Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.

Currently, an effective targeted therapy for pancreatitis is lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1R122H gene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1R122H protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1R122H mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1R122H mice with PRSS1WT mice, as well as enzymatically inactivated Dead-PRSS1R122H mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anticoagulation therapy, synergistically prevented progression to CP in PRSS1R122H mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.

Optimising the decellularization of human elastic cartilage with trypsin for future use in ear reconstruction.

Decellularized scaffolds can induce chondrogenic differentiation of stem cells. This study compares different methods to optimise the decellularization of auricular cartilage. The process consisted of an initial 12 hour dry freeze thaw which froze the cartilage specimens in an empty tube at -20 °C. Samples were allowed to thaw at room temperature followed by submersion in phosphate buffer solution in which they were frozen at -20 °C for a 12 hour period. They were then allowed to thaw at room temperature as before. Protocol A subsequently involved subjecting specimens to both deoxyribonuclease and sodium deoxycholate. Protocol B and C were adaptations of this using 0.25% trypsin (7 cycles) and a 0.5 molar solution of ethylenediaminetetraacetic acid (3 hours for each cycle) respectively as additional steps. Trypsin accelerated the decellularization process with a reduction in DNA content from 55.4 ng/µL (native) to 17.3 ng/µL (P-value < 0.05) after 14 days. Protocol B showed a faster reduction in DNA content when compared with protocol A. In comparison to protocol C after 14 days, trypsin also showed greater decellularization with a mean difference of 11.7 ng/µL (P-value < 0.05). Histological analysis with H&E and DAPI confirmed depletion of cells at 14 days with trypsin.

PRSS1 mutations and the proteinase/antiproteinase imbalance in the pathogenesis of pancreatic cancer.

This study aimed to investigate the mutations in the serine protease 1 gene (PRSS1) and the imbalance between trypsin and α1-antitrypsin in patients with pancreatic cancer. Polymerase chain reaction (PCR) was performed to amplify the sequences of PRSS1 from 65 patients with pancreatic cancer and 260 healthy controls, direct sequencing was performed, and the clinical features were analyzed. In addition, enzyme-linked immunosorbent assay (ELISA) was employed to detect serum trypsin and α1-antitrypsin in pancreatic cancer patients and healthy controls in the same period. Mutations were found at the promoter and exon 3 of the PRSS1 in patients with pancreatic cancer. That is, five patients had c.410 C > T mutation causing p.Thr 137 Met, and three patients had c. -338 T > G mutation at the promoter of the PRSS1. In patients with PRSS1 mutations, serum trypsin was 34.5 ± 18.3 ng/mL, which was significantly higher than that in normal controls (10.65 ± 6.03 ng/mL) and other pancreatic cancer (28.61 ± 8.96 ng/mL). What is more, in pancreatic cancer patients, serum α1-antitrypsin was 1.69 ± 0.86 g/L, which was comparable to that in normal controls (1.55 ± 0.53 g/L), while the ratio of serum trypsin to α1-antitrypsin was 1.46-fold to normal controls. The results presented here have provided a greater insight into the PRSS1 mutations and proteinase-inhibitor interactions occurring in pancreatic cancer.

Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy.

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.

Trypsin regulates meiotic initiation in the Japanese eel (Anguilla japonica) by promoting the uptake of taurine into germ cells during spermatogenesis.

Meiosis is a unique and critical process in reproduction. Although the key molecular components of meiosis have been identified, the molecular mechanisms regulating the entry into this pathway remain unclear. We previously demonstrated that a progestin in teleost fish, 17alpha, 20beta-dihydroxy-4-pregnen-3-one, is essential for meiotic initiation, and up-regulates taurine synthesis and the production of trypsin in Sertoli cells. In the present study, we found that trypsin promotes the uptake of taurine into germ cells through the up-regulation of solute carrier family 6 (neurotransmitter transporter, taurine), member 6 (Slc6a6) expression. We further found that this up-regulation of the taurine signal is required for Spo11a expression and meiotic initiation.

[Protease-dependent cell entry mechanism of coronaviruses].

Previous studies have demonstrated that the SARS-CoV S protein requires proteolytic cleavage by elastase, cathepsin or TMPRSS2 for S-mediated cell-cell or virus-cell membrane fusion. Activation of viral glycoprotein (GP) by protease also has been reported for influenza virus. The most distinctive difference between influenza virus and SARS-CoV is the stage during virus replication in which viral glycoproteins are cleaved by proteases. In influenza virus, the protease makes a simple cut in the GP during maturation. In contrast, SARS-CoV S protein is cleaved by the protease following receptor-induced conformational changes. The protease cleavage site in S protein is thought to be exposed only after receptor binding. In support of this model, we reported that the S protein of mouse hepatitis virus type 2 (MHV-2), which is highly similar to the S protein of SARS-CoV, requires two-step conformational changes mediated by sequential receptor binding and proteolysis to be activated for membrane fusion. Such a mechanism allows for tight temporal control over fusion by protecting the activating cleavage site from premature proteolysis yet allowing efficient cleavage upon binding to the receptor on target cells.

Trypsin reduces pancreatic ductal bicarbonate secretion by inhibiting CFTR Cl⁻ channels and luminal anion exchangers.

BACKGROUND & AIMS: The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS: PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS: PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS: Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.

PRSS3 promotes tumour growth and metastasis of human pancreatic cancer.

BACKGROUND AND AIMS: Metastasis accounts for the poor outcome of patients with pancreatic cancer. We recently discovered PRSS3 to be over-expressed in metastatic human pancreatic cancer cells. This study aimed to elucidate the role of PRSS3 in the growth and metastasis of human pancreatic cancer. METHODS: PRSS3 expression in human pancreatic cancer cell lines was detected by qPCR and immunoblotting. The effect of PRSS3 on cancer cell proliferation, migration and invasion in vitro, tumour growth and metastasis in vivo were investigated by manipulation of PRSS3 expression in human pancreatic cancer cell lines. VEGF expression was detected by ELISA, and the pathway through which PRSS3 regulates VEGF expression was investigated. The therapeutic effect of targeting this pathway on metastasis was assessed in vivo. Immunohistochemistry was employed to detect PRSS3 expression in human pancreatic cancer tissues. RESULTS: PRSS3 was over-expressed in the metastatic PaTu8988s cell line, but not in the non-metastatic PaTu8988t cell line. Over-expression of PRSS3 promoted pancreatic cancer cell proliferation as well as invasion in vitro, and tumour progression and metastasis in vivo. Stepwise investigations demonstrated that PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway. ERK inhibitor significantly delayed the progression of metastases of pancreatic cancer and prolonged the survival of animals bearing metastatic pancreatic cancer (p<0.05). 40.54% of human pancreatic cancers (n=74) were positive for PRSS3 protein. A significant correlation was observed between PRSS3 expression and metastasis (p<0.01). Multivariate Cox regression analysis indicated that patients with PRSS3 expression in their tumours had a shorter survival time compared to those without PRSS3 expression (p<0.05). CONCLUSION: PRSS3 plays an important role in the progression, metastasis and prognosis of human pancreatic cancer. Targeting the PRSS3 signalling pathway may be an effective and feasible approach for treatment of this lethal cancer.

Genetic aspects of pancreatitis.

Acute pancreatitis and chronic pancreatitis are complex inflammatory disorders of the pancreas with unpredictable severity, complications, and clinical courses. Growing evidence for genetic risk and modifying factors, plus strong evidence that only a minority of patients with these disorders are heavy alcohol drinkers, has revolutionized our concept of these diseases. Once considered a self-inflicted injury, pancreatitis is now recognized as a complex inflammatory condition like inflammatory bowel disease. Genetic linkage and candidate gene studies have identified six pancreas-targeting factors that are associated with changes in susceptibility to acute and/or chronic pancreatitis, including cationic trypsinogen (PRSS1), anionic trypsinogen (PRSS2), serine protease inhibitor Kazal 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR), chymotrypsinogen C (CTRC) and calcium-sensing receptor (CASR). Patients with mutations in these genes are at increased risk of pancreatitis caused by a variety of stresses including hyperlipidemia and hypercalcemia. Multiple studies are reporting new polymorphisms, as well as complex gene x gene and gene x environmental interactions.

Trypsin and chymotrypsin are involved in the progesterone-induced acrosome reaction in canine spermatozoa.

Acrosomal proteases allow the spermatozoon not only to cross the cumulus cells and penetrate the zona pellucida of the oocyte, but also they are needed for the acrosome reaction process (AR). The present study evaluated in vitro the role of trypsin and chymotrypsin in the acrosome reaction of canine spermatozoa by means of protease inhibitors. Spermatozoa obtained from the second fraction of the ejaculate and devoid of seminal plasma were re-suspended in canine capacitation medium (CCM) and incubated at 38.5 degrees C in 5% CO(2). After 2 h (period of sperm capacitation), aliquots of sperm suspension were incubated separately with trypsin inhibitor NPGB (p-nitrophenyl-p'-guanidino-benzoate); TI (Trypsin inhibitor I-S Type from soybean) and with chymotrypsin inhibitor TPCK (N-tosyl-L-phenylalanine-chloromethyl-ketone) for 30 min. The AR was induced with progesterone and evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. Acrosomal exocytosis levels were statistically significant higher in the samples treated with progesterone than in the control without inducer. However, the trypsin inhibitors NPGB, TI and the chymotrypsin inhibitor TPCK reduced the percentage of AR when compared with the control with progesterone and without inhibitor (p < 0.001), where the AR values were 45.63 +/- 3.8%, 51.63 +/- 2.8%, 58.38 +/-4.1% and 71.25 +/- 4.9%, respectively. These results show that trypsin and chymotrypsin inhibitors are effective in blocking the acrosome reaction induced by progesterone in canine; in addition, they suggest the participation of respective proteases in the AR process in this species.

Divergent roles of SPINK1 and PRSS2 variants in tropical calcific pancreatitis.

BACKGROUND/AIMS: Tropical calcific pancreatitis (TCP) refers to a type of idiopathic pancreatitis prevalent in Asia. The trypsin inhibitor (SPINK1) N34S variant partially explains the genetic susceptibility to TCP. As anionic trypsinogen (PRSS2) G191R protects against chronic pancreatitis in Europeans, we investigated whether this variant protects from TCP in Indians. METHODS: We enrolled 174 patients and 794 controls from two Indian tertiary care referral hospitals. We analyzed PRSS2 and SPINK1 variants by melting curve analysis, allele-specific discrimination assay, and sequencing. RESULTS: G191R was detected in 1 TCP patient (0.6%) compared to 13 controls (1.6%; OR 0.27, 95% CI 0.03-2.1; p = 0.33). SPINK1 N34S was enriched in the TCP population 67/174 (38.5%) compared to controls 10/234 (4.3%; OR 14, 95% CI 6.9-28.3; p < 0.001). CONCLUSION: G191R PRSS2 is a rare allele in the Indian population and the data suggest a nonsignificant trend towards a protective effect. N34S SPINK1 represents the major genetic risk factor in TCP.

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes.

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.

Trypsin activity and bile acid concentrations in the esophagus after distal gastrectomy.

The pathogenesis of reflux esophagitis is not well understood and remains controversial. Distal gastrectomy serves as a model to assess the role of duodenal reflux with low gastric acidity in the development of reflux esophagitis. We investigated the relationship between the severity of esophagitis and gastroduodenal juice reflux, with particular focus on trypsin and bile acids after distal gastrectomy reconstructed with Billroth I anastomosis. Twenty-eight patients with gastroesophageal reflux disease after distal gastrectomy were enrolled. Esophageal and duodenal contents were aspirated under endoscopical examination, and their trypsin activity and bile acid concentrations were measured. The grade of reflux esophagitis was assessed by endoscopy and the symptoms were scored. Moreover, the grade of infiltration of inflammatory cells and the expression of COX-2 mRNA in the esophageal epithelium were evaluated. Patients with severe esophagitis had a higher amount of trypsin activity and bile acid concentrations in the esophagus, but not in the duodenum, compared to patients with mild esophagitis (P < 0.05). There was a strong positive correlation between the trypsin activity and the bile acid concentrations in the esophagus (r = 0.743, P = 0.0001). Moreover, the COX-2 mRNA expression and the grade of infiltrating inflammatory cells in the esophageal mucosa significantly correlated with the trypsin activity and bile acid concentrations in the esophagus. Thus, duodenogastroesophageal reflux with low gastric acidity is one of the pathogeneses in the development of reflux esophagitis from the present clinical study with patients after distal gastrectomy reconstructed with Billroth I anastomosis.

CD166 expression, characterization, and localization in salivary epithelium: implications for function during sialoadenitis.

CD166 is an Ig superfamily molecule that binds homotypically to itself and heterotypically to CD6. Interactions between CD6 and CD166 are important during immune development and in alloreactivity. CD166 is expressed at increased levels in selected cancers and in rheumatoid arthritis synovium. Knowledge that CD166 was expressed in normal human salivary epithelium led to these studies of CD166 and CD6 in diseased mouse salivary glands, that resemble pathology seen in the human disease, Sjögren's syndrome. We showed that in mouse salivary epithelium CD166 was expressed but that expression of CD166 did not necessarily predict its function. Recombinant soluble CD6-Ig bound to CD6 ligands (CD6L) on transformed and freshly isolated salivary epithelial cells. Cross-blocking studies showed that binding of CD6-Ig to salivary epithelium was in part dependent on CD166, but that CD6-Ig binding may also involve additional CD6L. Binding of CD6-Ig was sensitive to trypsin digestion but resistant to digestion by collagenase and sialidase. Anti-CD166 ab precipitated CD166 from salivary epithelium pre- and post-treatment with the pro-inflammatory cytokine IFN-gamma. In contrast CD6-Ig only precipitated CD166 from IFN-gamma treated cells. More extensive colocalization between CD166 and the actin cytoskeleton was observed in sialoadenitis epithelium compared to control. We conclude that during sialoadenitis, CD166 undergoes a gain of function, resulting in closer association with the actin cytoskeleton and increased capacity to bind CD6. We suggest that altered CD166 function may contribute to the pro-inflammatory milieu during sialoadenitis seen in Sjögren's syndrome.

Expression of protease activated receptor-2 (PAR-2) in gastric cancer.

BACKGROUND AND OBJECTIVES: Recently, the four-kind cloning of the protease-activated receptor (PAR) had been carried out. PAR-2 is activated by trypsin and it is supposed that PAR-2 participated in proliferation of the endothelial cell or in neovascularization. We considered whether the expression of PAR-2 has relevance to progression in gastric cancer. METHODS: Immunohistochemical study by the envision method was carried out on 183 samples of gastric cancer in the first department of surgery, University of Fukui, using anti-PAR-2 mouse monoclonal antibody and on 95 samples of them that were pointed out advanced gastric cancers by pathological diagnosis using anti-trypsin rabbit polyclonal antibody. Tissues, which were stained more than 20% of the tumor cells, were classified as PAR-2 protein-positive. Correlation with immunostainings and clinicopathological factors was analyzed statistically. RESULTS: There were 77 (42.1%) carcinomas positive for PAR-2 expression. The PAR-2 expression was intensely strong on the cell membrane of primary cancer tissues. The expression of PAR-2 correlated with the depth of wall invasion, lymphatic invasion, venous invasion, and liver metastasis. The patients with PAR-2 expression-positive tumors had a significant poorer prognosis than those with expression-negative tumors. Univariate analyses identified PAR-2 expression as negative predictors. Multivariate analyses indicated that PAR-2 expression was not an independent factor. A positive reaction for trypsin was obtained in 45 (47.4%) patients. We found a significant correlation between PAR-2 immunostaining and trypsin immunostaining. CONCLUSION: The results of this study lead us to believe that expression of PAR-2 is concerned with progression of gastric cancer.

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells.

Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.

Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction.

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.

Proinflammatory role of trypsin and protease-activated receptor-2 in a rat model of acute pancreatitis.

OBJECTIVES: The pathophysiology of acute pancreatitis is strongly associated with autoactivation of trypsin. The biologic activity of trypsin on cells is attributed to the activation of protease-activated receptor-2 (PAR-2). We hypothesize that trypsin may activate acinar cells or inflammatory cells through PAR-2 signals in acute pancreatitis. METHODS: We immunochemically analyzed the expression of PAR-2 in the rat acinar cell line, ARIP, and the rat pancreas, using anti-rat PAR-2 cleavage site (PCS) and anti-rat PAR-2 N-terminal fragment (PNF) antibodies. Plasma levels of PNF were determined. Furthermore, the effects of the anti-rat PCS antibody and nafamostat mesylate, a potent trypsin inhibitor, on PAR-2 activation during acute pancreatitis were also analyzed. RESULTS: ARIP cells expressed PAR-2, which was activated by exogenous trypsin activity. We also showed that PAR-2 is strongly expressed in pancreatic acinar and duct cells and that it is activated in rat cerulein-induced acute pancreatitis. The anti-rat PCS antibody and nafamostat mesylate reduced interleukin-6 and interferon gamma production and alleviated distant organ injury. CONCLUSIONS: These results suggest that trypsin and its specific receptor, PAR-2, play an important role in cytokine production and the resultant development of distant organ injury during rat acute pancreatitis.

Trypsin stimulates the phosphorylation of p42,44 mitogen-activated protein kinases via the proteinase-activated receptor-2 and protein kinase C epsilon in human cultured prostate stromal cells.

BACKGROUND: The pathogenesis of benign prostatic hyperplasia (BPH) is not well understood. It involves the proliferation of prostate stromal cells. The proteinase-activated receptor subtype 2 (PAR-2) receptor is expressed by human prostate tissue and can be stimulated by serine proteases. Prostate epithelial cells secrete serine proteases such as trypsin, prostate specific antigen (PSA), and human glandular kallikrein (hK2). The p42,44 mitogen activated protein kinase (MAP kinase) pathway regulates cell proliferation. Trypsin can stimulate this pathway via the PAR-2 receptor and protein kinase C (PKC) in other tissues. Serine proteases secreted by prostate epithelial cells may interact with PAR-2 receptors expressed by prostate stromal cells causing them to proliferate. The aim of the present study was to establish whether functional PAR-2 receptors are expressed by human prostate stromal cells (HPSCs) and to determine whether PAR-2 stimulation can activate p42,44 MAP kinase via a pathway involving PKC. METHODS: HPSCs were cultured from patients undergoing trans urethral resection of the prostate (TURP). HPSCs were stimulated with PAR agonists. Immunoblotting of HPSC lysate with anti-p42,44 MAP kinase and -PKC isoforms. Data were analyzed with densitometry. RESULTS: Trypsin and the PAR-2 synthetic peptide SLIGKV caused significant increases in MAP kinase phosphorylation and calcium mobilization in HPSCs. The MAP kinase response was attenuated by pertussis toxin (PTX), phorbol 12,13 dibutyrate, Go6983, and Ro 318220. The PKC isoforms alpha, delta, epsilon, and zeta were detected in HPSCs. Trypsin caused the translocation of PKC(epsilon) from the cytosol to the membrane in HPSCs and was able to stimulate cellular proliferation. CONCLUSIONS: The PAR-2 selective serine protease trypsin activates p42,44 MAP kinase phosphorylation via PKC(epsilon). This may be an important mechanism of BPH pathophysiology.

Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity.

Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.