Activated alpha(2)-macroglobulin induces cell proliferation and mitogen-activated protein kinase activation by LRP-1 in the J774 macrophage-derived cell line

The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor of activated forms of the proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*). It has been proposed that alpha(2)M* and LRP-1 modulate diverse cellular processes, including cell adhesion, proliferation, and migration, which are involved in inflammation and tumor progression. However, relatively little is known about the role of alpha(2)M*/LRP-1 interaction on these processes. In this work, we demonstrate that alpha(2)M* binding to LRP-1 induces cell proliferation and MAPK activation in the J774 macrophage-derived cell line, which were blocked by RAP, an antagonist of LRP-1-binding ligands, and by PD980059, a specific inhibitor for the Mek1-ERK1/2 pathway. In addition, we demonstrate that LPS, a bacterial product that it is known to down-regulate the LRP-1 expression on macrophage, abrogated the signaling activity triggered by alpha(2)M* on LPS-treated J774 cells. These results suggest that alpha(2)M*/LRP-1 interaction constitutes a key role in the macrophage functioning during inflammation and cancer.     

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae)

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self-sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south-east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark-based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishes.     

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Direct seeding of Brazilian savanna trees: effects of plant cover and fertilization on seedling establishment and growth

Direct seeding is a promising technique for ecological restoration, but it has been poorly studied in neotropical savannas. Different types of plant cover (no cover, crops, or green manure) and fertilization (unfertilized, synthetic fertilizer, or poultry litter) were used to verify if survival and growth of different tree species after direct seeding could be enhanced by the use of any combination of these techniques. Seedling emergence, establishment, and growth were followed for 2 years for six savanna tree species sown in an agricultural field in Central Brazil. Germination was high (52%, on average) for Anacardium occidentale, Aspidosperma macrocarpon, Hymenaea stigonocarpa, Dipteryx alata, Eugenia dysenterica, and Magonia pubescens. Six additional species were planted, but less than 5% of these seeds germinated. Crops (60% shade) did not affect seedling survival and biomass compared with the control treatment, supporting the use of this strategy during the initial phase of restoration to involve farmers in the process. In contrast, the excessive shading (95%) from the green manure treatment decreased the survival of two species and the growth in biomass and diameter of five species, especially when combined with fertilization. Seedling growth was very slow throughout the experiment, requiring extended weed management. This study supports the use of direct seeding of the studied species for savanna restoration, but methods could be improved to include a larger number of species.     

Muscle-bone properties after prolonged voluntary wheel running in a mouse model of dominant severe osteogenesis imperfecta

Objective:The objective of the current study is to assess the effect of a seven-week voluntary wheel running intervention on muscles and bones properties in a mouse model mimicking dominant severe osteogenesis imperfecta (OI).Methods:Female wild-type (WT) and OI (Col1a1^Jrt/+) mice either performed voluntarily wheel-running exercise for 7-weeks or remained sedentary. Running distance and speed, forelimb grip strength, isolated muscle force and fatigability as well as bone morphology and mechanical properties were assessed.Results:We demonstrate that female WT and OI mice voluntarily performed exercise, although OI mice exercised less than WT littermates. The exercise regimen increased soleus muscle masses in WT and OI but increased relative grip strength in WT mice only. Specific muscle force and fatigability were similar between WT and OI mice and did not improve with exercise. Furthermore, the exercise regimen did not improve the femoral architectural and biomechanical properties in OI mice.Conclusion:Our study suggests that voluntary wheel running is not appropriate to assess the effects of exercise in a mouse model of OI. Findings from exercising OI mice model studies may not necessarily be transferable to humans.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.Dissemination of tumor cells is strictly linked to their ability to attach to and move within the extracellular matrix (ECM) in a three-dimensional (3D) environment. The use of 3D experimental model systems revealed that a higher complexity in cell migration and adaptation responses exists in the 3D model than in the classical 2D model (10, 16, 41, 49). A striking example is given by the fact that only in 3D could individually migrating cells use different mechanisms such as mesenchymal and amoeboid motility (16, 17). The relative slow mesenchymal migration is characterized by a fibroblast-like spindle shape and is dependent on integrin-mediated adhesion and on protease function (16). The amoeboid motility can in some cases represent a less adhesive, integrin-independent type of movement. Cells use a propulsive mechanism and are highly deformable, and rather than degrade the matrix, they are able to squeeze through it (16). As a result, the cells that use the amoeboid motility can potentially move faster than cells that use a mesenchymal strategy. Mesenchymal and amoeboid movements are also characterized by a different involvement of small GTPases of the Rho family. A high RhoA activity is associated mainly with the amoeboid motility, while the mesenchymal migration needs a high Rac activity at the leading edge to promote the extension of cellular protrusions (41, 48). Under certain circumstances, cancer cells can undergo conversion from a mesenchymal toward an amoeboid motility, an event referred as mesenchymal-amoeboid transition (MAT) (50). MAT represents a putative escape mechanism in tumor cell dissemination that could be induced by inhibition of pericellular proteolysis (50) or by increased membrane-associated RhoA activity (18, 40).Key mediators of cell motility through ECM substrates are the members of the Src family kinases. The prototype of Src family kinases, c-Src (14), is activated following cell-ECM adhesion and contributes to regulate the focal adhesion turnover and the cytoskeletal modifications necessary for normal cell adhesion and motility (52). The c-Src gene is the proto-oncogene of the transforming gene v-src of Rous sarcoma virus, and its elevated protein level and activity have been found in many human tumors (20, 28, 27, 34). Despite the accumulation of information and new molecular understanding of how Src is controlled, there is still an incomplete picture about its role in the generation of the malignant phenotype. v-Src shows higher levels of the kinase activity and transforming ability than c-Src (14, 15, 52). It induces normal cells to acquire a variety of transformed features, including alteration of morphology and increase of invasion ability due to its role in focal adhesion remodeling (7, 9, 13).Many data suggest that there is a close relationship between cell-ECM interaction and the proliferation and movements in both normal and tumor cells (5, 38, 43). Accordingly, Src activation may influence not only cell motility but also cell cycle progression by targeting the cell cycle inhibitor p27^kip1 to proteasomal degradation (22, 39). Recent evidences indicated that p27^kip1 (hereafter called p27) can also regulate cell migration, even though its role still remains controversial since it has been reported to either block or stimulate cell movements (1, 4, 11, 19, 21, 23, 29, 45).Based on these notions, we tested the possible contribution of p27 to the growth and motility phenotypes induced by v-src transformation, with special regard to those cellular invasive features that can be observed in 3D environments. By studying in vitro and in vivo the behavior of wild-type (WT) and p27-null fibroblasts transformed with v-src, we highlight a new role for p27 in the regulation of cellular plasticity that can ultimately drive tumor cell shape, motility, and invasion.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptor TRAIL-R3 is up-regulated by p53 in breast tumor cells through a mechanism involving an intronic p53-binding site

Tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (TRAIL-R3) is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors. We studied the regulation of TRAIL-R3 gene expression in breast tumor cells treated with the genotoxic drug doxorubicin (DXR). The breast tumor cell line MCF-7 (p53 wild type) responded to DXR with a marked elevation of TRAIL-R3 expression at the mRNA, total protein, and cell surface levels. In contrast, in EVSA-T cells (p53 mutant) DXR did not induce increased expression of TRAIL-R3. In MCF-7 cells overexpressing the human papillomavirus protein E6, which causes p53 degradation, DXR-induced TRAIL-R3 expression was notably reduced. Furthermore, in MCF-7 cells overexpressing a temperature-sensitive p53 mutant (Val135), shifting the cultures to the permissive temperature was sufficient to induce the expression of TRAIL-R3. We also cloned and characterized a p53 consensus element located within the first intron of the human TRAIL-R3 gene. This element binds p53 and confers responsiveness to genotoxic damage to constructs of the TRAIL-R3 promoter in transient transfection experiments. Our results indicate that genotoxic treatments such as DXR, frequently used in cancer therapy, may also induce genes such as TRAIL-R3 that potentially have antiapoptotic actions and thus interfere with the TRAIL signaling system. This is particularly important in view of the proposed use of TRAIL in antitumor therapy.