Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

Identification of corticosteroid-regulated genes in cardiomyocytes by serial analysis of gene expression.

Corticosteroids (aldosterone, cortisol/corticosterone) exert direct functional effects on cardiomyocytes. However, gene networks activated by corticosteroids in cardiomyocytes, as well as the involvement of the mineralocorticoid receptor (MR) vs the glucocorticoid receptor (GR) in these effects, remain largely unknown. Here we characterized the corticosteroid-dependent transcriptome in primary culture of neonatal mouse cardiomyocytes treated with 10(-6) M aldosterone, a concentration predicted to occupy both MR and GR. Serial analysis of gene expression revealed 101 aldosterone-regulated genes. The MR/GR specificity was characterized for one regulated transcript, namely ecto-ADP-ribosyltransferase-3 (Art3). Using cardiomyocytes from GR(null/null) or MR(null/null) mice we demonstrate that in GR(null/null) cardiomyocytes the response is abrogated, but it is fully maintained in MR(null/null) cardiomyocytes. We conclude that Art3 expression is regulated exclusively via the GR. Our study identifies a new set of corticosteroid-regulated genes in cardiomyocytes and demonstrates a new approach to studying the selectivity of MR- vs GR-dependent effects.

Steady-state expression of self-regulated genes.

MOTIVATION: Regulatory gene networks contain generic modules such as feedback loops that are essential for the regulation of many biological functions. The study of the stochastic mechanisms of gene regulation is instrumental for the understanding of how cells maintain their expression at levels commensurate with their biological role, as well as to engineer gene expression switches of appropriate behavior. The lack of precise knowledge on the steady-state distribution of gene expression requires the use of Gillespie algorithms and Monte-Carlo approximations. METHODOLOGY: In this study, we provide new exact formulas and efficient numerical algorithms for computing/modeling the steady-state of a class of self-regulated genes, and we use it to model/compute the stochastic expression of a gene of interest in an engineered network introduced in mammalian cells. The behavior of the genetic network is then analyzed experimentally in living cells. RESULTS: Stochastic models often reveal counter-intuitive experimental behaviors, and we find that this genetic architecture displays a unimodal behavior in mammalian cells, which was unexpected given its known bimodal response in unicellular organisms. We provide a molecular rationale for this behavior, and we implement it in the mathematical picture to explain the experimental results obtained from this network.

Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes.

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8+ T cells and natural killer (NK) cells expressing high levels as compared to CD4+ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4-8- and CD4+8+ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4+ subset but present in the more mature (HSAlow) fraction of CD8+ cells. Furthermore, while thymic CD4-8- T cell receptor (TCR) gamma delta cells have little P-gly activity, a minor subset of CD4-8- or CD4+ TCR alpha beta + thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.

Induction of tolerogenic vs immunogenic dendritic cells (DCs) in the presence of GM-CSF is regulated by the strength of signaling from monophosphoryl lipid A (MPLA) in association with glutathione and fetal hemoglobin gamma-chain.

Previous studies showed a fetal sheep liver extract (FSLE), in association with monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS), could interact to induce the development of dendritic cells (DCs) which regulated production of Foxp3+ Treg. This interaction was associated with an altered gene expression both of distinct subsets of TLRs and of CD200Rs. Prior studies had suggested that major interacting components within FSLE were gamma-chain of fetal hemoglobin (Hgbgamma) and glutathione (GSH). We investigated whether differentiation/maturation of DCs in vitro in the presence of either GM-CSF or Flt3L to produce preferentially either immunogenic or tolerogenic DCs was itself controlled by an interaction between MPLA, GSH and Hgbgamma. At low (approximately 10 microg/ml) Hgbgamma concentrations, DCs developing in culture with GSH and MPLA produced optimal stimulation of allogeneic CTL cell responses in vitro (and enhanced skin graft rejection in vivo). At higher concentrations (>40 microg/ml Hgbgamma) and equivalent concentrations of MPLA and GSH, the DCs induce populations of Treg which can suppress the induction of allogeneic CTL and graft rejection in vivo. These different populations of DCs express different patterns of mRNAs for the CD200R family. Addition of anti-TLR or anti-MD-1 mAbs to DCs developing in this mixture (Hgbgamma+GSH+MPLA), suggests that one effect of (GSH+Hgbgamma) on MPLA stimulation may involve altered signaling through TLR4.

Light-regulated plant growth and development.

Plants are sessile and photo-autotrophic; their entire life cycle is thus strongly influenced by the ever-changing light environment. In order to sense and respond to those fluctuating conditions higher plants possess several families of photoreceptors that can monitor light from UV-B to the near infrared (far-red). The molecular nature of UV-B sensors remains unknown, red (R) and far-red (FR) light is sensed by the phytochromes (phyA-phyE in Arabidopsis) while three classes of UV-A/blue photoreceptors have been identified: cryptochromes, phototropins, and members of the Zeitlupe family (cry1, cry2, phot1, phot2, ZTL, FKF1, and LKP2 in Arabidopsis). Functional specialization within photoreceptor families gave rise to members optimized for a wide range of light intensities. Genetic and photobiological studies performed in Arabidopsis have shown that these light sensors mediate numerous adaptive responses (e.g., phototropism and shade avoidance) and developmental transitions (e.g., germination and flowering). Some physiological responses are specifically triggered by a single photoreceptor but in many cases multiple light sensors ensure a coordinated response. Recent studies also provide examples of crosstalk between the responses of Arabidopsis to different external factors, in particular among light, temperature, and pathogens. Although the different photoreceptors are unrelated in structure, in many cases they trigger similar signaling mechanisms including light-regulated protein-protein interactions or light-regulated stability of several transcription factors. The breath and complexity of this topic forced us to concentrate on specific aspects of photomorphogenesis and we point the readers to recent reviews for some aspects of light-mediated signaling (e.g., transition to flowering).

15-hydroxyprostaglandin dehydrogenase is down-regulated in gastric cancer.

PURPOSE: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer. EXPERIMENTAL DESIGN: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1beta (IL-1beta) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines. RESULTS: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2'-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1beta repressed expression of 15-PGDH mRNA and protein. This effect of IL-1beta was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E(2) production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210). CONCLUSIONS: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1beta, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

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.