A downstream AP-1 element regulates in vitro lung transcription from the human pulmonary surfactant protein B promoter.

We have used the human lung surfactant protein B (SP-B) gene as a template for in vitro transcription studies. Transcription factors were provided by nuclear extracts from a cultured line of human lung (type II-like) cells. Elements upstream of -50 had essentially no effect on the efficiency of the SP-B promoter in vitro. However, a deletion of the region from +8 to +8 reduced in vitro transcription by a factor of 10. The only factor whose binding was detected between +1 and +100 by footprinting, and between +12 and +38 by electrophoretic mobility shift analysis (EMSA), was a member of the AP-1 family. Mutation of 4 of 7 bases of the AP-1 site reduced transcription two-fold and ablated the AP-1 EMSA binding complex observed on the SP-B downstream region (+12 to +38). Competition with unlabeled AP-1 consensus oligonucleotide abolished the downstream footprint over the AP-1 site. Thus, the SP-B promoter is one of a very small class of RNA polymerase II promoters that are strongly dependent in vitro on sequence elements downstream of the transcription start site, and, in this case, the AP-1 consensus element and surrounding sequences.

Activation of adenosine A3 receptors on macrophages inhibits tumor necrosis factor-alpha.

Murine macrophage-derived tumor necrosis factor alpha (TNF-alpha) gene expression has been shown to be dramatically induced by bacterial lipopolysaccharide, and to be dependent upon nuclear factor-kappa B (NF-kappa B) binding sites in its promoter for the lipopolysaccharide induction. Murine J774.1 macrophage cells were found to predominantly express the adenosine A3 receptor RNA relative to adenosine A1 receptor or adenosine A2 receptor RNA. Adenosine receptor agonists, in a dose-dependent manner characteristic of the adenosine A3 receptor, blocked the endotoxin induction of the TNF-alpha gene and TNF-alpha protein expression in the J774.1 macrophage cell line. The adenosine A3 receptor antagonist BW-1433 dose-dependently reversed this adenosine inhibitory effect on TNF-alpha gene expression. Thus, the binding of adenosine receptor agonists to the adenosine A3 receptor interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF-alpha, revealing a novel cross-talk between the murine adenosine A3 receptor and the endotoxin CD14 receptor in J774.1 macrophages.

Alpha-1 adrenergic signaling in a cardiac murine atrial myocyte (HL-1) cell line.

Activation of alpha-1 adrenergic receptors in the heart has been shown to result in increased contractile activity, cardiac fetal gene re-expression, and myocyte hypertrophy. Three alpha-1 adrenergic receptors have been identified through molecular cloning. Due to the limited selectivities of the currently available alpha-1 adrenergic receptor antagonists, the signaling pathways activated by specific subtypes in the heart remain unresolved. To resolve this dilemma, we have used a molecular approach to identify the signaling pathways and downstream genes that are engaged in response to activation of individual alpha-1 adrenergic subtypes in cardiac cells. We have transfected constitutively active alpha-1 adrenergic receptors (alpha1a-S290/293-AR [1] or the alpha1b-S288/294-AR [2]) subtypes into the cardiac murine myocyte cell line (HL-1) and studied the signal transduction pathway(s) and cardiac gene(s) activated by them. In this study, we demonstrate that the alpha1a-S290/293 -AR [1] subtype preferentially couples to cardiac-specific atrial natriuretic factor (ANF) gene expression, while the alpha1b-S288/294-AR preferentially couples to activation of mitogen-activated protein kinase (MAPK), Ets-like transcription factor-1 (Elk1) and serum response element (SRE) signaling pathways. Endogenous alpha-1 adrenergic receptors are expressed, and stimulate phosphatidylinositol-hydrolysis upon activation with the alpha-1 agonist, phenylephrine.

Angiotensin II activates Stat5 through Jak2 kinase in cardiac myocytes.

Angiotensin II (Ang II) treatment was recently shown to activate Jak2, Stat1, and Stat3 proteins in cardiac myocytes. Angiotensin-converting enzyme (ACE) inhibitors have been shown to be an effective clinical treatment following myocardial infarction, implying that inhibition of Ang II production is beneficial in this pathological condition. Some of the effects of Ang II in cardiac myocytes may be mediated by the JAK-STAT signaling pathway. The AT1 receptor was the first G-protein-coupled-receptor reported to activate the JAK-STAT pathway. Recently, however, another G-protein-coupled-receptor (i.e. serotonin) was also shown to signal through the JaK2 and STAT proteins in myoblasts. We hypothesized that Ang II treatment might also activate Stat5 transcription factors in cardiac myocytes. In this study, we provide evidence that the G-protein-coupled, Ang II type I (AT1) receptor couples to activation of Stat5 through Jak2 kinase in neonatal rat ventricular myocytes. Angiotensin II induces a 1.5- to 10-fold increase in a Stat5 transcription complex, which binds to the prolactin-inducing element (PIE). By Western analysis, Stat5 protein levels were shown to be tyrosine phosphorylated two- to three-fold over control, following. Ang II treatment of cardiac myocytes. Phosphorylation of Stat5a and Stat5b proteins was rapid and sustained (30-60 min), and Jak2 kinase co-immunoprecipitated with activated Stat5 proteins. In cardiac myocytes, Stat5 proteins co-immunoprecipitated with the AT1 receptor. Selective inhibition of Jak2 kinase with AG-490 blocked formation of prolactin-inducing factor (PIF) complexes by Ang II, suggesting that Jak2 kinase was required for the tyrosine phosphorylation of Stat5 in cardiac myocytes.

Adenosine A3 receptor agonists inhibit murine macrophage tumor necrosis factor-alpha production in vitro and in vivo.

Adenosine and related analogs have been shown to regulate a variety of cell functions through different classes of adenosine receptors. Murine J774.1 macrophage cells were found to predominantly express adenosine A3 receptor RNA relative to adenosine A1 receptor or adenosine A2 receptor RNA. Adenosine receptor agonists, in a dose-dependent manner characteristic of the adenosine A3 receptor, blocked endotoxin-induction of the TNF-alpha gene and TNF-alpha protein expression in the J774.1 macrophage cell line. The adenosine A3 receptor antagonist BW-1433 dose-dependently reversed this adenosine receptor agonist inhibitory effect on TNF-alpha gene expression. Thus, the binding of adenosine receptor agonists to the adenosine A3 receptor interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF-alpha, revealing a novel cross-talk between the murine adenosine A3 receptor and the endotoxin CD14 receptor in J774.1 macrophages.

The type I angiotensin II receptor couples to Stat1 and Stat3 activation through Jak2 kinase in neonatal rat cardiac myocytes.

The octapeptide, angiotensin II, has a modulatory role on cardiac cellular growth associated with hypertension and in compensatory remodeling following myocardial infarction. The molecular signal transduction pathways that participate in these and other cellular actions in response to angiotensin II are presently being elucidated. The signal transducers and activators of transcription (STAT) pathway directly links cytokine and growth factor receptors with transcriptional activity. We provide evidence that the G protein-linked, angiotensin II, AT1-receptor couples to activation of the STAT pathway in neonatal rat cardiac myocytes. Angiotensin II induces primarily sis-inducing factor (SIF) B and to a lesser extent SIF-C and SIF-A. The EC50 of this response was 40 nM and Stat1 and Stat3 proteins were identified as components of the SIF complexes. Stat1 and Stat3 were tyrosine phosphorylated five-fold and three-fold, respectively, over control levels following angiotensin II treatment of cardiac myocytes. Phosphorylation of Stat1 and Stat3 proteins was rapid (5 min) and sustained (60 min). Jak2 was also tyrosine phosphorylated eight-fold by angiotensin II treatment, and phosphorylated Stat1 and Stat3 proteins co-immunoprecipitated with activated Jak2 kinase. Selective inhibition of Jak2 kinase with AG-490 blocked formation of angiotensin II induced SIF complexes, suggesting that Jak2 kinase is required for cardiomyocyte SIF induction. In addition, Jak2, Stat1 and Stat3 proteins co-immunoprecipitated with the AT1-receptor. These are the first data to demonstrate coupling of a G-protein coupled receptor, AT1, to the JAK-STAT pathway in primary cultured cardiac myocytes and suggest that this pathway may be involved in transcriptional regulation by angiotensin II.

Specific transcriptional inhibition of bone marrow-derived macrophage tumor necrosis factor-alpha gene expression and protein production using novel enantiomeric carbocyclic nucleoside analogues.

Tumor necrosis factor-alpha (TNF-alpha) is a powerful macrophage-derived proinflammatory cytokine, via both direct effects on host tissues as well as indirectly through the induction of other proinflammatory mediators, including interleukin- (IL) 1 beta and IL-6. Activation of murine bone marrow-derived macrophages (BMDM phi) with lipopolysaccharide (LPS) causes rapid expression of TNF-alpha, which as an autocrine factor enhances BMDM phi function through IL-1 beta and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDM phi TNF-alpha using novel enantiomeric carbocyclic nucleoside analogues. BMDM phi were derived in vitro from murine bone marrow progenitors using colony stimulating factor-1 and treated with combinations of LPS (1-100 nG/ml) and the enantiomeric carbocyclic nucleoside (10-100 microM) analogues MDL 201, 112 (9-[(1S,3R)-cis-cyclopentan-3-ol]adenine); MDL 201,451 (9-[1R,3S)-cis-cyclopentan-3-ol]adenine); MDL 201,449 (9-[(1R,3R)-trans-cyclopentan-3-ol]adenine) and MDL 201,484 (9-[(1S,3S)-trans-cyclopentan-3-ol]adenine). Northern blot analysis showed that MDL 201,449 was the most effective agent in vitro at selectively inhibiting TNF-alpha. MDL 201,449 reduced TNF-alpha mRNA levels by nearly 50% for up to 4 hr after the simultaneous addition of LPS and the synthetic agent. In contrast, mRNA and secreted protein levels for IL-1 beta (measured by the D10.S bioassay) and mRNA for TNF-alpha receptor p60 and TNF-alpha receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDM phi up-to 2 hr after LPS treatment and at concentrations as low as 10 microM. Regulation of BMDM phi IL-6 by carbocyclic nucleoside analogues in response to LPS appears to be both concentration and time dependent, because IL-6 mRNA and secreted protein levels were inhibited at only high drug concentrations (100 microM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that M phi-derived proinflammatory cytokine gene expression is differentially, rather than coordinately, regulated by selective signal transduction and/or molecular pathways. Enantiomeric carbocyclic nucleoside analogues that specifically inhibit TNF-alpha may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-alpha has been shown to have an important role in initiating the early stages of disease.

Molecular mechanisms of angiotensin II in modulating cardiac function: intracardiac effects and signal transduction pathways.

Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system (RAS), regulates volume and electrolyte homeostasis and is involved in cardiac and vascular cellular growth in humans and other species. This system, which has been conserved throughout evolution, plays an important role in cardiac and vascular pathology associated with hypertension, coronary heart disease, myocarditis and congestive heart failure. The traditional RAS is viewed as a system in which circulating Ang II is delivered to target organs and cells. However, in the past decade, a local RAS has been described in cardiac cells, providing evidence for autocrine and paracrine pathways by which biological actions of Ang II could be mediated. The critical actions of Ang II are mediated primarily through the AT1, G-protein (guanylyl nucleotide binding protein) coupled receptor. In addition to coupling to conventional G-protein signal transduction pathways, the AT1 receptor was recently shown to increase the tyrosine phosphorylation of several intracellular substrates, including the STAT (Signal Transducers and Activators of Transcription) family of novel transcription factors, in rat cardiac fibroblasts, myocytes and vascular smooth muscle cells, and AT1 receptor transfected CHO cells. It has been shown that Ang II stimulates the tyrosine phosphorylation and nuclear translocation of Stat1 (Stat 91) and Stat3 (Stat 92). Angiotensin II acting directly through the AT1 receptor, induces the formation of a complex of STAT proteins termed SIF (sis-inducing factor) which binds the DNA sequence, SIE (sis-inducing element) present in the promotor element of many genes. This provides evidence for a direct role of Ang II in mediating inflammatory and remodeling responses through the JAK-STAT pathway. Thus, it is likely that the JAK-STAT pathway has an important role in Ang II-mediated effects on gene transcription, cardiac and vascular cellular growth/development, and inflammatory responses.