-
International Journal of Cancer May 1996Serum concentrations of trypsinogen-2 and trypsin-2-alpha(1)-antitrypsin (trypsin-2-AAT) were determined in 145 patients with malignant and 61 with benign... (Comparative Study)
Comparative Study
Serum trypsinogen-2 and trypsin-2-alpha(1)-antitrypsin complex in malignant and benign digestive-tract diseases. Preferential elevation in patients with cholangiocarcinomas.
Serum concentrations of trypsinogen-2 and trypsin-2-alpha(1)-antitrypsin (trypsin-2-AAT) were determined in 145 patients with malignant and 61 with benign digestive-tract diseases. The validity of these tests for detection of cancer was compared with that of CA 19-9 and CEA. Elevated levels of trypsinogen-2 (>90 micrograms/l) and trypsin-2-AAT (>25 micrograms/l) were found in 46% and 42%, respectively, of patients with malignant disease and the levels of trypsinogen-2 were significantly higher than in those with benign disease (p<0.005). High trypsinogen-2 and trypsin-2-AAT concentrations were found most often in patients with biliary and pancreatic cancer, but also in benign obstructive biliary disease. Our results suggest that trypsinogen-2 and trypsin-2-AAT are new potential markers for cholangiocarcinomas.
Topics: Amylases; Bile Duct Neoplasms; Bilirubin; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cholangiocarcinoma; Digestive System Diseases; Digestive System Neoplasms; False Positive Reactions; Humans; Liver Neoplasms; Pancreatic Neoplasms; Trypsin; Trypsinogen; alpha 1-Antitrypsin
PubMed: 8621252
DOI: 10.1002/(SICI)1097-0215(19960503)66:3<326::AID-IJC10>3.0.CO;2-9 -
Biochimica Et Biophysica Acta.... Dec 2022Acute pancreatitis (AP) was initiated within pancreatic parenchymal cells and sustained by uncontrolled inflammatory responses. AXL and MERTK receptor tyrosine kinases...
BACKGROUND
Acute pancreatitis (AP) was initiated within pancreatic parenchymal cells and sustained by uncontrolled inflammatory responses. AXL and MERTK receptor tyrosine kinases play a crucial role in negatively regulating the innate immunity. Therefore, this study aimed to investigate the role and underlying mechanism of AXL and MERTK in AP.
METHODS
Experimental AP was induced by ten hourly intraperitoneal administration of caerulein in global, hematopoietic- and pancreas-specific Axl and Mertk deficient mice. Pancreatitis severity was assessed biochemically and histologically. Pancreatic transcriptomics and pancreatic infiltrating immune cells were profiled. Some mice were given R428, an antagonist of AXL and MERTK. AXL and MERTK in peripheral leukocytes were measured by flow cytometry.
FINDINGS
The levels of AXL and MERTK in pancreatic tissue and pancreatic CD45 cells were dynamically altered at 6 h and 12 h after the 1st injection of caerulein. Global and hematopoietic-specific, but not pancreas-specific deletion of Axl and Mertk protected against pancreatic necrosis and trypsinogen activation. Pancreatic transcriptomic analysis revealed that differentially expressed gene signatures were mainly related to metabolic and inflammatory pathways. Furthermore, deletion or inhibition of Axl and Mertk selectively inhibited pancreatic neutrophil infiltration, which was primarily related to CXCL2 secreted by pro-inflammatory macrophages. Increased levels of MERTK in peripheral leukocytes were correlated with more severe form of AP.
INTERPRETATION
Our findings reveal that specific AXL/MERTK antagonist may be a novel and potential early treatment for AP and the levels of MERTK in peripheral leukocytes may be a promising biomarker for predicting pancreatic severity in patients with AP.
FUNDING
National Natural Science Foundation of China, Shanghai Natural Science Foundation, a Shanghai Young Talent Award and a Shanghai Young Orient Scholar Award.
RESEARCH IN CONTEXT
Evidence before this study Acute pancreatitis (AP) is a common inflammatory disorder of the exocrine pancreas, the severity of which was determined by the extent of pancreatic necrosis, with no targeted therapy. AP was initiated by signals within pancreatic parenchymal cells and sustained by uncontrolled innate immune responses. One of the three crucial regulatory roles for AXL and MERTK is to negatively regulate innate immune responses. Added value of this study Global and hematopoietic-, but not pancreas-specific Axl and Mertk deficiency protected against pancreatitis, primarily pancreatic necrosis. Deletion of Axl and Mertk selectively inhibited pancreatic neutrophil infiltration that was related to CXCL2 secreted by pro-inflammatory macrophages. AXL and MERTK antagonist similarly reduced pancreatitis severity via limiting CXCL2-mediated pancreatic neutrophil infiltration. Higher levels of MERTK, but not AXL in peripheral leukocytes were correlated with more severe form of acute pancreatitis. Implications of all the available evidence A specific AXL/MERTK antagonist may be a novel and potential early treatment for AP. The level of MERTK on peripheral leukocytes may be a promising biomarker for predicting disease severity in patients with AP.
Topics: Acute Disease; Animals; Ceruletide; Chemokine CXCL2; China; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pancreatitis, Acute Necrotizing; Trypsinogen; Tyrosine; c-Mer Tyrosine Kinase
PubMed: 35841983
DOI: 10.1016/j.bbadis.2022.166490 -
Molecular Medicine Reports Nov 2019Acute pancreatitis (AP) exhibits high morbidity and mortality rates. The onset of AP is characterized by early trypsinogen activation.The present study aimed to...
Acute pancreatitis (AP) exhibits high morbidity and mortality rates. The onset of AP is characterized by early trypsinogen activation.The present study aimed to investigate the expression of microRNA (miR)‑92a‑3p and early growth response protein 1 (Egr1), and the effect of miR‑92a‑3p on trypsinogen activation in the pancreatic exocrine cell line AR42J. mRNA and miRNA microarrays were used to identify differentially expressed mRNAs and miRNAs in AR42J cells. A miRNA‑mRNA network was constructed using bioinformatics software, and Egr1 and its regulated miRNA subnetworks were identified by reviewing previous literature. The results suggested that miR‑92a‑3p could bind to Egr1 3'untranslated region sequence. Subsequently, miR‑92a‑3p mimic and inhibitor were used to transfect AR42J cells. Following transfection, reverse transcription‑quantitative PCR and western blotting were performed to detect Egr1 expression. Furthermore, AR42J cells were cotransfected with miR‑92a‑3p inhibitor and small interfering (si)‑Egr1. The trypsinogen activation rate of AR42J cells was measured by flow cytometry. Microarrays and bioinformatics results indicated that Egr1 may be a target gene of miR‑92a‑3p. In addition, the present study suggested that miR‑92a‑3p downregulated Egr1 in vitro and that miR‑92a‑3p and Egr1 expression was associated with trypsinogen activation. Furthermore, miR‑92a‑3p inhibitor reversed the effect of si‑Egr1 on trypsinogen activation. In conclusion, miR‑92a‑3p may negatively regulate the activation of trypsinogen in AR42J cells via Egr1.
Topics: Cell Line; Computational Biology; Early Growth Response Protein 1; Gene Expression Profiling; Gene Expression Regulation; MicroRNAs; Nucleic Acid Conformation; RNA Interference; RNA, Messenger; Transcriptome; Trypsinogen
PubMed: 31545429
DOI: 10.3892/mmr.2019.10673 -
Scientific Reports Jun 2019Intra-pancreatic activation of trypsin is an early event in pancreatitis. Trypsinogen can be activated to trypsin either through autoactivation (trypsin-mediated...
Intra-pancreatic activation of trypsin is an early event in pancreatitis. Trypsinogen can be activated to trypsin either through autoactivation (trypsin-mediated trypsinogen activation) or by the lysosomal protease cathepsin B (CTSB). Experimental separation of CTSB-mediated activation from autoactivation in mice is possible through knocking in mutations that render trypsinogen sensitive to CTSB but resistant to trypsin. Here we present biochemical studies on novel mouse cationic trypsinogen (isoform T7) mutants engineered for selective CTSB activation. First, we demonstrated that mutation K24G, which alters the activation site Lys in T7 trypsinogen, abolished autoactivation while activation by CTSB was stimulated 4-fold at pH 4.0. Interestingly, CTSB-mediated activation of the K24G mutant became more sensitive to inhibition by increasing pH. Next, Ala-scanning of the five Asp residues preceding the activation site Lys revealed that mutation D22A accelerated CTSB-mediated activation by 2-fold. Finally, combination of mutations D22A and K24G resulted in a trypsinogen mutant that exhibited 14-fold increased activation by CTSB and normal pH sensitivity. We conclude that we successfully engineered a mouse T7 trypsinogen mutant (D22A,K24G), which is robustly activated by CTSB but cannot undergo autoactivation. These studies set the stage for the generation of a preclinical mouse model of CTSB-dependent pancreatitis.
Topics: Animals; Cathepsin B; Cathepsin L; Enzyme Activation; Humans; Liver; Mice; Mutation; Protein Engineering; Trypsinogen
PubMed: 31235832
DOI: 10.1038/s41598-019-45631-z -
American Journal of Physiology.... Dec 2021The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation...
The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen paralogs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased the autoactivation of T7 trypsinogen threefold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side chains are preferred for maximal autoactivation, and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by NH-terminal truncation with chymotrypsin C reduced the autoactivation of the D23del mutant. Homozygous mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable with that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in mice relative to C57BL/6N mice. Furthermore, when the allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the polyaspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens. Unwanted autoactivation of the digestive protease trypsinogen can result in pancreatitis. The trypsinogen activation peptide contains a polyaspartate motif that suppresses autoactivation. This study demonstrates that evolutionary expansion of these aspartate residues in mouse cationic trypsinogen further inhibits autoactivation and enhances protection against pancreatitis.
Topics: Amino Acid Motifs; Animals; Evolution, Molecular; Mice; Mice, Inbred C57BL; Mutation; Oligopeptides; Pancreatitis; Peptides
PubMed: 34643096
DOI: 10.1152/ajpgi.00265.2021 -
The American Journal of Pathology Dec 2000Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon...
Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.
Topics: Blotting, Northern; Genitalia, Male; Humans; Immunohistochemistry; In Situ Hybridization; Isoenzymes; Male; Prostate-Specific Antigen; Semen; Trypsin; Trypsinogen; Zinc
PubMed: 11106574
DOI: 10.1016/S0002-9440(10)64840-7 -
Biochimica Et Biophysica Acta.... Nov 2022Alcohol abuse, a main cause of pancreatitis, has been known to augment NF-κB activation and cell necrosis in pancreatitis. However, the underlying mechanisms are...
BACKGROUND
Alcohol abuse, a main cause of pancreatitis, has been known to augment NF-κB activation and cell necrosis in pancreatitis. However, the underlying mechanisms are unclear. We recently reported that inhibition of protein kinase D (PKD) alleviated NF-κB activation and severity of experimental pancreatitis. Here we investigated whether PKD signaling mediated the modulatory effects of alcohol abuse on pathological responses in alcoholic pancreatitis.
METHODS
Alcoholic pancreatitis was provoked in two rodent models with pair-feeding control and ethanol-containing Lieber-DeCarli diets for up to 8 weeks followed by up to 7 hourly intraperitoneal injections of cerulein at 1 μg/kg (rats) or 3 μg/kg (mice). Effects of PKD inhibition by PKD inhibitors or genetic deletion of pancreatic PKD isoform (PKD3Δpanc mice) on alcoholic pancreatitis parameters were determined.
RESULTS
Ethanol administration amplified PKD signaling by promoting expression and activation of pancreatic PKD, resulted in augmented/promoted pancreatitis responses. Pharmacological inhibition of PKD or with PKD3Δpanc mice prevented the augmenting/sensitizing effect of ethanol on NF-κB activation and inflammatory responses, cell necrotic death and the severity of disease in alcoholic pancreatitis. PKD inhibition prevented alcohol-enhanced trypsinogen activation, mRNA expression of multiple inflammatory molecules, the receptor-interacting protein kinase activation, ATP depletion, and downregulation of pro-survival Bcl-2 protein in alcoholic pancreatitis. Furthermore, PKD inhibitor CID755673 or CRT0066101, administrated after the induction of pancreatitis in mouse and rat alcoholic pancreatitis models, significantly mitigated the severity of pancreatitis.
CONCLUSION
PKD mediates effect of alcohol abuse on pathological process of pancreatitis and constitutes a novel therapeutic target to treat this disease.
Topics: Adenosine Triphosphate; Alcoholism; Animals; Ceruletide; Ethanol; Mice; NF-kappa B; Necrosis; Pancreatitis, Alcoholic; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Rats; Trypsinogen
PubMed: 35835415
DOI: 10.1016/j.bbadis.2022.166486 -
American Journal of Physiology.... Jun 2020Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA)...
Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP. The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.
Topics: Animals; Ceruletide; Dopamine Agonists; Gene Expression Regulation; HSP72 Heat-Shock Proteins; Heat Shock Transcription Factors; Lysosomes; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Phosphorylation; Protein Phosphatase 2; Quinpirole; Receptors, Dopamine D2; Trypsinogen; Up-Regulation
PubMed: 32308041
DOI: 10.1152/ajpgi.00354.2019 -
Molecular Medicine Reports Sep 2020Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response...
Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3‑sulfate (TLC‑S)‑treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNA‑transfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNA‑transfected cells, indicating that in TLC‑S‑induced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNA‑microRNA (miRNA/miR)‑long non‑coding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)‑miR‑214‑3p and rno‑miR‑764‑5p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.
Topics: Animals; Cell Line; Computational Biology; Early Growth Response Protein 1; Enzyme Activation; Gene Expression Profiling; Gene Regulatory Networks; MicroRNAs; Models, Biological; Oligonucleotide Array Sequence Analysis; Pancreatitis; RNA, Long Noncoding; RNA, Small Interfering; Rats; Taurolithocholic Acid; Trypsinogen; Up-Regulation
PubMed: 32705196
DOI: 10.3892/mmr.2020.11316 -
Molecular and Cellular Biology Oct 1985In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to...
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; DNA; Dogs; Gene Expression Regulation; Isoelectric Point; Isoenzymes; Nucleic Acid Conformation; Pancreas; Protein Biosynthesis; Protein Sorting Signals; RNA, Messenger; Trypsinogen
PubMed: 3841794
DOI: 10.1128/mcb.5.10.2669-2676.1985