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The epithelial-derived type II transmembrane serine protease epithin has been shown to be upregulated in a variety of cancer cell lines and tumor tissues, and its upregulation correlates well with tumor progression in many cases. However, little is known regarding the regulation of its expression and the mechanism of its roles in tumor progression. Here, we show that transforming growth factor-beta (TGF-beta), a potent inducer of epithelial-mesenchymal transition (EMT) in tumor progression, upregulates epithin, and that epithin plays a critical role in TGF-beta-induced EMT. Forced overexpression of epithin induced EMT to exhibit characteristic morphological changes, alternations in EMT-related proteins and enhanced cell motility. Conversely, shRNA-mediated knockdown of endogenous epithin inhibited TGF-beta-induced expression of mesenchymal markers and morphological changes. Furthermore, TGF-beta-induced cell migration and invasion were significantly impaired by epithin knockdown. In addition, we demonstrate that TGF-beta upregulates epithin transcriptionally via the Smad2/Smad4-mediated pathway. These results suggest that epithin is a key mediator of TGF-beta-induced EMT in tumor progression.

Epithin, a mouse type II transmembrane serine protease, is processed at Gly149 and released from the membrane. Here, we report the identification of an epithin isoform, epithin(Delta), containing a 66 amino acid deletion from the full-length epithin, which is missing the 4th LDLRA domain and the protease activation sequence. This truncated isoform showed the same characteristic N-terminal processing at Gly149 as the full-length form, however, no protease activity was detected. The N-terminal processed epithin(Delta) short form (Epi(Delta)-S) was not released into the medium under conditions in which the processed epithin short form (Epi-S) is released. This type of epithin shedding was also prevented when serine protease inhibitors were added to cells expressing the full-length form. These results strongly suggest that the serine protease activity is involved in the shedding process. The presence of epithin(Delta) message was detected in multiple tissues and its significance is discussed.

We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/Ly110 expressing subpopulation can be identified among the CDR1 + or 6C3 + cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1 + 6C3 + Ly110 + , CDR1 + 6C3 − / low Ly110 + , CDR1 + Ly110 − . Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1 + or 6C3 + epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17 164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon α inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.

A three-stranded DNA that is a putative intermediate of homologous recombination is a novel DNA triplex, R -form DNA. In R - form DNA the third strand includes both purines and pyrimidines and is parallel to the identical strand of the duplex. To text and refine our previously proposed R -form base triplets we have used two approaches: (1) dimethyl sulfate protection of R -form DNA; and (2) thermal dissociation of R -form DNAs in which the duplex strands were substituted in a strand-specific manner with either 7-deaza-guanine or 7-deaza-adenine. Together, the footprinting and isosteric substitution results demonstrate that the third strand in R -form DNA is in contact with the purines in the N 7 position in the major groove of the Watson–Crick duplex in three ((GC):G, (AT):A and (TA):T out of the four possile triplets. Furthermore, these reults suggest that the N 7 positions of the duplex play a significant role in stabilizing the DNA–DNA contacts during the homology recognition process.