence (REGL) is also located in 2p12 and expressed in the pancreas. Here we describe the physical mapping of these genes within a 100-kb genomic region. A YAC clone was converted into an ordered cosmid contig. We constructed a restriction map of the cosmid contig and localized the loci corresponding to the genes. A third REG-related sequence also maps to this region. Although this sequence was previously described as a pseudogene, we show here that it is also expressed in the pancreas.

A gene homologous to the reg gene is expressed in the human pancreas.

We have determined the nucleotide sequence of regl a human genomic DNA fragment homologous to the reg gene which is expressed in the exocrine pancreas and regenerating islets. Sequence comparisons of reg and regl suggested similar exon-intron organisation. Based on this assumption, specific oligonucleotides for regl exons were used to demonstrate expression of the regl gene in pancreas and liver. The proteins encoded by reg and regl comprise 166 amino acids and differ by 22 amino acids only.

Cloning cDNAs corresponding to the 5' end of messenger RNAs using specific DNA primers.

A method is described to clone cDNAs corresponding to the 5' end of specific mRNAs which are absent from cDNA libraries. A segment is excised from the 5' end of a previously cloned truncated cDNA and hybridized to total polyadenylated RNA. The hybridized primer is extended using reverse transcriptase, tailed with deoxycytidine, and the second strand synthesized using oligo (dG)10-16 as a primer. The primer-extended double-stranded cDNA is cloned into pBR322 by dC-dG homopolymeric tailing. This method has been successful for cloning a 398 bp cDNA fragment corresponding to the entire 5' end of rat pancreatic elastase I mRNA, a prominent pancreatic mRNA, and a 510 bp fragment encoding the 5' end of kallikrein mRNA, a lower abundant pancreatic mRNA.

The human pancreatic stone protein.

Chronic calcifying pancreatitis (CCP) is characterized by the presence of stones in pancreatic ducts. Calcium carbonate (CaCO3) is the main constituent of stones, to which is associated an organic matrix consisting primarily of one protein of Mr 14,000, the pancreatic stone protein or PSP. PSP is not present as such in pancreatic juice, but in polymorphic forms with higher molecular weights. These secretory forms (PSP S2-5, Mr 16-19,000) are synthesized in the acinar cells of the pancreas and secreted along the same secretory pathway as the exocrine enzymes. The heterogeneity of the forms of higher Mr (PSP S2-5) is probably due to different glycosylation patterns. PSP and PSP S1 are generated by the cleavage of an Arg-Ile bond in the N-terminal part of PSP S2-5. The N-terminal sequence of PSP (40 amino acids) is identical to that of PSP S1, whose complete sequence (133 amino acids) has been determined. Yet, the two proteins differ by their pI. Pancreatic juice is normally supersaturated in CaCO3, suggesting the presence of a stabilizer preventing CaCO3 precipitation. The PSP S could play that role, since an activity inhibiting the nucleation and growth in vitro of CaCO3 crystals was found in pancreatic juice, associated with these proteins. Moreover, PSP S concentration was significantly lower in the pancreatic juice of patients with CCP than in control patients. Proteins homologous to PSP S were also found in the dog, rat, swine, monkey and ox. They constitute a new family of pancreatic secretory proteins, whose biological role would be to maintain pancreatic juice in a stable state towards CaCO3.