TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

Background

Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.

Methods

TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.

Results

The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.

Conclusions

TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.     

A taxonomy of organ-specific breast cancer metastases based on a protein-protein interaction network

We carried out a systems-level study of the mechanisms underlying organ-specific metastases of breast cancer. We followed a network-based approach using microarray expression data from human breast cancer metastases to select organ-specific proteins that exert a range of functions allowing cell survival and growth in the microenvironment of distant organs. MinerProt, a home-made software application, was used to group organ-specific signatures of brain (1191 genes), bone (1623 genes), liver (977 genes) and lung (254 genes) metastases by function and select the most differentially expressed gene in each function. As a result, we obtained 19 functional representative proteins in brain, 23 in bone, 15 in liver and 9 in lung, with which we constructed four organ-specific protein-protein interaction networks. The network taxonomy included seven proteins that interacted in brain metastasis, which were mainly associated with signal transduction. Proteins related to immune response functions were bone specific, while those involved in proteolysis, signal transduction and hepatic glucose metabolism were found in liver metastasis. No experimental protein-protein interaction was found in lung metastasis; thus, computationally determined interactions were included in this network. Moreover, three of these selected genes (CXCL12, DSC2 and TFDP2) were associated with progression to specific organs when tested in an independent dataset. In conclusion, we present a network-based approach to filter information by selecting key protein functions as metastatic markers or therapeutic targets.     

Functional clustering of metastasis proteins describes plastic adaptation resources of breast-cancer cells to new microenvironments

To examine the molecular mechanisms underlying breast cancer metastasis in liver and search for potential markers of metastatic progression in soft-tissue, we analyzed metastatic variants developed from the highly metastatic MDA-MB 435 cell line through in vivo stepwise selection in the athymic mice. Comparative proteomic analysis using two-dimensional electrophoresis (2DE-DIGE) revealed that 74 protein spots were reproducibly more than doubled in liver metastatic cells compared to parental counterpart. From 22 proteins identified by MALDI-TOF, belonging to intermediate filaments, intracellular transport and ATP synthesis, we generated a protein-protein interaction network containing 496 nodes, 12 of which interacted. GRP 75 was connected with four other proteins: prohibitin, HSP 27, elongin B and macropain delta chain. After functional classification, we found that pathways including hepatocyte growth factor receptor (p = 0.014), platelet-derived growth factor (p = 0.018), vascular endothelial growth factor (p = 0.021) and epidermal growth factor (p = 0.050) were predominant in liver metastatic cells, but not in lung metastatic cells. In conclusion, we suggest that GRP 75 is involved in cell proliferation, tumorigenesis and stress response in metastatic cells by recruiting signals in which the transmembrane receptor protein tyrosine kinase signaling pathway (p-value FDR = 1.71 x 10(-2)) and protein amino acid phosphorylation (p-value FDR = 3.28 x 10(-2)) might be the most significant biological process differentially increased in liver metastasis.     

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.     

Transforming Growth Factor β1Induces Squamous Carcinoma Cell Variants with Increased Metastatic Abilities and a Disorganized Cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial–fibroblastic conversion when cultured in the presence of TGF-β₁. This conversion is associatedin vivowith a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-β₁. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell–cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell–cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.     

Cells previously desensitized to type 1 interferons display different mechanisms of activation of stat-dependent gene expression from naïve cells

Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferon-activated gene expression in na?ve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In na?ve cells, the ISG54 gene is activated via IFN beta-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN beta weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in na?ve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in na?ve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.     

Foot infections associated with Arcanobacterium pyogenes in free-living fallow deer (Dama dama)

We describe foot infection associated with Arcanobacterium pyogenes in three adult male free-living fallow deer (Dama dama) from Sueve Regional Hunting Reserve (Principality of Asturias, Spain). Affected fallow deer were culled in November 1997 and 1998 during the hunting season. Necropsy, radiography, and microbiologic analysis were carried out for each animal. Unilateral swelling of one extremity at the coronary band was observed in all three cases. Areas of bone loss, severe periosteal reaction, and soft tissue swelling were seen on radiography. Lead fragments were observed in one fallow deer. Seven bacterial species were isolated, but only Arcanobacterium pyogenes was routinely found. Weather conditions in the area (mild temperatures and high humidity), the land (alternating pasture land and rock), the animal population density (both fallow deer and domestic herds of cows, horses, sheep, and goats, live side by side in the same areas), and hunting activities could be related to the frequency of these infections.     

Influence of maternal stress on metal-induced pre- and postnatal effects in mammals: a review

Studies in rodents have shown that, during pregnancy, maternal stress from restraint, noise, light, and heat among other factors may be associated with adverse effects on embryo/fetal and postnatal development. Moreover, it is also well known that exposure to certain metal levels during gestation can also cause maternal and developmental toxicity. Because potentially, pregnant women may be concurrently exposed to metals and various types of stress, the influence of maternal stress on the metal-induced adverse pre- and postnatal effects has been investigated for a number of elements. This influence is reviewed here. It is concluded that maternal stress enhances the metal-induced embryo/fetal and developmental toxicity only at doses of the metal which are also clearly toxic to the dam.