Elevated CREB activity in embryonic fibroblasts of gene-targeted histamine deficient mice.

OBJECTIVES: Histamine is a known inducer of cAMP-responsive element binding protein (CREB), which plays a key role in initiation of adipogenesis. Our present goal was to study how histamine deficiency impacts CREB signalling and adipogenesis. METHODS: We used a histidine-decarboxylase gene-targeted (HDC KO) mice model lacking endogenous histamine. We measured CREB activity and expression by EMSA, Western blot and real-time RT-PCR, as well as cAMP levels by ELISA in primary embryonic fibroblasts derived from WT and HDC KO mice. The ability of these cells to form adipocytes was also tested in preliminary experiments. RESULTS: We found that in the absence of the histamine, cells show higher constitutive CREB activity and greatly increased intracellular cAMP levels, as well as that in contrast to WT cells, HDC KO fibroblasts are more prone to differentiate into adipocytes. CONCLUSION: These data suggest a newly recognised inhibitory role for histamine in CREB activity and draws attention to the potential role of histamine in adipocyte differentiation.

Tumour necrosis factor-alpha inhibits adipogenesis via a beta-catenin/TCF4(TCF7L2)-dependent pathway.

Tumour necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is a potent negative regulator of adipocyte differentiation. However, the mechanism of TNF-alpha-mediated antiadipogenesis remains incompletely understood. In this study, we first confirm that TNF-alpha inhibits adipogenesis of 3T3-L1 preadipocytes by preventing the early induction of the adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). This suppression coincides with enhanced expression of several reported mediators of antiadipogenesis that are also targets of the Wnt/beta-catenin/T-cell factor 4 (TCF4) pathway. Indeed, we found that TNF-alpha enhanced TCF4-dependent transcriptional activity during early antiadipogenesis, and promoted the stabilisation of beta-catenin throughout antiadipogenesis. We analysed the effect of TNF-alpha on adipogenesis in 3T3-L1 cells in which beta-catenin/TCF signalling was impaired, either via stable knockdown of beta-catenin, or by overexpression of dominant-negative TCF4 (dnTCF4). The knockdown of beta-catenin enhanced the adipogenic potential of 3T3-L1 preadipocytes and attenuated TNF-alpha-induced antiadipogenesis. However, beta-catenin knockdown also promoted TNF-alpha-induced apoptosis in these cells. In contrast, overexpression of dnTCF4 prevented TNF-alpha-induced antiadipogenesis but showed no apparent effect on cell survival. Finally, we show that TNF-alpha-induced antiadipogenesis and stabilisation of beta-catenin requires a functional death domain of TNF-alpha receptor 1 (TNFR1). Taken together these data suggest that TNFR1-mediated death domain signals can inhibit adipogenesis via a beta-catenin/TCF4-dependent pathway.

Histamine deficiency suppresses murine haptoglobin production and modifies hepatic protein tyrosine phosphorylation.

Histidine decarboxylase (HDC) synthesizes endogenous histamine from histidine in mammals. HDC-deficient mice (HDC-/-), if kept on a histamine-free diet, have no histamine in their tissues. HDC-/- mice show multiple phenotypes. In this study we show that both the constitutively expressed and turpentine-induced level of an acute-phase protein, haptoglobin, is significantly lower in the serum of HDC-/- mice compared to that of wild-type animals. This effect was abolished if HDC gene-targeted mice received histamine-rich food. No differences were found when lipopolysaccharide (LPS) was used to induce the acute-phase reaction. Using specific antibodies to phosphorylated tyrosine, we showed that protein tyrosine phosphorylation (Y-P) of approximately 50- and 26- to 27-kDa liver proteins is significantly decreased in HDC-/- mice, but that the difference was largely diminished if the animals were kept on a histamine-rich diet, suggesting that the phenotype with lower haptoglobin production is diet inducible. Upon in vivo treatment with LPS, Y-P band intensity decreased, regardless of the presence or absence of histamine. Identification of elements of the signalling pathway with decreased phosphorylation may elucidate the molecular background of the effect of endogenous histamine in the hepatic acute-phase reaction.

Soluble interleukin-6 receptor in plasma and in lymphocyte culture supernatants of healthy individuals and patients with systemic lupus erythematosus and rheumatoid arthritis.

The soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist since its complex with IL-6 binds to gp130 making IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. We measured the sIL-6R levels by ELISA sandwich technology in sera and in supernatants of lymphocyte cultures without and after incubations with dexamethasone. Our results indicate, that the sIL-6R levels in sera of patients with inactive systemic lupus erythematosus (SLE) and active rheumatoid arthritis (RA) were higher than those of the control group, active SLE and inactive RA. In vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, however it strongly suppressed sIL-6R in both RA groups. At mRNA level, we found that in SLE both the mRNA coding the cell-bound and an alternatively spliced variant corresponding to soluble IL-6R transcript increases, however the strong decrease of sIL6R protein in RA was not found at mRNA level.

Effect of a polyanion macromolecule on mitochondria DNA synthesis in isolated rat liver mitochondria.

The incorporation of (3H) thymidine into isolated rat liver mitochondria in the presence of 0--0.5 mM ATP was stimulated by a polyanion with an average molecular weight of 10 000 daltons (at 50--200 micrograms/ml concentration) and containing in a proportion of 1 : 2 : 3 methacrylate, maleate and styrene. The above concentrations of the polyanion inhibit the incorporation of (3H) thymidine in the presence of 2 mM ATP and have no substantial effect on (14C)isoleucine incorporation in vitro. The mtDNA synthesis stimulatory effect of the polyanion is independent of its inhibitory effect on adenylate translocase. The polyanion does not enhance the nucleotide transport across the inner membrane of the mitochondria and the changes of thymidine phosphorylating activity are not involved in the increased mtDNA synthesis. It is supposed that the polyanion attached to the inner membrane of the mitochondria alters the binding of mtDNA to the inner membrane of the mitochondria. Thus, the polyanion might be an effective tool in studying the functional significance of mtDNA-membrane association in mtDNA replication.