Crosstalk between peroxisome proliferator-activated receptor delta and VEGF stimulates cancer progression.

Peroxisome proliferator-activated receptor (PPAR) delta is a member of the nuclear hormone receptor superfamily. PPARdelta may ameliorate metabolic diseases such as obesity and diabetes. However, PPARdelta's role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPARdelta decreases intestinal adenoma growth in Apc(Min/+) mice and inhibits tumor-promoting effects of a PPARdelta agonist GW501516. More importantly, we found that activation of PPARdelta up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPARdelta agonists for treatment of metabolic disorders in patients who are at high risk for colorectal cancer, but also support the rationale for developing PPARdelta antagonists for prevention and/or treatment of cancer.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Species-Specific Recognition of Aspergillus fumigatus by Toll-like Receptor 1 and Toll-like Receptor 6.

Background. Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown. Methods. Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs. Results. Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6. Conclusions. Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties.

BAFF is a B cell survival factor that binds to three receptors BAFF-R, TACI and BCMA. BAFF-R is the receptor triggering naïve B cell survival and maturation while BCMA supports the survival of plasma cells in the bone marrow. Excessive BAFF production leads to autoimmunity, presumably as the consequence of inappropriate survival of self-reactive B cells. The function of TACI has been more elusive with TACI(-/-) mice revealing two sides of this receptor, a positive one driving T cell-independent immune responses and a negative one down-regulating B cell activation and expansion. Recent work has revealed that the regulation of TACI expression is intimately linked to the activation of innate receptors on B cells and that TACI signalling in response to multimeric BAFF and APRIL provides positive signals to plasmablasts. How TACI negatively regulates B cells remains elusive but may involve an indirect control of BAFF levels. The discovery of TACI mutations associated with common variable immunodeficiency (CVID) in humans not only reinforces its important role for humoral responses but also suggests a more complex role than first anticipated from knockout animals. TACI is emerging as an unusual TNF receptor-like molecule with a sophisticated mode of action.

An olfactory receptor for food-derived odours promotes male courtship in Drosophila.

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRU(M)). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRU(M)-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased--in an IR84a-dependent manner--in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fru(M) courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.

Association with coregulators is the major determinant governing peroxisome proliferator-activated receptor mobility in living cells.

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells.

The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor gamma (PPARgamma) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARalpha, beta, and gamma in RT4 cells and only by PPARbeta in T24 cells via a transcriptional activation of the VEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor.

cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy.

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Teichoic acids are not required for Streptococcus pneumoniae and Staphylococcus aureus cell walls to trigger the release of tumor necrosis factor by peripheral blood monocytes.

In gram-negative bacteria, the outer membrane lipopolysaccharide is the main component triggering cytokine release from peripheral blood mononuclear cells (PBMCs). In gram-positive bacteria, purified walls also induce cytokine release, but stimulation requires 100 times more material. Gram-positive walls are complex megamolecules reassembling distinct structures. Only some of them might be inflammatory, whereas others are not. Teichoic acids (TA) are an important portion (> or =50%) of gram-positive walls. TA directly interact with C3b of complement and the cellular receptor for platelet-activating factor. However, their contribution to wall-induced cytokine-release by PBMCs has not been studied in much detail. In contrast, their membrane-bound lipoteichoic acids (LTA) counterparts were shown to trigger inflammation and synergize with peptidoglycan (PGN) for releasing nitric oxide (NO). This raised the question as to whether TA are also inflammatory. We determined the release of tumor necrosis factor (TNF) by PBMCs exposed to a variety of TA-rich and TA-free wall fragments from Streptococcus pneumoniae and Staphylococcus aureus. TA-rich walls from both organisms induced measurable TNF release at concentrations of 1 microg/ml. Removal of wall-attached TA did not alter this activity. Moreover, purified pneumococcal and staphylococcal TA did not trigger TNF release at concentrations as high as > or =100 microg/ml. In contrast, purified LTA triggered TNF release at 1 microg/ml. PGN-stem peptide oligomers lacking TA or amino-sugars were highly active and triggered TNF release at concentrations as low as 0.01 microg/ml (P. A. Majcherczyk, H. Langen, et al., J. Biol. Chem. 274:12537-12543,1999). Thus, although TA is an important part of gram-positive walls, it did not participate to the TNF-releasing activity of PGN.

The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism.

The peroxisome proliferator-activated receptor alpha is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPARalpha mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPARalpha, we searched for genes regulated by PPARalpha using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPARalpha null mice, it was found that PPARalpha decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPARalpha influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPARalpha using the synthetic PPARalpha ligand WY14643 decreased mRNA levels of these genes, suggesting that PPARalpha is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPARalpha in vivo. It is concluded that in addition to oxidation of fatty acids, PPARalpha also regulates metabolism of amino acids in liver, indicating that PPARalpha is a key controller of intermediary metabolism during fasting.

Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.