Peroxisome proliferator-activated receptors: three isotypes for a multitude of functions.

The peroxisome proliferator-activated receptors (PPARs) are fatty acid and eicosanoid inducible nuclear receptors, which occur in three different isotypes. Upon activator binding, they modulate the expression of various target genes implicated in several important physiological pathways. During the past few years, the identification of both PPAR ligands, natural and synthetic, and PPAR targets and their associated functions has been one of the most important achievements in the field. It underscores the potential therapeutic application of PPAR-specific compounds on the one side, and the crucial biological roles of endogenous PPAR ligands on the other.

Initiation and limitation of Ly-49A NK cell receptor acquisition by T cell factor-1.

The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by approximately 20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A(+) NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator beta-catenin. Indeed, the proximal Ly-49A promoter was beta-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.

Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Tissue-based immune monitoring I: tumor core needle biopsies allow in-depth interrogation of the tumor microenvironment.

We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of 7 consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8, and FoxP3. A portion of the cores was used to isolate RNA for 1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGF-beta, 2) multiplexed PCR-based T cell receptor (TCR) CDR3 Vβ region spectratyping, and 3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3ε, CD8A, and FoxP3 gene expression, respectively, by qPCR (r=0.61, 0.86, and 0.89; all p< 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR Vβ chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters.

The anaerobic transcriptional regulator ANR induces the arginine deiminase and denitrification pathways in Pseudomonas aeruginosa during oxygen limitation. The homologous activator FNR of Escherichia coli, when introduced into an anr mutant of P. aeruginosa, could functionally replace ANR for anaerobic growth on nitrate but not for anaerobic induction of arginine deiminase. In an FNR-positive E. coli strain, the ANR-dependent promoter of the arcDABC operon, which encodes the enzymes of the arginine deiminase pathway, was not expressed. To analyse systematically these distinct induction patterns, a lacZ promoter-probe, broad-host-range plasmid containing various -40 regions (the ANR/FNR recognition sequences) and -10 promoter sequences was constructed. These constructs were tested in P. aeruginosa and in E. coli expressing either ANR or FNR. In conjunction with the consensus -10 hexamer of E. coli sigma 70 RNA polymerase (TATAAT), the consensus FNR site (TTGAT ..... ATCAA) was recognized efficiently by ANR and FNR in both hosts. By contrast, when promoters contained the Arc box (TTGAC .... ATCAG), which is found in the arcDABC promoter, or a symmetrical mutant FNR site (CTGAT .... ATCAG), ANR was a more effective activator than was FNR. Conversely, an extended 22 bp, fully symmetrical FNR site allowed better activation with FNR than with ANR. Combination of the arc promoter -10 sequence (CCTAAT) with the Arc box or the consensus FNR site resulted in good ANR-dependent expression in P. aeruginosa but gave practically no expression in E. coli, suggesting that RNA polymerase of P. aeruginosa differs from the E. coli enzyme in -10 recognition specificity. In conclusion, ANR and FNR are able to activate the RNA polymerases of P. aeruginosa and E. coli when the -40 and -10 promoter elements ae identical or close to the E. coli consensus sequences.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.

Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta.

Cyclooxygenase-derived prostaglandin E(2) (PGE(2)) is the predominant prostanoid found in most colorectal cancers (CRC) and is known to promote colon carcinoma growth and invasion. However, the key downstream signaling pathways necessary for PGE(2)-induced intestinal carcinogenesis are unclear. Here we report that PGE(2) indirectly transactivates PPARdelta through PI3K/Akt signaling, which promotes cell survival and intestinal adenoma formation. We also found that PGE(2) treatment of Apc(min) mice dramatically increased intestinal adenoma burden, which was negated in Apc(min) mice lacking PPARdelta. We demonstrate that PPARdelta is a focal point of crosstalk between the prostaglandin and Wnt signaling pathways which results in a shift from cell death to cell survival, leading to increased tumor growth.

IB1 reduces cytokine-induced apoptosis of insulin-secreting cells.

IB1/JIP-1 is a scaffold protein that interacts with upstream components of the c-Jun N-terminal kinase (JNK) signaling pathway. IB1 is expressed at high levels in pancreatic beta cells and may therefore exert a tight control on signaling events mediated by JNK in these cells. Activation of JNK by interleukin 1 (IL-1beta) or by the upstream JNK constitutive activator DeltaMEKK1 promoted apoptosis in two pancreatic beta cell lines and decreased IB1 content by 50-60%. To study the functional consequences of the reduced IB1 content in beta cell lines, we used an insulin-secreting cell line expressing an inducible IB1 antisense RNA that lead to a 38% IB1 decrease. Reducing IB1 levels in these cells increased phosphorylation of c-Jun and increased the apoptotic rate in presence of IL-1beta. Nitric oxide production was not stimulated by expression of the IB1 antisense RNA. Complementary experiments indicated that overexpression of IB1 in insulin-producing cells prevented JNK-mediated activation of the transcription factors c-Jun, ATF2, and Elk1 and decreased IL-1beta- and DeltaMEKK1-induced apoptosis. These data indicate that IB1 plays an anti-apoptotic function in insulin-producing cells probably by controlling the activity of the JNK signaling pathway.

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0.

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

Peroxisome proliferator-activated receptor agonists.

The peroxisome proliferator-activated receptors are a family of three ligand-activated transcription factors. Fibrate antihyperlipidemic drugs and thiazolidinedione antihyperglycemic drugs were recently identified as synthetic ligands for these receptors. In addition, certain unsaturated fatty acids and eicosanoids were shown to bind the receptors, and thus represent naturally occurring PPAR ligands. The synthetic and natural ligands have proven to be powerful tools in dissecting the biology of these orphan receptors.

Spatio-temporal sequence of cross-regulatory events in root meristem growth.

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

Functions of the peroxisome proliferator-activated receptor (PPAR) alpha and beta in skin homeostasis, epithelial repair, and morphogenesis.

The three peroxisome proliferator-activated receptors (PPAR alpha, PPAR beta, and PPAR gamma) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. They are regarded as being sensors of physiological levels of fatty acids and fatty acid derivatives. In the adult mouse skin, they are found in hair follicle keratinocytes but not in interfollicular epidermis keratinocytes. Skin injury stimulates the expression of PPAR alpha and PPAR beta at the site of the wound. Here, we review the spatiotemporal program that triggers PPAR beta expression immediately after an injury, and then gradually represses it during epithelial repair. The opposing effects of the tumor necrosis factor-alpha and transforming growth factor-beta-1 signalling pathways on the activity of the PPAR beta promoter are the key elements of this regulation. We then compare the involvement of PPAR beta in the skin in response to an injury and during hair morphogenesis, and underscore the similarity of its action on cell survival in both situations.