Regulation of lymphocyte proliferation and death by FLIP.

Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Tight control of apoptosis is essential for immune function, because its altered regulation can result in cancer and autoimmunity. Signals from members of the tumour-necrosis-factor receptor (TNF-R) family, such as Fas and TNF-R1, activate the caspase cascade and result in lymphocyte death by apoptosis. Anti-apoptotic proteins, such as FLIP (also known as FLICE/caspase-8 inhibitory protein) have recently been identified. FLIP expression is tightly regulated in T cells and might be involved in the control of both T-cell activation and death. Abnormal expression of FLIP might have a role not only in autoimmune diseases, but also in tumour development and cardiovascular disorders.

Heat perception and signalling in plants: a tortuous path to thermotolerance.

An accurate assessment of the rising ambient temperature by plant cells is crucial for the timely activation of various molecular defences before the appearance of heat damage. Recent findings have allowed a better understanding of the early cellular events that take place at the beginning of mild temperature rise, to timely express heat-shock proteins (HSPs), which will, in turn, confer thermotolerance to the plant. Here, we discuss the key components of the heat signalling pathway and suggest a model in which a primary sensory role is carried out by the plasma membrane and various secondary messengers, such as Ca(2+) ions, nitric oxide (NO) and hydrogen peroxide (H(2) O(2) ). We also describe the role of downstream components, such as calmodulins, mitogen-activated protein kinases and Hsp90, in the activation of heat-shock transcription factors (HSFs). The data gathered for land plants suggest that, following temperature elevation, the heat signal is probably transduced by several pathways that will, however, coalesce into the final activation of HSFs, the expression of HSPs and the onset of cellular thermotolerance.

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

Mapping cell-matrix stresses during stretch reveals inelastic reorganization of the cytoskeleton.

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actin-myosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.

Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis.

Rhizobacteria-induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

The tumor suppressive role of WIF1 in glioblastoma

Expression based prediction of gene alterations identified WNT inhibitory factor I (WIF1) as a new candidate tumor suppressor gene involved in glioblastoma. WIF1 encodes a secreted WNT antagonist and it is strongly down-regulated in most glioblastoma as compared to normal brain both by genomic deletion and WIF1 promoter hypermethylation. WIF1 expression in glioblastoma cell lines inhibited cell proliferation in vitro and in vivo and strongly reduced migration capability. Interestingly, WIF1 expression induced a senescence-like phenotype characterized by the appearance of enlarged, flattened and multinucleated cells positive for the presence of senescence associated ß-galactosidase, a late marker of senescence. It is of note that WIF1 induced senescence, in glioma cell lines, is independent of either p53 or pRB, two pathways that have been widely associated with this process. The analysis of the signaling pathways downstream of WIF1 brought some interesting results. WIF1 expression inhibited the canonical pathway but alteration of this pathway alone couldn't explain all the WIFl-induced effects. Some WIF1-related changes were attributed to inhibition of the non-canonical pathway, as we could prove by downregulation of WNT5a, the main ligand of the non-canonical WNT pathway. For example, a drastic reduction of phosphorylation of both ERK and p38 was detected when either overexpressing WIF1 or downregulating WNT5a. Due to the complexity of the non-canonical pathway is difficult to define the precise mechanism of signal transduction. We have excluded the involvement of the WNT5a-JNK-APl pathway and preliminary results suggest the implication of the WNT-calcium signaling, but further evidence is needed. Moreover, from the analysis of the gene expression profile of WIF1 expressing cells we could select a very interesting candidate: MALATI, a non-coding RNA widely associated with migratory capability in many different types of tumors. We found MALATI to be overexpressed in glioblastoma specimens compared to normal brain and to be associated with total tumor volume. The downregulation of MALATI by RNAi (RNA interference] drastically impairs migration, thus it is a very interesting potential target in the context of invasive tumors such as glioblastoma. Résumé WIFl a été sélectionné en tant que putatif suppresseur de tumeurs dans le cadre des glioblastomes par une analyse qui a était conduit à partir des données d'expression de gènes provenant d'environ 80 glioblastomes. WIF1 code pour une protéine destinée à la sécrétion qui antagonise la voie de WNT et son expression est fortement sous-exprimé dans la plupart des glioblastome par rapport à tissu cérébral normal. Cette sous-expression est due à deux mécanismes différents: à la délétion de la partie génomique codant pour WIF1 et à l'hyper méthylation de son promoteur. La surexpression de WIF1 réduit la capacité de prolifération des cellules de glioblastome in vitro ainsi que in vivo et elle réduit aussi leur capacité migratoire. Il est intéressant de remarquer que l'espression de WIF1 induit un phénotype sénescent caractérisé par l'apparition de cellules aplaties, multi nucléées et positives pour l'activité de l'enzyme ß-galactosidase associée à la sénescence, un marqueur tardif de la sénescence. Il est à noter que le phénotype sénescent qui est induit par WIF1 est indépendant de p53 et pRB, deux voies qui ont été largement associées à ce processus. L'analyse des les voies de signalisation en aval de WIFl a apporté des résultats intéressants. L'expression de WIF1 inhibe la voie canonique de WNT, mais l'altération de cette voie seule ne pouvait pas expliquer tous les effets induits par WIF1. Nous avons pu prouver que certains changements sont liés à l'inhibition de la voie non-canonique qui est activée par WNT5cc. Par exemple, une réduction drastique de la phosphorylation de ERK et p38 à la fois a été détectée lorsque WIFl a été surexprimé ou WNT5a sous- exprimé. En raison de la complexité de la voie non-canonique, il est difficile de définir le mécanisme précis de la transduction du signal. Nous avons exclu l'implication de la voie JNK-WNT5a-APl et les résultats préliminaires suggèrent l'implication de la voie de signalisation appelée WNT-calcium. En plus, l'analyse du profil d'expression génique de cellules sur-exprimant WIF1 nous a permis d'identifier un candidat très intéressant: MALATI, un ARN non- codants largement associés à la capacité migratoire dans nombreux types de tumeurs. Nous avons trouvé que MALATI est surexprimé dans les échantillons de glioblastome par rapport à tissu cérébral normal et il est associé au volume total de la tumeur. La sous-expression de MALATI altère considérablement la migration des cellules tumorales. Donc, MALATI, est une cible potentielle très intéressante dans le cadre d'une tumeur invasive telle que le glioblastome.

Mechanisms of apoptosis in retinitis pigmentosa.

Mutations in humans are associated with several forms of inherited retinal dystrophies, such as Retinitis Pigmentosa which lead to retinal cell death and irreversible loss of vision. Genes involved in affected patients mainly encode proteins related to vision physiology including visual cycle and light-dependent phototransduction cascade. As reported in spontaneous and genetically engineered mouse models, apoptosis is a common fate in retinal degeneration, although the triggered signals to retinal apoptosis remain largely unraveled. Several studies highlighted that many of the molecular pathways involved in ocular diseases rely on caspase-dependent or -independent apoptotic mitochondrial pathway involving the Bcl-2 family of proteins. Anti- and pro-apoptotic Bcl-2 members are present in retinal tissues and are thought to play a role in the pathogenesis of several retinal disorders. Since almost no efficient treatments are available so far, it remains a great challenge to decipher the molecular pathways involved in retinal dystrophies and to develop alternative therapies to prevent or inhibit eye defect. Toward this goal, mutation-independent strategies such as molecular therapy provides promising and exciting approaches to deliver anti-apoptotic molecules targeting the Bcl-2 pathway through the use of cell permeable transport peptides. Modulation of common apoptotic signaling pathways may be of outstanding potential to target multiple retinal dystrophies regardless of the primary genetic defect.

Myeloid differentiation factor-88/interleukin-1 signaling controls cardiac fibrosis and heart failure progression in inflammatory dilated cardiomyopathy.

RATIONALE: The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self-antigen-presenting cells and promotes autoreactive CD4(+) T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. OBJECTIVE: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. METHODS AND RESULTS: Using alpha-myosin heavy chain peptide (MyHC-alpha)-loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88(-/-) mice with similar distributions of heart-infiltrating cell subsets and comparable CD4(+) T-cell responses. Injection of complete Freund's adjuvant (CFA) or MyHC-alpha/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88(-/-) mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow-derived cells was critical for development of cardiac fibrosis during progression to heart failure. CONCLUSIONS: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Calcineurin inhibitors activate the proto-oncogene Ras and promote protumorigenic signals in renal cancer cells.

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1(-/-) mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal cells plasticity to regulate their function.

The unfolded protein response: integrating stress signals through the stress sensor IRE1α.

Stress induced by accumulation of unfolded proteins at the endoplasmic reticulum (ER) is a classic feature of secretory cells and is observed in many tissues in human diseases including cancer, diabetes, obesity, and neurodegeneration. Cellular adaptation to ER stress is achieved by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that transmits information about the protein folding status at the ER to the nucleus and cytosol to restore ER homeostasis. Inositol-requiring transmembrane kinase/endonuclease-1 (IRE1α), the most conserved UPR stress sensor, functions as an endoribonuclease that processes the mRNA of the transcription factor X-box binding protein-1 (XBP1). IRE1α signaling is a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble, here referred to as the UPRosome. Here we provide an overview of the signaling and regulatory mechanisms underlying IRE1α function and discuss the emerging role of the UPR in adaptation to protein folding stress in specialized secretory cells and in pathological conditions associated with alterations in ER homeostasis.