Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis.

Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) analysis demonstrated ectopic expression of KLK6 in human colon adenocarcinomas but not in normal epithelia. Importantly, high levels of KLK6 protein were detected in the ascites of CRC patients with peritoneal metastasis, but not in benign ascites. These data indicate that KLK6 overexpression is associated with aggressive CRC, and may be applied to differentiate between benign and malignant ascites.

Molecular Pathways Associated with Kallikrein 6 Overexpression in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The high mortality of CRC is related to its ability to metastasize to distant organs. The kallikrein-related peptidase Kallikrein 6 (KLK6) is overexpressed in CRC and contributes to cancer cell invasion and metastasis. The goal of this study was to identify KLK6-associated markers for the CRC prognosis and treatment. Tumor Samples from the CRC patients with significantly elevated KLK6 transcript levels were identified in the RNA-Seq data from Cancer Genome Atlas (TCGA) and their expression profiles were evaluated using Gene Ontology (GO), Phenotype and Reactome enrichment, and protein interaction methods. KLK6-high cases had a distinct spectrum of mutations in titin (TTN), APC, K-RAS, and MUC16 genes. Differentially expressed genes (DEGs) found in the KLK6-overexpressing CRCs were associated with cell signaling, extracellular matrix organization, and cell communication regulatory pathways. The top KLK6-interaction partners were found to be the members of kallikrein family (KLK7, KLK8, KLK10), extracellular matrix associated proteins (keratins, integrins, small proline rich repeat, S100A families) and TGF-ß, FOS, and Ser/Thr protein kinase signaling pathways. Expression of selected KLK6-associated genes was validated in a subset of paired normal and tumor CRC patient-derived organoid cultures. The performed analyses identified KLK6 itself and a set of genes, which are co-expressed with KLK6, as potential clinical biomarkers for the management of the CRC disease.

Insights into the activity control of the kallikrein-related peptidase 6: small-molecule modulators and allosterism.

The activity of kallikrein-related peptidase 6 (KLK6) is deregulated in various diseases such as cancer and neurodegenerative diseases. KLK6 is thus considered as an attractive therapeutical target. In this short report, we depict some novel findings on the regulation of the KLK6 activity. Namely, we identified mechanism-based inhibitors (suicide substrates) from an in-house library of 6-substituted coumarin-3-carboxylate derivatives. In addition, a molecular dynamics study evidenced the allosteric behavior of KLK6 similar to that previously observed for some trypsin-like serine proteases. This allosteric behavior together with the coumarinic scaffold bring new opportunities for the design of KLK6 potent activity modulators, useful as therapeutics or activity-based probes.

Advanced high-grade serous ovarian cancer: inverse association of KLK13 and KLK14 mRNA levels in tumor tissue and patients' prognosis.

PURPOSE: Gene expression of a variety of the 15 members of the KLK serine protease family is dysregulated in ovarian cancer. We aimed at determining the clinical relevance of KLK13 and KLK14 mRNA expression in tumor tissues of a homogeneous patient cohort afflicted with advanced high-grade serous ovarian cancer (FIGO stage III/IV). METHODS: mRNA expression levels of KLK13 and KLK14 were assessed by quantitative PCR in tumor tissue of 91 patients and related with clinical factors and patients' outcome. RESULTS: There was no significant association of KLK13 and KLK14 mRNA expression with the clinical factors ascitic fluid volume or residual tumor mass. In univariate Cox regression analysis, elevated KLK13 mRNA levels were significantly linked with shorter progression-free (PFS; hazard ratio [HR] = 1.97, P = 0.020) and overall survival (OS; HR = 1.81, P = 0.041). High KLK14 mRNA levels were significantly associated with prolonged PFS (HR = 0.44, P = 0.017) and showed a trend towards significance for OS (HR = 0.55, P = 0.070). In multivariable analysis, including the factors age, residual tumor mass, ascitic fluid volume, KLK13, and KLK14, both KLKs, apart from residual tumor mass, remained statistically independent predictive markers: patients with high KLK13 mRNA expression levels displayed a more than twofold increase risk for shorter PFS (HR = 2.14, P = 0.020) as well as OS (HR = 2.05, P = 0.028), whereas elevated KLK14 mRNA values were found to be significant for both, prolonged PFS (HR = 0.36, P = 0.007) and OS (HR = 0.46, P = 0.037). CONCLUSION: These results indicate that in advanced high-grade serous ovarian cancer KLK13 may become proficient for tumor-supporting functions, whereas KLK14 may have adopted tumor-suppressing activity.

Kallikrein-related peptidase 7 overexpression in melanoma cells modulates cell adhesion leading to a malignant phenotype.

We recently reported that human melanoma cells, but not benign melanocytes, aberrantly express kallikrein-related peptidase 7 (KLK7). Here, we show a KLK7 overexpression-mediated decrease of cell adhesion to extracellular matrix binding proteins, associated with downregulation of α5/ß1/αv/ß3 integrin expression. We also report an up-regulation of MCAM/CD146 and an increase in spheroid formation of these cells. Our results demonstrate that aberrant KLK7 expression leads to a switch to a more malignant phenotype suggesting a potential role of KLK7 in melanoma invasion. Thus, KLK7 may represent a biomarker for melanoma progression and may be a potential therapeutic target for melanoma.

Aberrant expression of kallikrein-related peptidase 7 is correlated with human melanoma aggressiveness by stimulating cell migration and invasion.

Members of the tissue kallikrein-related peptidase (KLK) family not only regulate several important physiological functions, but aberrant expression has also been associated with various malignancies. Clinically, KLKs have been suggested as promising biomarkers for diagnosis and prognosis in many types of cancer. As of yet, expression of KLKs and their role in skin cancers are, however, poorly addressed. Malignant melanoma is an aggressive disease associated with poor prognosis. Hence, diagnostic biomarkers to monitor melanoma progression are needed. Herein, we demonstrate that although mRNA of several KLKs are aberrantly expressed in melanoma cell lines, only the KLK7 protein is highly secreted in vitro. In line with these findings, ectopic expression of KLK7 in human melanomas and its absence in benign nevi were demonstrated by immunohistochemistry in vivo. Interestingly, overexpression of KLK7 induced a significant reduction in melanoma cell proliferation and colony formation. Moreover, KLK7 overexpression triggered an increase in cell motility and invasion associated with decreased expression of E-cadherin and an upregulation of MCAM/CD146. Our results demonstrate, for the first time, that aberrant KLK7 expression leads to a switch from proliferative to invasive phenotype, suggesting a potential role of KLK7 in melanoma progression. Thus, we hypothesize that KLK7 may represent a potential biomarker for melanoma progression.

Kallikrein-related peptidase 7 (KLK7) is a proliferative factor that is aberrantly expressed in human colon cancer.

Emerging evidence indicates that serine proteases of the tissue kallikrein-related peptidases family (KLK) are implicated in tumorigenesis. We recently reported the ectopic expression of KLK4 and KLK14 in colonic cancers and their signaling to control cell proliferation. Human tissue kallikrein-related peptidase 7 (KLK7) is often dysregulated in many cancers; however, its role in colon tumorigenesis has not yet been established. In the present study, we analyzed expression of KLK7 in 15 colon cancer cell lines and in 38 human colonic tumors. In many human colon cancer cells, KLK7 mRNA was observed, which leads to KLK7 protein expression and secretion. Furthermore, KLK7 was detected in human colon adenocarcinomas, but it was absent in normal epithelia. KLK7 overexpression in HT29 colon cancer cells upon stable transfection with a KLK7 expression plasmid resulted in increased cell proliferation. Moreover, subcutaneous inoculation of transfected cells into nude mice led to increased tumor growth that was associated with increased tumor cell proliferation as reflected by a positive Ki-67 staining. Our results demonstrate the aberrant expression of KLK7 in colon cancer cells and tissues and its involvement in cell proliferation in vitro and in vivo. Thus, KLK7 may represent a potential therapeutic target for human colon tumorigenesis.

Kallikrein-related peptidase signaling in colon carcinoma cells: targeting proteinase-activated receptors.

We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.

Kallikrein-related peptidase 14 acts on proteinase-activated receptor 2 to induce signaling pathway in colon cancer cells.

Serine proteinases participate in tumor growth and invasion by cleaving and activating proteinase-activated receptors (PARs). Recent studies have implicated PAR-1 and PAR-4 (activated by thrombin) and PAR-2 (activated by trypsin but not by thrombin) in human colon cancer growth. The endogenous activators of PARs in colon tumors, however, are still unknown. We hypothesize that the kallikrein-related peptidase (KLK) family member KLK14, a known tumor biomarker, is produced by colonic tumors and signals to human colon cancer cells by activating PARs. We found that i) KLK14 mRNA was present in 16 human colon cancer cell lines, ii) KLK14 protein was expressed and secreted in colon cancer cell lines, and iii) KLK14 (0.1 µmol/L) induced increases in intracellular calcium in HT29, a human colon cancer-derived cell line. KLK14-induced calcium flux was associated with internalization of KLK14-mediated activation of PAR-2. Furthermore, KLK14 induced significant extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and HT29 cell proliferation, presumably by activating PAR-2. A PAR-2 cleavage and activation-blocking antibody dramatically reduced KLK14-induced ERK1/2 signaling. Finally, ectopic expression of KLK14 in human colon adenocarcinomas and its absence in normal epithelia was demonstrated by IHC analysis. These results demonstrate, for the first time, the aberrant expression of KLK14 in colon cancer and its involvement in PAR-2 receptor signaling. Thus, KLK14 and its receptor, PAR-2, may represent therapeutic targets for colon tumorigenesis.

Kallikrein-related peptidase 4: a new activator of the aberrantly expressed protease-activated receptor 1 in colon cancer cells.

Certain serine proteases are considered to be signaling molecules that act through protease-activated receptors (PARs). Our recent studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. Here we analyzed the expression of KLK4, a member of the kallikrein-related peptidase (KLK) family of serine proteases and explored whether this member can activate PAR1 and PAR2 in human colon cancer cells. Immunohistochemistry showed KLK4 expression in human colon adenocarcinomas and its absence in normal epithelia. KLK4 (1 micromol/L) initiated loss of PAR1 and PAR2 from the HT29 cell surface as well as increased intracellular calcium transients in HT29 cells. This KLK4-induced Ca2+ flux was abrogated after an initial challenge of the cells with TRAP (SFLLR-NH2; 100 micromol/L), which is known to desensitize PAR1 and PAR2. Interestingly, PAR1 blocking antibody, which inhibits cleavage and activation by thrombin, dramatically reduced KLK4-induced Ca2+ influx, but blocking cleavage of PAR2 failed to attenuate the KLK4-induced Ca2+ flux. Consistently, desensitization with AP1 (TFFLR-NH2), targeting PAR1, attenuated most of the Ca2+ flux induced by KLK4. KLK4 also induced a rapid and significant ERK1/2 phosphorylation in HT29 cells. Our results demonstrate, for the first time, that KLK4 is aberrantly expressed in colon cancer and capable of inducing PAR1 signaling in cancer cells. These data suggest that KLK4 signaling via PAR1 may represent a novel pathway in colon tumorigenesis.

Aberrant expression of proteinase-activated receptor 4 promotes colon cancer cell proliferation through a persistent signaling that involves Src and ErbB-2 kinase.

Thrombin is now recognized as an important factor in many cancers. Here, we examined the expression and role of the recently discovered thrombin receptor PAR4, in human colon cancer cells. PAR4 mRNA was found in 10 out of 14 (71%) human colon cancer cell lines tested but not in epithelial cells isolated from normal human colon. This finding is in line with immunostaining results of PAR4 in human colon tumors and its absence in normal human colonic mucosa. Investigation of the functional significance of the aberrant expression of PAR4 in colon cancer cells revealed (i) a prompt increase in intracellular calcium concentration on challenge with PAR4-specific agonist AP4 (100 microM) and (ii) marked mitogenic response (2.5-fold increase in cell number) in a dose-dependent manner on treatment with AP4 (0.1-300 microM). Analysis of the signaling pathways downstream of PAR4 activation in HT29 cells showed (i) a sustained phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and (ii) the involvement of epidermal growth factor receptor B-2 (ErbB-2) but not of epidermal growth factor receptor in PAR4-induced mitogen-activated protein kinase activation. Tyrphostin AG1478, the ErbB inhibitor, reversed the action of AP4 on ERK1/2 and ErbB-2 phosphorylation and HT29 cell growth. Finally, the Src inhibitor PP2 abrogated ErbB-2 and ERK phosphorylation and HT29 cell proliferation, suggesting the essential role of Src activity in PAR4-induced phosphorylation of ErbB-2. These data highlight the role of PAR4 as a new important player in the control of colon tumors and underline the critical role of ErbB-2 transactivation.

PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation.

PAR-2 (protease-activated receptors-2) are G protein-coupled receptors whose action on mucin secretion by intestinal epithelial cells is still unknown. The aim of this study was to examine the effect of PAR-2 activation on mucin secretion in the human colonic goblet cell line HT29-Cl.16E and the intracellular pathways involved. We found that PAR-2 mRNA was constitutively expressed by HT29-Cl.16E cells as well as by isolated human normal colonocytes. The PAR-2-activating peptide SLIGKV-NH(2) elicited rapid mucin secretion in HT29-Cl.16E, which was partially inhibited by calcium chelator BAPTA. Inhibitors of MAPK activation (PD98059) and EGFR tyrosine kinase activity (AG1478) abrogated PAR-2-induced ERK1/2 and EGFR tyrosine phosphorylation, respectively, and subsequent mucin secretion. Finally, PAR-2-induced EGFR transactivation was involved upstream of ERK1/2 activation. Our results show that the activation of PAR-2 expressed by human intestinal epithelial cells enhances mucin secretion, a component of the intestinal innate defence, via a pathway involving EGFR transactivation.

Leptin and Ob-Rb receptor isoform in the human digestive tract during fetal development.

CONTEXT: Leptin, partially produced by the stomach, is a hormone involved in energy balance and regulation of food intake. It also regulates some digestive functions through its functional receptor Ob-Rb expressed by gastrointestinal epithelial cells. OBJECTIVE: The objective of the study was to investigate the temporal and spatial appearance of Ob-Rb in the human digestive tract and leptin in the stomach. DESIGN: The esophagus, stomach, and intestine samples of 7- to 24-wk-old human fetuses and adult mucosae were studied by RT-PCR, immunohistochemistry, and Western blot. Leptin was measured by RIA in amniotic fluids at 16-33 wk gestation. RESULTS: All mucosae expressed Ob-Rb (mRNA and/or protein) between 7 and 9 wk gestation. Leptin protein appeared by 8 wk in the gastric mucosa, whereas leptin mRNA was detected around 11 wk. Leptin levels in amniotic fluids were significantly higher during the second than during the third trimester. Overall, Ob-Rb immunoreactivity was higher in young fetuses, during the period corresponding to the formation of gastric buds and primitive intestinal crypts and the beginning of differentiation of epithelial cell types, than in the oldest. Leptin added to culture medium of gastrointestinal explants from 10- to 12-wk-old fetuses appeared to affect DNA synthesis as compared with controls, indicating that leptin receptor functionality was developing. CONCLUSIONS: The strong expression of leptin, in amniotic fluid when fetuses begin swallowing then in the gastric mucosa, and the early presence of Ob-Rb in mucosae suggest a possible role for leptin, exerted endoluminally and in a paracrine pathway, in the developmental process (growth and/or maturation) of the human digestive tract.

Mast cell tryptase controls paracellular permeability of the intestine. Role of protease-activated receptor 2 and beta-arrestins.

Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves protease-activated receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.

Activation of proteinase-activated receptor 1 promotes human colon cancer cell proliferation through epidermal growth factor receptor transactivation.

Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by the fact that stimulation of cell proliferation and ERK1/2 on activation of PAR1 is reversed by the MMP inhibitor batimastat, TGF-alpha neutralizing antibodies, EGFR ligand binding domain blocking antibodies, and the EGFR tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGFR seems to be a major mechanism whereby activation of PAR1 results in colon cancer cell growth. Finally, PAR1 activation induces Src phosphorylation, which is reversed by using the Src tyrosine kinase inhibitor PP2, suggesting that Src activation plays a permissive role for PAR1-mediated ERK1/2 activation and cell proliferation probably acting downstream of the EGFR. These data explain how thrombin exerts robust trophic action on colon cancer cells and underline the critical role of EGFR transactivation.

Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation.

Several lines of evidence suggest that tumor-derived trypsin contributes to the growth and invasion of cancer cells. We have recently shown that trypsin is a potent growth factor for colon cancer cells through activation of the G protein-coupled receptor protease-activated receptor 2 (PAR2). Here, we analyzed the signaling pathways downstream of PAR2 activation that lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events upon activation of PAR2 by the serine protease trypsin or the specific PAR2-activating peptide (AP2): (i) a matrix metalloproteinase-dependent release of transforming growth factor (TGF)-alpha, as demonstrated with TGF-alpha-blocking antibodies and measurement of TGF-alpha in culture medium; (ii) TGF-alpha-mediated activation of epidermal growth factor receptor (EGF-R) and subsequent EGF-R phosphorylation; and (iii) activation of ERK1/2 and subsequent cell proliferation. The links between these events are demonstrated by the fact that stimulation of cell proliferation and ERK1/2 upon activation of PAR2 is reversed by the metalloproteinase inhibitor batimastat, TGF-alpha-neutralizing antibodies, EGF-R ligand binding domain-blocking antibodies, and the EGF-R tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGF-R appears to be a major mechanism whereby activation of PAR2 results in colon cancer cell growth. By using the Src tyrosine kinase inhibitor PP2, we further showed that Src plays a permissive role for PAR2-mediated ERK1/2 activation and cell proliferation, probably acting downstream of the EGF-R. These data explain how trypsin exerts robust trophic action on colon cancer cells and underline the critical role of EGF-R transactivation.

Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells.

The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.

Trypsin is produced by and activates protease-activated receptor-2 in human cancer colon cells: evidence for new autocrine loop.

In this work, we showed that human colon cancer cell lines produce trypsin which can activate a receptor for trypsin, the protease-activated receptor-2 (PAR-2), in these cells. RT-PCR experiments showed that trypsinogen transcripts were present in four colon cancer cell lines: T84, Caco-2, HT-29 and C1.19A. By Western blot analysis we found a 25 kDa immunoreactive band identified as trypsinogen I in cell lysates and in the corresponding culture media. Concentrations of trypsin in cell media were found in nanomolar range, thus compatible with activation of protease-activated receptor 2 (PAR-2). This was further demonstrated in a colon cancer cell line (H-29) Ca2+i assay since increases in Ca2+i were observed in response to media from T84, Caco-2 or C1.19A cells that were similar to that observed with 2-5 nM trypsin and were abolished by trypsin inhibitor. Altogether, these data show that colon cancer cell lines produce and secrete trypsin at concentrations compatible with activation of PAR-2. They support possible autocrine/paracrine regulation of PAR-2 activity by trypsin in colon cancer cells.

Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel.

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.