RESUMO
The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of nuclear factor (NF)κB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Receptor PAR-2/metabolismo , Serina Endopeptidases/metabolismo , Proteínas ras/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Progressão da Doença , Células Epiteliais/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Receptor PAR-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas ras/genéticaRESUMO
Corticosteroid binding globulin, a member of the serpin family, was previously shown to be secreted mainly apically from MDCK cells in an N-glycan independent manner [Larsen et al. (1999) FEBS Lett. 451, 19-22]. Apart from N-glycosylation, serpins are not known to carry any other posttranslational modifications, suggesting the presence of a proteinaceous apical sorting signal. In the present study we have expressed four other members of the serpin family: alpha1-antitrypsin, C1 inhibitor, plasminogen activator inhibitor-1 and antithrombin in MDCK cells. Tight monolayers of transfected cells were grown on filters and the amounts of recombinantly expressed serpins in the apical and the basolateral media were determined. alpha1-Antitrypsin and C1 inhibitor were found mainly in the apical medium whereas plasminogen activator inhibitor-1 and antithrombin were found in roughly equal amounts in the apical and basolateral media. Control experiments showed that all four serpins are transported along the exocytotic pathway in an uncomplicated way that does not involve transcytosis or differences in stability on the two sides of the cells. We conclude that some members of the serpin family including corticosteroid binding globulin, alpha1-antitrypsin and C1 inhibitor are secreted mainly apically from MDCK cells whereas plasminogen activator inhibitor-1 and antithrombin are secreted in a non-polarized manner.
Assuntos
Polaridade Celular/fisiologia , Inibidores de Serina Proteinase/metabolismo , Serpinas/metabolismo , Sequência de Aminoácidos , Antitrombinas/química , Antitrombinas/metabolismo , Linhagem Celular , Proteínas Inativadoras do Complemento 1/química , Proteínas Inativadoras do Complemento 1/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Serpinas/química , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismoRESUMO
Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.
Assuntos
Transdução de Sinais , Sequência de Aminoácidos , Animais , Receptor de Asialoglicoproteína , Antígenos CD13/química , Antígenos CD13/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , DNA Complementar , Cães , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/química , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
It has been suggested that N-glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96-98]. Human corticosteroid binding globulin contains six consensus sites for N-glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild-type corticosteroid binding globulin is N-glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N-glycosylation sites, and a mutant lacking all six N-glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N-glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells.
Assuntos
Polissacarídeos/metabolismo , Transcortina/metabolismo , Animais , Linhagem Celular , Cães , Glicosilação , Humanos , CoelhosRESUMO
Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.
Assuntos
Gastroenterite Suína Transmissível/etiologia , Macrolídeos , Vírus da Gastroenterite Transmissível/fisiologia , Vírus da Gastroenterite Transmissível/patogenicidade , Cloreto de Amônio/farmacologia , Animais , Antibacterianos/farmacologia , Antígenos CD13/genética , Antígenos CD13/metabolismo , Compartimento Celular , Linhagem Celular , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Cães , Endocitose , Inibidores Enzimáticos/farmacologia , Gastroenterite Suína Transmissível/patologia , Gastroenterite Suína Transmissível/virologia , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Fusão de Membrana , Microscopia Eletrônica , Inibidores da Bomba de Prótons , Receptores de Superfície Celular/fisiologia , Suínos , Vírus da Gastroenterite Transmissível/ultraestrutura , Replicação Viral/efeitos dos fármacosRESUMO
To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.
Assuntos
Cistatinas/metabolismo , Organelas/metabolismo , Animais , Brefeldina A , Células CHO , Células Cultivadas , Células Clonais , Cricetinae , Cicloeximida/farmacologia , Ciclopentanos/farmacologia , Cistatina C , Cistatinas/biossíntese , Cistatinas/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Dimerização , Retículo Endoplasmático/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Lisossomos/metabolismo , Organelas/efeitos dos fármacos , Papaína/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Pele/citologia , Pele/metabolismo , TransfecçãoRESUMO
In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.
Assuntos
Antígenos CD13/metabolismo , Membrana Celular/enzimologia , Animais , Transporte Biológico , Antígenos CD13/química , Antígenos CD13/genética , Linhagem Celular , DNA Complementar/genética , Glicosilação , Humanos , Polissacarídeos/análise , Suínos , TransfecçãoAssuntos
Antígenos CD13/genética , Éxons , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD13/biossíntese , Humanos , Mucosa Intestinal/enzimologia , Íntrons , Rim/enzimologia , Microvilosidades/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Suínos , Transcrição GênicaRESUMO
In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N was observed. This indicates that transcytosis of aminopeptidase N from the basolateral to the apical membrane does not occur by default transport but involves an active sorting mechanism.
Assuntos
Antígenos CD13/metabolismo , Células CACO-2/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos CD13/biossíntese , Membrana Celular/enzimologia , Complexo de Golgi/enzimologia , Humanos , Cinética , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Suínos , TransfecçãoRESUMO
A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.
Assuntos
Endométrio/metabolismo , Rim/metabolismo , Uteroglobina/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , DNA , Cães , Endométrio/química , Endométrio/citologia , Feminino , Rim/citologia , Coelhos , Uteroglobina/genéticaRESUMO
Human aminopeptidase N carries an apical sorting signal on its ectodomain necessary for its correct transport to the apical membrane in Madin-Darby canine kidney cells. To determine whether the apical sorting signal is localized in the serine/threonine rich stalk or in the catalytic head group, anchor/stalk-minus aminopeptidase N, consisting of the hemagglutinin signal peptide and the catalytic head group of human aminopeptidase N, was expressed in MDCK cells. Anchor/stalk-minus aminopeptidase N was secreted mainly to the apical side. The catalytic head group of human aminopeptidase N thus carries an apical sorting signal.
Assuntos
Aminopeptidases/metabolismo , Sequência de Aminoácidos , Aminopeptidases/genética , Animais , Western Blotting , Antígenos CD13 , Catálise , Células Cultivadas , DNA , Cães , Humanos , Dados de Sequência Molecular , Testes de PrecipitinaRESUMO
Coronaviruses, like many animal viruses, are characterized by a restricted host range and tissue tropism. Transmissible gastroenteritis virus (TGEV), a major pathogen causing a fatal diarrhoea in newborn pig, replicates selectively in the differentiated enterocytes covering the villi of the small intestine. To investigate the molecular determinants of the infection, we characterized the surface molecule used by the virus for binding and entry into host cells. Here we report that aminopeptidase N, an ectoenzyme abundantly expressed at the apical membrane of the enterocytes, serves as a receptor for TGEV. Monoclonal antibodies were selected for their ability to block infection by TGEV of porcine cell lines. They recognized a brush-border membrane protein of M(r) 150K, which was identified as aminopeptidase N by amino-terminal sequencing. Two lines of evidence supported the view that the peptidase itself acts as a receptor. First, virions bound specifically to aminopeptidase N that was purified to homogeneity. Second, recombinant expression of aminopeptidase N conferred infectivity by TGEV to an otherwise non-permissive cell line.
Assuntos
Aminopeptidases/fisiologia , Intestinos/enzimologia , Receptores Virais/fisiologia , Vírus da Gastroenterite Transmissível/fisiologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Animais , Anticorpos Monoclonais , Western Blotting , Antígenos CD13 , Linhagem Celular , Clonagem Molecular , DNA/genética , Técnicas de Imunoadsorção , Intestinos/microbiologia , Dados de Sequência Molecular , Receptores Virais/química , Proteínas Recombinantes/fisiologia , SuínosRESUMO
In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.