When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.

Different c-Jun N-terminal kinases (JNKs) are activated by a plethora of signals and phosphorylate substrates such as c-Jun, which is required for efficient cell cycle progression. Although JNK1 and JNK2 were shown to differentially regulate fibroblast proliferation, the underlying mechanistic basis remains unclear. We found that Jnk2-/- fibroblasts exit G1 and enter S phase earlier than wild-type counterparts, while Jnk1-/- cells show the inverse phenotype. Moreover, Jnk2-/- erythroblasts also exhibit a proliferative advantage. JNK2 deficiency results in elevated c-Jun phosphorylation and stability, whereas the absence of JNK1 reduces c-Jun phosphorylation and stability. Re-expression of JNK2 in Jnk2-/- cells reverses the JNK2 null phenotype, whereas ectopic expression of JNK1 augments it. JNK2 is preferentially bound to c-Jun in unstimulated cells, thereby contributing to c-Jun degradation. In contrast, JNK1 becomes the major c-Jun interacting kinase after cell stimulation. These data provide mechanistic insights into the distinct roles of different JNK isoforms.

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

The glucocorticoid receptor (GR) coordinates a multitude of physiological responses in vivo. In vitro, glucocorticoids are required for sustained proliferation of erythroid progenitors (ebls). Here, we analyze the impact of the GR on erythropoiesis in vivo, using GR-deficient mice or mice expressing a GR defective for transactivation. In vitro, sustained proliferation of primary ebls requires an intact GR. In vivo, the GR is required for rapid expansion of ebls under stress situations like erythrolysis or hypoxia. A particular, GR-sensitive progenitor could be identified as being responsible for the stress response. Thus, GR-mediated regulation of ebl proliferation is essential for stress erythropoiesis in vivo.

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropinreleasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a K(D) value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.

Glucocorticoids (GCs IntroductionUpon triggering of their T-cell receptor (TCR), resting naive T lymphocytes are activated to elicit an immune response. In contrast, preactivated T cells undergo activation-induced cell death (AICD) in response to the same signal. [1][2][3] Apoptosis by AICD is an important mechanism to down-regulate the number of reactive T cells and to terminate the immune response. 4 The main apoptotic pathway of AICD is mediated by the CD95 (apoptosis antigen-1 [APO-1]/Fas) death receptor system since cell death by TCR restimulation can be blocked almost completely in the presence of reagents that inhibit CD95/CD95L interaction. 1-3 CD95 ligand (CD95L, CD178) is induced upon TCR/CD3 cross-linking and triggers CD95 signaling and subsequently apoptosis. 5 Deregulation of CD95L expression results in several diseases, such as autoimmunity and uncontrolled lymphoproliferation. [6][7][8] The transcriptional regulation of the CD95L promoter upon TCR/CD3 stimulation has been studied extensively. Various transcription factors and regulatory elements have been identified, such as several binding sites for nuclear factor of activated T cells (NF-AT), nuclear factor B (NF-B), activator protein 1 (AP-1), early growth response (Egr) protein, and interferon regulatory factor 1 (IRF-1). 9 The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that acts as a major modulator of the immune system due to its anti-inflammatory and immunosuppressive activities, thus serving a function that is frequently put to use in the treatment of autoimmune disorders, inflammatory diseases, and allergy. In the absence of ligand, the receptor is retained in the cytoplasm in an inactive state forming a large complex that includes chaperones. 10 Upon hormone binding, the GR dissociates from the cytoplasmic complex and translocates into the nucleus. Within the nucleus it acts as a transcriptional activator by binding as a homodimer with high affinity to glucocorticoid responsive DNA elements (GREs). In addition to enhancing the rate of transcription, the GR can also act as a transcriptional repressor by binding to different DNA sequences called negative GREs (nGREs). 11,12 Besides direct DNA binding, the GR can also modulate transcription as a monomer via protein-protein interactions as documented by repressive effects on AP-1 and NF-B 13-15 and activation of signal transducer and activator of transcription 5 (Stat-5). 16 Glucocorticoid (GC) hormones exert their potent antiinflammatory action mainly by inhibition of cytokine gene expression, and hence effectively block the activation of the immune system. 15,17,18 In addition, GCs are potent inducers of apoptosis in thymocytes and in some T-cell hybridomas by DNA bindingdependent transcriptional regulation, whereas peripheral T cells are only weakly sensitive toward GC-induced cell death. Although both GC and TCR stimulation can induce apoptosis in T-cell hybridomas, surprisingly, cell death is strongly reduced when both stimuli are provided simultaneously. 19...

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