Signal Transducer And Activator Of Transcription 5 Expression Research Articles

Effects of High-Dose IFNα2b on Regional Lymph Node Metastases of Human Melanoma: Modulation of STAT5, FOXP3, and IL-17

Signal transducer and activator of transcription 5 (STAT5) and STAT3 oppose one another in regulation of the reciprocal development of CD4+CD25+FOXP3+ regulatory T cells (Treg) and T helper 17 (Th17). A reduction in STAT3 is associated with up-regulation of Treg, and STAT5 activation promotes Treg differentiation or function while constraining Th17 generation. The effects of IFNalpha on STAT signaling in relation to tumor tissue Treg and Th17 have not been documented in humans beyond the observations that IFNalpha2b down-regulates STAT3. Following diagnostic biopsy and before definitive surgery, 20 doses of high-dose IFNalpha2b (HDI) were administered to patients with stage IIIB melanoma who gave written informed consent. Lymph node biopsies, in which both total STAT3 and phosphorylated STAT3 were down-regulated by HDI, were probed with STAT5, FOXP3, CD4, and interleukin 17 (IL-17) with immunohistochemistry and/or immunofluorescence techniques. The percentage of FOXP3+ lymphocytes determined by immunohistochemistry was up-regulated from 3.06 +/- 0.65% to 9.86 +/- 1.27% (n = 13, P = 0.0002), and this observation was confirmed by immunofluorescence evaluation of CD4+FOXP3+ Tregs. HDI induced STAT5 up-regulation (five cases observed) in melanoma cells and lymphocytes but did not induce the generation of IL-17-expressing lymphocytes. Increased STAT5 expression was associated with increased FOXP3 expression among lymphocytes, and STAT5 was constitutively activated among both melanoma cells and lymphocytes. IFNalpha2b up-regulates STAT5 and down-regulates STAT3, in conjunction with up-regulation of Treg and inhibition of IL-17-expressing lymphocytes in melanoma tissues. These findings suggest that the effects of IFNalpha may be potentiated through interference with the response of Tregs and/or STAT5.

Insulin Decreases Inflammatory Signal Transcription Factor Expression in Primary Human Liver Cells after LPS Challenge

Hepatic homeostasis is essential for survival in critically ill and burned patients. Insulin administration improves survival and decreases infections in these patients. To determine the molecular mechanisms, the aim of the present study was to establish a stress model using primary human hepatocytes (PHHs) and to study the effects of insulin on the hepatic inflammatory signaling cascade. Liver tissue was obtained from general surgical patients, and PHHs were isolated and maintained in culture. Primary hepatocyte cultures were challenged with various doses of lipopolysaccharide (LPS), and the inflammatory signal transcription cascade was determined by real-time PCR. In subsequent experiments, primary hepatocyte cultures were challenged with LPS and insulin was added in various doses. Glucose was determined by colorimetric assays. PHHs treated with 100 microg/mL LPS showed a profound inflammatory reaction with increased expression of interleukin (IL)-6, IL-10, IL-1beta, tumor necrosis factor (TNF), and signal transducer and activator of transcription 5 (STAT-5). Insulin at 10 IU/mL significantly decreased IL-6, TNF, and IL-1beta at pretranslational levels, an effect associated with decreased STAT-5 mRNA expression (P < 0.05). Glucose concentration and cellular metabolic activity were not different between controls and insulin-treated cells. Based on our results, we suggest that primary hepatocyte cultures can be used to study the effect of LPS on the inflammatory cascade. Insulin decreases hepatic cytokine expression, which is associated with decreased STAT-5 expression.

Short Communication: A Milk Trait-Associated Polymorphism in the Bovine Growth Hormone Receptor Gene Does Not Affect Receptor Signaling

Growth hormone (GH), also known as somatotropin, stimulates milk production in cows. At the tissue level, the action of GH is mediated by the GH receptor (GHR) and the receptor-activated intracellular signaling pathway involving Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). A T/A nucleotide variation in exon 8 of the bovine GHR gene, resulting in a phenylalanine to tyrosine change in the transmembrane domain of the GHR protein, has been reported to be associated with a major effect on milk yield in cows. The objective of this study was to determine whether the 2 versions of GHR differ in mediating GH-induced STAT5 activation of gene expression. We created cDNA expression plasmids for the 2 versions of GHR and cotransfected each of them with a STAT5 expression plasmid and a luciferase reporter gene construct containing STAT5 binding sites into 2 different cell lines. Treatment of the transfected cells with various concentrations of GH triggered a dose-dependent increase in luciferase activity. However, the GH-induced luciferase activity was not different between the 2 GHR expression plasmids, indicating that the 2 GHR forms did not differ in mediating GH-induced STAT5 activation of gene expression. Thus, if the T/A polymorphism in exon 8 of the GHR gene has a causative effect on milk production, this effect is unlikely to be mediated by the JAK2-STAT5 pathway, the currently known major signaling pathway from the growth hormone receptor.

Naturally Occuring N-Terminal Truncated STAT5 Isoforms Are Widely Expressed, Functional, and Have Enhanced Nuclear Localization.

Signal transducer and activator of transcription 5(STAT5) plays major roles in hematopoietic stem cell and progenitor cell development. Our previous studies have shown that mast cells derived from STAT5ab−/− mice in which the first two exons were deleted, had severe defects in interleukin (IL)-3 and stem cell factor (SCF) induced proliferation and survival signaling. However, expression of an endogenous hypomorphic N-terminal truncated form of STAT5 through cryptic start codons found at position 102 and 136 was uncovered, suggesting the possibility of compensation for STAT5-mediated functions. ΔN isoforms of STAT5 were also identified in different wild-type and STAT5ab−/− mouse tissues including significant expression in brain, liver, spleen, lung, kidney, but not in heart. Here we describe that endogenous mast cell STAT5ΔN had surprisingly increased nuclear localization in STAT5ab−/− mast cells even without cytokine stimulation but which increased in response to IL-3 and SCF. In general, the N-terminus is responsible for tetramerization and regulates receptor recognition and phosphatase recruitment. Others have reported that N-terminal truncated mutants of STAT1 and STAT4 failed to migrate into nuclei upon cytokine stimulation. Therefore, the N-terminus of STAT5 functions differently. To further characterize this function of STAT5aΔN136, we co-transfected STAT5aΔN136 with mouse erythropoietin receptor (EPOR) into 293T cells, and subsequently, stimulated the cells with EPO (20 μg/ml). Consistent with the mast cell studies, immunostaining revealed that STAT5aΔN136 had enhanced nuclear retention relative to full-length STAT5a. Both STAT5a and STAT5aΔN136 migrated to the nucleus upon EPO stimulation for 30 minutes. EPO stimulation also showed a similar pattern of phosphorylation, however, STAT5aΔN136 phosphorylation was weaker and more transient, compared to full-length STAT5a. A known constitutively active STAT5aS711F mutant used as a control, showed extremely high and persistent phosphorylation levels. To test for functional activity in mast cells, we used MSCV-based retroviral vectors expressing either STAT5a or STAT5aΔN136 upstream of GFP and transduced STAT5ab−/− mast cells. Initial transduction efficiencies of 20–40% were obtained with all vectors. After 10–25 days of culture, %GFP+ cells rose to 85–100% for both STAT5a and STAT5aΔN136, while the mast cells transduced with an MSCV control vector remained about 20% GFP+. Notably, in mast cells and GP+E86 ecotropic producer cells, overexpession of retroviral full-length STAT5a resulted in detectable STAT5aΔN136 co-expression from the same vector, indicating that effects due to STAT5 expression are potentially modulated by ΔN isoforms generated through alternative internal start codons. Overall, this work indicates that ΔN isoforms of STAT5 are capable of enhanced nuclear localization and can promote growth in STAT5ab−/− mast cells, suggesting that they likely participate in normal signaling functions. Studies to assess transcriptional activity of these isoforms using STAT promoter luciferase constructs is ongoing. A better understanding of the regulation of these natural isoforms of STAT5a and STAT5b and their physiological significance may lead to novel insights into JAK/STAT signaling.

Aryl hydrocarbon receptor-mediated suppression of GH receptor and Janus kinase 2 expression in mice

Differential mRNA display revealed that a cDNA encoding the major urinary protein 2 (MUP2) that belongs to the lipocalin superfamily was absent in livers of mice treated with 3-methylcholanthrene (MC). The expression of MUP2 is known to be stimulated by growth hormone (GH), through the GH receptor (GHR), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signal transduction pathway. Since MC is an aryl hydrocarbon receptor (AhR) ligand, the effects of MC treatment on the expression of GHR, JAK2 or STAT5 in the livers of wild-type or AhR-null mice were examined. The result indicated that the expression of GHR and JAK2 mRNA was greatly decreased by MC in wild-type mice but not in AhR-null mice. In addition, the binding activity of STAT5 bound to STAT5-binding element was reduced after MC treatment in wild-type mice but not in AhR-null mice. Based on these results, we conclude that the suppression of MUP2 mRNA expression by MC is caused by the AhR-mediated disruption of the GH signaling pathway. Possible mechanism(s) by which exposure to aromatic hydrocarbons causes a decrease in the body weight of mice, which has been referred to as wasting syndrome, will also be discussed.