Enhanced venous thrombus resolution in plasminogen activator inhibitor type-2 deficient mice.

BACKGROUND: The resolution of deep vein thrombosis requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. OBJECTIVE: To investigate the role of PAI-2 in venous thrombus formation and resolution. METHODS: Venous thrombus resolution was compared in wild-type C57BL/6, PAI-2(-/-) , and PAI-1(-/-) mice using the stasis model of deep vein thrombosis. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. RESULTS: We found that the absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2(-/-) mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2-deficient thrombi had increased levels of the neutrophil chemoattractant CXCL2, which was associated with early enhanced neutrophil recruitment. CONCLUSIONS: These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution.

Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo.

Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.

Hypermethylation of the 5' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis.

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 5' CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.

Characterisation of PAUSE-1, a powerful silencer in the human plasminogen activator inhibitor type 2 gene promoter.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. The PAI-2 promoter tightly regulates PAI-2 gene expression in a cell-specific manner and this control is mediated, in part, by the upstream silencer element, PAUSE-1. Here we have defined PAUSE-1 and investigated its activity as a silencer. A series of mutations were generated within the PAUSE-1 element and analysed for transcription factor binding and transcriptional silencing activity. These studies have defined the minimal functional PAUSE-1 element as TCTN(x)AGAN(3)T(4), where x = 0, 2 or 4. Examination of related elements present in other promoters, such as the human IFNbeta promoter, suggests that PAUSE-1 is a member of a family of universal silencers with the consensus sequence TCTN(x)AGA. UV crosslinking analyses determined that the PAUSE-1 binding protein was approximately 67 kDa. Insertion of PAUSE-1 into the heterologous (SV40) or the minimal PAI-2 promoters silenced transcription by 2.5-fold. These data show that PAUSE-1 acts as a powerful silencer of PAI-2 gene transcription and is likely to be important in the silencing of other genes as well.

An orthotopic xenograft model of human nonseminomatous germ cell tumour.

We have established the first example of an orthotopic xenograft model of human nonseminomatous germ cell tumour (NSGCT). This reproducible model exhibits many clinically relevant features including metastases to the retroperitoneal lymph nodes and lungs, making it an ideal tool for research into the development and progression of testicular germ cell tumours.

Human trypsinogen in colorectal cancer.

Trypsinogen (TRY), the precursor to the serine protease trypsin, is found in the pancreas and mediates digestive proteolysis in the small intestine. Differential display of cDNAs expressed by human colorectal tumor tissues compared with adjacent normal colonic mucosa identified an isoform of TRY (TRY2) up-regulated in colorectal cancers. Northern blot analysis of RNA isolated from a series of 28 malignant colon tumors and corresponding normal mucosa showed that TRY transcripts were up-regulated 2- to 33-fold in 29% of tumors. Further, TRY mRNA was expressed in 6 colorectal cancer cell lines, with highest levels detected in the metastatic tumor lines SW620 and HT29. Immunostaining for TRY protein expression showed intense immunoreactivity in the supranuclear cytoplasm of colon tumors in 16% of tissue specimens. To evaluate the relative contributions of 2 isoforms of TRY, TRY1 and TRY2, to total TRY mRNA expression, a semi-quantitative multiplex RT-PCR assay was developed. TRY2 mRNA was detected in all 6 colorectal tumor cell lines, whereas TRY1 mRNA was expressed only in the metastatic tumor lines, showing that the high levels of TRY expression in the metastatic tumor lines are likely due to up-regulation of TRY1. Evaluation of TRY1 and TRY2 mRNA expression by multiplex RT-PCR in a series of 20 colon tumor tissues representative of the range of tumor progression showed that TRY2 mRNA was expressed much more commonly than TRY1 mRNA in normal mucosa (26% vs. 6%) as well as in primary tumor tissues (65% vs. 15%). These data demonstrate that TRY2 is the dominant TRY in colon tissue and suggest that up-regulation of TRY1 expression in colon tumors may be associated with a metastatic phenotype.

Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis.

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans approximately 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at approximately day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.

Localization of the mosaic transmembrane serine protease corin to heart myocytes.

Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125-135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Cloning of the cDNA and localization of the gene encoding human NRBP, a ubiquitously expressed, multidomain putative adapter protein.

Adapter proteins modulate multiple signaling pathways by regulating the aggregation of other factors into signaling complexes. Here we have identified a novel human cDNA encoding NRBP, a multidomain putative adapter protein containing (i) two putative nuclear receptor binding motifs (LXXLL), (ii) a putative binding domain for Src homology-2 (SH2) domain containing proteins, (iii) a kinase-like domain, (iv) a bipartite nuclear localization signal, and (v) three sequences rich in glutamic acid, serine, proline, and threonine (PEST) residues. The NRBP mRNA transcript, of approximately 2.4 kb, was ubiquitously expressed in a wide range of normal human tissues and 15 human tumor cell lines. The NRBP cDNA is predicted to encode a polypeptide of 535 amino acids with a molecular mass of 59.8 kDa. Translation of NRBP mRNA in vitro reveals three translation products of 60, 51, and 43 kDa, suggesting that translation of NRBP may initiate at multiple sites. The NRBP gene was localized to human chromosome 2p23, near the location of the NCOA1 gene encoding the nuclear receptor coactivator, steroid receptor coactivator-1 (SRC-1). The features of NRBP predict a function as an adapter protein potentially linking signaling pathways involving nuclear receptors and SH2 domain containing proteins.

Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif.

Induction of human plasminogen activator inhibitor type-2 (PAI-2) gene transcription is the response of macrophages to inflammatory stimuli, such as the pleiotropic cytokine, tumour necrosis factor-alpha (TNFalpha). Here we have examined whether PAI-2 gene transcription in response to TNFalpha may be mediated through a regulatory pathway involving the transcription factor, NF-kappaB. We have tested the function of two potential NF-kappaB-like sites present in the PAI-2 proximal promoter for responsiveness to TNFalpha using chloramphenicol acetyl transferase reporter gene deletion and mutation analyses. While no evidence was found for TNFalpha regulation of the PAI-2 gene through either of these two sites, one of the NF-kappaB-like motifs, transcriptional regulatory motif (TRM), present at position -400 was found to be essential for constitutive PAI-2 transcription, as mutation of this motif abolished basal PAI-2 promoter activity in both monocyte-like U937 cells and HT1080 fibrosarcoma cells. Competition electrophoretic mobility shift assays identified four TRM-binding proteins present in U937, HT1080 and HeLa cell extracts, which bound to this motif but were not components of the NF-kappaB regulatory complex. Expression screening of a HeLa cell cDNA library using the -400 TRM as a probe identified two cDNAs encoding partial peptides which specifically bound the TRM motif. DNA sequence analysis revealed that one cDNA was novel, and the second cDNA encoded exon 5 of the nephroblastoma overexpressed (novH) proto-oncogene, suggesting a new role for this peptide in gene regulation. Taken together, these findings identify a new regulatory element required for constitutive PAI-2 transcription, and identify potential DNA-binding proteins associated with this element that may play a role in PAI-2 gene regulation.

Two novel mucin genes down-regulated in colorectal cancer identified by differential display.

Epithelial mucins are large, secreted and cell surface glycoproteins involved in epithelial cell protection, adhesion modulation, and signaling. Using differential display, we have identified two novel mucin cDNAs (dd34 and dd29), hereafter designated MUC11 and MUC12, respectively, that are down-regulated in colorectal cancers. Northern blots demonstrated polydisperse signals characteristic of mucin transcripts in RNA from normal colon that were absent in colorectal cancer. Both cDNAs were mapped by fluorescence in situ hybridization to chromosome band 7q22, the location of the MUC3 mucin gene, thus suggesting that there may be a cluster of mucin genes at this locus. The sequences of both differential display clones were extended by a combination of screening libraries and PCR. The 2.8-kb MUC11 cDNA composite encoded 35 serine/threonine-rich, mucin-like degenerate 28 amino acid tandem repeats. The MUC12 cDNA composite encoded a putative transmembrane mucin containing two extracellular cysteine-rich, EGF-like domains, a coiled-coil region, and a mucin-like domain consisting of 28 amino acid degenerate tandem repeats. Distinct patterns of expression of MUC11, MUC12, and MUC3 mRNAs were observed in a range of normal human tissues. MUC12 mRNA was not expressed in any of six colorectal cancer cell lines examined and was down-regulated or absent in 6 of 15 (40%) tumors compared with matched normal colonic tissue. In contrast, MUC11 showed a different pattern of mRNA expression, with four of these lines showing low levels and the other two lines showing relatively high levels of MUC11 transcripts. Expression of MUC11 was down-regulated in the tumors of 12 of 15 (80%) paired samples. Structural homology of MUC12 with rat, mouse, and human MUC3 and human and rat MUC4/ASGP2 indicate that there is a distinct subfamily of transmembrane mucins with conserved epidermal growth factor domains. The homology of MUC12 with epidermal growth factor-like growth factors and its down-regulation in colorectal cancers, together with known interactions between rat MUC4 and c-erbB-2 growth factor receptors, suggests that MUC12 may be involved in epithelial cell growth regulation.

Picornavirus receptor down-regulation by plasminogen activator inhibitor type 2.

Therapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy. Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple human picornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulation of the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the major group of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses), and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackieviruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactor protein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or related serpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeutic interventions against ICAM-1-mediated respiratory inflammation.

Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin.

We investigate the regulation of plasminogen activator inhibitor-2 (PAI-2) in murine macrophages. PAI-2 mRNA was inducible by bacterial lipopolysaccharide (LPS) in primary cells and macrophage-like cell lines. Evidence is presented for a role for autocrine factors, including cyclooxygenase products but not the cytokines tumor necrosis factor alpha or interferon-beta (IFN-beta). PAI-2 mRNA levels generally varied inversely from those of its target, urokinase-type plasminogen activator (uPA), and the macrophage growth factor CSF-1, which induces uPA, inhibited PAI-2 expression in cells treated subsequently with LPS. Expression of PAI-2 was distinct from that of other LPS-inducible genes in terms of induction time course, LPS dose response, and sensitivity to co-stimulation with IFN-gamma. Induction of PAI-2 mRNA in subclones of the cell line RAW 264 was not uniform, reflecting heterogeneous expression in the parent line. The expression pattern of PAI-2 is discussed in terms of a possible role in LPS-induced pathology such as septicemia.

Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.

The alpha v beta 6 integrin induces gelatinase B secretion in colon cancer cells.

In human cancers, the co-operative role between cell-adhesion receptors and proteases capable of degrading matrix barriers remains poorly understood. We have previously reported that the epithelium-restricted integrin alpha(v)beta6 becomes highly expressed in colon cancer compared with normal mucosa and that heterologous expression of alpha(v)beta6 in colon cancer cells is associated with enhanced cell growth. Herein, we report that alpha(v)beta6 expression in colon cancer cells leads to a relative increase in secretion of the matrix metalloproteinase gelatinase B over its respective inhibitor and that this secretion parallels the level of cell-surface beta6 expression. The alpha(v)beta6-mediated gelatinase B secretion is associated with increased proteolysis of denatured collagen at the cell surface, and inactivation of gelatinase B in beta6-expressing tumour cells inhibits cell spreading and proliferation within 3-dimensional collagen matrices. Our findings suggest that alpha(v)beta6-mediated gelatinase B secretion is important in the progression of human colon cancer.

Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro.

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell-cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/beta2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/beta2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1beta reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.