Francisco Portillo

The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.

The Snail family of transcription factors has previously been implicated in the differentiation of epithelial cells into mesenchymal cells (epithelial–mesenchymal transitions) during embryonic development. Epithelial–mesenchymal transitions are also determinants of the progression of carcinomas, occurring concomitantly with the cellular acquisition of migratory properties following downregulation of expression of the adhesion protein E-cadherin. Here we show that mouse Snail is a strong repressor of transcription of the E-cadherin gene. Epithelial cells that ectopically express Snail adopt a fibroblastoid phenotype and acquire tumorigenic and invasive properties. Endogenous Snail protein is present in invasive mouse and human carcinoma cell lines and tumours in which E-cadherin expression has been lost. Therefore, the same molecules are used to trigger epithelial–mesenchymal transitions during embryonic development and in tumour progression. Snail may thus be considered as a marker for malignancy, opening up new avenues for the design of specific anti-invasive drugs.

Transcriptional regulation of cell polarity in EMT and cancer

The epithelial-to-mesenchymal transition (EMT) is a crucial process in tumour progression providing tumour cells with the ability to escape from the primary tumour, to migrate to distant regions and to invade tissues. EMT requires a loss of cell–cell adhesion and apical–basal polarity, as well as the acquisition of a fibroblastoid motile phenotype. Several transcription factors have emerged in recent years that induce EMT, with important implications for tumour progression. However, their effects on cell polarity remain unclear. Here, we have re-examined the data available related to the effect of EMT related transcription factors on epithelial cell plasticity, focusing on their impact on cell polarity. Transcriptional and post-transcriptional regulatory mechanisms mediated by several inducers of EMT, in particular the ZEB and Snail factors, downregulate the expression and/or functional organization of core polarity complexes. We also summarize data on the expression of cell polarity genes in human tumours and analyse genetic interactions that highlight the existence of complex regulatory networks converging on the regulation of cell polarity by EMT inducers in human breast carcinomas. These recent observations provide new insights into the relationship between alterations in cell polarity components and EMT in cancer, opening new avenues for their potential use as therapeutic targets to prevent tumour progression.

A molecular role for lysyl oxidase‐like 2 enzyme in Snail regulation and tumor progression

The transcription factor Snail controls epithelial–mesenchymal transitions (EMT) by repressing E‐cadherin expression and other epithelial genes. However, the mechanisms involved in the regulation of Snail function are not fully understood. Here we show that lysyl‐oxidase‐like 2 and 3 (LOXL2 and LOXL3), two members of the lysyl‐oxidase gene family, interact and cooperate with Snail to downregulate E‐cadherin expression. Snails lysine residues 98 and 137 are essential for Snail stability, functional cooperation with LOXL2/3 and induction of EMT. Overexpression of LOXL2 or LOXL3 in epithelial cells induces an EMT process, supporting their implication in tumor progression. The biological importance of LOXL2 is further supported by RNA interference of LOXL2 in Snail‐expressing metastatic carcinoma cells, which led to a strong decrease of tumor growth associated to increased apoptosis and reduced expression of mesenchymal and invasive/angiogenic markers. Taken together, these results establish a direct link between LOXL2 and Snail in carcinoma progression.

Genetic Profiling of Epithelial Cells Expressing E-Cadherin Repressors Reveals a Distinct Role for Snail, Slug, and E47 Factors in Epithelial-Mesenchymal Transition

The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of Madin-Darby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion.

Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells

Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (Igp)‐containing vacuoles. Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable Igp‐containing filamentous structures that connect with the Salmonella‐containing vacuoles. Salmonella typhimurium SL1344::Tn 10dCm mutant strains unable to induce these structures were isolated. All contained insertions within a novel Salmonella induced filament gene A (sifA). sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38kDa and an apparent molecular mass of 35kDa. sifA is flanked by ∼300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases. sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines. Fourteen‐base‐pair direct repeats mark the probable site of integration of sifA and its flanking DNA at the 3 end of potB. sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52–54%). Deletion mutant strains

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation.

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis‐(3′,5′) cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient‐deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB‐G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di‐GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.

Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence.

During infection of their hosts, Gram‐positive bac‐teria express surface proteins that serve multiple biological functions. Surface proteins harbouring a C‐terminal sorting signal with an LPXTG motif are covalently linked to the cell wall peptidoglycan by a transamidase named sortase. Two genes encoding putative sortases, termed srtA and srtB, were identified in the genome of the intracellular pathogenic bacterium Listeria monocytogenes. Inactivation of srtA abolishes anchoring of the invasion protein InlA to the bacterial surface. It also prevents the proper sorting of several other peptidoglycan‐associated LPXTG proteins. Three were identified by a mass spectrometry approach. The ΔsrtA mutant strain is defective in entering epithelial cells, similar to a ΔinlA mutant. In contrast to a ΔinlA mutant, the ΔsrtA mutant is impaired for colonization of the liver and spleen after oral inoculation in mice. Thus, L. monocytogenes srtA is required for the cell wall anchoring of InlA and, presumably, for the anchoring of other LPXTG‐containing proteins that are involved in listerial infections.

Regulation of plasma membrane H+-ATPase in fungi and plants

The plasma membrane H+-ATPase from fungi and plants is a proton pump which plays a key role in the physiology of these organisms controlling essential functions such as nutrient uptake and intracellular pH regulation. In fungal and plant cells the activity of the proton pump is regulated by a large number of environmental factors at both transcriptional and post-translational levels. During the last years the powerful tools of molecular biology have been successfully used in fungi and plants allowing the cloning of a wide diversity of H+-ATPase genes and rapid progress on the molecular basis of reaction mechanism and regulation of the proton pump. This review focuses on recent results on regulation of plasma membrane H+-ATPase obtained by molecular approaches.

Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters.

ABSTRACT The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1(YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H+-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein

By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip–Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E‐cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection.