The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Taxonomic hypotheses regarding the genus Gerbillus (Rodentia,Muridae, Gerbillinae) based on molecular analyses of museum specimens

Methodological improvements now allow routine analyses of highly degraded DNA samples as found in museum specimens. Using these methods could be useful in studying such groups as rodents of the genus Gerbillus for which i) the taxonomy is still highly debated, ii) collection of fresh specimens may prove difficult. Here we address precise taxonomic questions using a small portion of the cytochrome b gene obtained from 45 dry skin/skull museum samples (from 1913 to 1974) originating from two African and three Asian countries. The specimens were labelled Gerbillus gerbillus, Gerbillus andersoni, Gerbillus nanus, Gerbillus amoenus, Gerbillus perpallidus and Gerbillus pyramidum, and molecular results mostly confirmed these assignations. The close relationship between Gerbillus nanus (Asian origin) and Gerbillus amoenus (African origin) confirmed that they represent vicariant sibling species which differentiated in allopatry on either side of the Red Sea. In the closely related Gerbillus perpallidus and Gerbillus pyramidum, specimens considered as belonging to one Gerbillus pyramidum subspecies (Gerbillus pyramidum floweri) appeared closer to Gerbillus perpallidus suggesting that they (Gerbillus pyramidum floweri and Gerbillus perpallidus) may represent a unique species, distributed on both sides of the Nile River, for which the correct name should be Gerbillus floweri. Furthermore, the three other Gerbillus pyramidum subspecies grouped together with no apparent genetic structure suggesting that they may not yet represent genetically differentiated lineages. This study confirms the importance of using these methods on museum samples, which can open new perspectives in this particular group as well as in other groups of interest.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.     

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.