Analysis of the Upstream Regulatory Region of the Enhancer of Split m7 gene in Drosophila

The Notch pathway is one of the vital signaling pathways used during Drosophila development. Present in many organisms and extensively studied in D. melanogaster, this pathway serves to transduce signals between neighboring developing cells and inhibits neuronal differentiation by lateral inhibition. The primary targets of Notch are the Enhancer of split (E(spl)) genes. Although the upstream regulatory regions of the E(spl) genes contain biding sites for Suppressor of Hairless, Proneural, and E(spl) proteins, their expression patterns are not identical. There is a hidden complexity in the regulatory regions of these genes that may help explain the conservation of the overall organization of the E(spl) complex between different species of Drosophila. We are interested in determining what is responsible for this variation in expression, and have investigated these genes using different approaches. First, using the upstream regulatory sequence of D. melanogaster and D. pseudoobscura as reference sequences, we compared the upstream regions of the E(spl) m7 gene to D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. grimshawi, D. virilis, and D. mojavensis using BLAT (Kent 2002) and EvoPrinter (Odenwald et al. 2005) applications. Second, we isolated and sequenced part of the regulatory region of D. pseudoobscura m7 to confirm previous published results and gain more insight on the functionality of the region. The differences and similarities in upstream sequences of the E(spl) genes are being used as a tool to help further determine if functions of these genes are conserved. This approach will give insight into which regulatory sites are essential to proper Drosophila development after millions of years of species divergence

Regulation of Signal Transducer and Activator of Transcription Signaling by the Tyrosine Phosphatase PTP-BL

SummarySignal Transducer and Activator of Transcription (STAT) proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation after cytokine stimulation. One mechanism by which STAT signaling is regulated is by dephosphorylation through the action of protein tyrosine phosphatases (PTP). We have identified PTP-Basophil like (PTP-BL) as a STAT PTP. PTP-BL dephosphorylates STAT proteins in vitro and in vivo, resulting in attenuation of STAT-mediated gene activation. In CD4+ T cells, PTP-BL deficiency leads to increased and prolonged activation of STAT4 and STAT6, and consequently enhanced T helper 1 (Th1) and Th2 cell differentiation. Taken together, our findings demonstrate that PTP-BL is a physiologically important negative regulator of the STAT signaling pathway

Mechanisms of the Hepatic Acute-Phase Response during Bacterial Pneumonia▿

The acute-phase response is characterized by increased circulating levels of acute-phase proteins (APPs) generated by the liver. During bacterial pneumonia, APPs correlate with the severity of disease, serve as biomarkers, and are functionally significant. The kinetics and regulatory mechanisms of APP induction in the liver during lung infection have yet to be defined. Here we show that APP mRNA transcription is induced in the livers of mice whose lungs are infected with either Escherichia coli or Streptococcus pneumoniae, and that in both cases this induction occurs in tandem with activation in the liver of the transcription factors signal transducer and activator of transcription 3 (STAT3) and NF-κB RelA. Interleukin-6 (IL-6) deficiency inhibited the activation of STAT3 and the induction of select APPs in the livers of pneumonic mice. Furthermore, liver RelA activation and APP induction were reduced for mice lacking all signaling receptors for tumor necrosis factor alpha and IL-1. In a murine hepatocyte cell line, knockdown of either STAT3 or RelA by small interfering RNA inhibited cytokine induction of the APP serum amyloid A-1, demonstrating that both transcription factors were independently essential for the expression of this gene. These data suggest that during pneumonia caused by gram-negative or gram-positive bacteria, the expression of APPs in the liver depends on STAT3 activation by IL-6 and on RelA activation by early-response cytokines. These signaling axes may be critical for integrating systemic responses to local infection, balancing antibacterial host defenses and inflammatory injury during acute bacterial pneumonia

Alveolar Epithelial STAT3, IL-6 Family Cytokines, and Host Defense during Escherichia coli Pneumonia

While signal transducer and activator of transcription (STAT) 3 signaling has been linked to multiple pathways influencing immune function and cell survival, the direct influence of this transcription factor on innate immunity and tissue homeostasis during pneumonia is unknown. Human patients with dominant-negative mutations in the Stat3 gene develop recurrent pneumonias, suggesting a role for STAT3 in pulmonary host defense. We hypothesized that alveolar epithelial STAT3 is activated by IL-6 family cytokines and is required for effective responses during gram-negative bacterial pneumonia. STAT3 phosphorylation was increased in pneumonic mouse lungs and in murine lung epithelial (MLE)-15 cells stimulated with pneumonic bronchoalveolar lavage fluid (BALF) through 48 hours of Escherichia coli pneumonia. Mice lacking active STAT3 in alveolar epithelial cells (Stat3Δ/Δ) had fewer alveolar neutrophils and more viable bacteria than control mice early after intratracheal E. coli. By 48 hours after E. coli infection, however, lung injury was increased in Stat3Δ/Δ mice. Bacteria were cleared from lungs of both genotypes, albeit more slowly in Stat3Δ/Δ mice. Of the IL-6 family cytokines measured in lungs from infected C57BL/6 mice, IL-6, oncostatin M, leukemia inhibitory factor (LIF), and IL-11 were significantly elevated. Neutralization studies demonstrated that LIF and IL-6 mediated BALF-induced STAT3 activation in MLE-15 cells. Together, these results indicate that during E. coli pneumonia, select IL-6 family members activate alveolar epithelial STAT3, which functions to promote neutrophil recruitment and to limit both infection and lung injury

Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia

Neutropenia and related infections are the most important dose-limiting toxicities in anticancer chemotherapy and radiotherapy. In this study, we explored a new strategy for augmenting host defense in neutropenia-related pneumonia. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) signaling in neutrophils was elevated by depleting PTEN, a phosphatidylinositol 3′-phosphatase that hydrolyzes PtdIns(3,4,5)P3. In myeloid-specific PTEN knockout mice, significantly more neutrophils were recruited to the inflamed lungs during neutropenia-associated pneumonia. Using an adoptive transfer technique, we demonstrated that this enhancement could be caused directly by PTEN depletion in neutrophils. In addition, disruption of PTEN increased the recruitment of macrophages and elevated proinflammatory cytokines/chemokine levels in the inflamed lungs, which could also be responsible for the enhanced neutrophil recruitment. Depleting PTEN also significantly delayed apoptosis and enhanced the bacteria-killing capability of the recruited neutrophils. Finally, we provide direct evidence that enhancement of neutrophil function by elevating PtdIns(3,4,5)P3 signaling can alleviate pneumonia-associated lung damage and decrease pneumonia-elicited mortality. Collectively, these results not only provide insight into the mechanism of action of PTEN and PtdIns(3,4,5)P3 signaling pathway in modulating neutrophil function during lung infection and inflammation, but they also establish PTEN and related pathways as potential therapeutic targets for treating neutropenia-associated pneumonia