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Inflammatory bowel diseases are characterized by chronic, progressive and destructive inflammation of the gastrointestinal tract. The two main forms of inflammatory bowel diseases (IBD), Crohn s disease and Ulcerative Colitis, currently affect over 1 million Americans including military personnel, and the incidence among aging veterans is rising. Compromised intestinal barrier function underlies much of the pathology associated with many inflammatory bowel diseases. Matriptase is a membrane-anchored serine protease encoded by the Suppression of Tumorigenicity-14 (ST14) gene that is required for epithelial barrier homeostasis. The project uses the St14 hypomorphic mouse model of matriptase deficiency to 1) determine molecular mechanisms that mediate matriptase protection during DSS-induced experimental inflammatory colitis, 2) define molecular mechanisms by which matriptase becomes decreased during inflammatory colitis, and 3) investigate the importance of matriptase to cytokine induced barrier loss using an in vitro model of intestinal barrier repair. Our data demonstrate that the matriptase barrier forming pathway is down-regulated at the transcriptional and protein levels by cytokines produced during inflammatory colitis. Further, matriptase acts downstream of prostasin to mediate barrier formation and both of these proteases are coordinately regulated. The loss of this pathway is likely to facilitate intestinal barrier disruption in human IBD., The original document contains color images.

Inflammatory bowel diseases are characterized by chronic, progressive and destructive inflammation of the gastrointestinal tract. The two main forms of inflammatory bowel diseases, Crohn s disease and Ulcerative Colitis, currently affect over 1 million Americans including military personnel, and the incidence among aging veterans is rising. Compromised intestinal barrier function underlies much of the pathology associated with many inflammatory bowel diseases. Matriptase is a membrane-anchored serine protease encoded by the Suppression of Tumorigenicity-14 (ST14) gene that is required for epithelial barrier homeostasis. Here, we are investigating matriptase dysregulation and its contribution to the pathogenesis of acute colitis using the St14 hypomorphic mouse model of matriptase deficiency. The project uses a mouse model that is genetically deficient in matriptase and an experimental model of inflammatory colitis, to determine molecular processes by which matriptase protects barrier function in inflamed mucosa, and to define the mechanisms by which matriptase becomes decreased during inflammation associated with inflammatory bowel diseases. In the first year of this grant, our findings support a critical protective role for matriptase in restoring barrier function to injured intestinal mucosa, such that its down-regulation by IL-13 likely enhances excessive activation of the immune system., The original document contains color images.

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 5P-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.

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.

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.