GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.     

Glucose diffusion in pancreatic islets of Langerhans.

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.     

异丙肾上腺素对小鼠心肌MAPK、NFκB和JAK/STAT信号转导途径的激活效应

为研究异丙肾上腺素(isoproterenol,ISO)诱导心肌肥厚或心肌重塑的分子机制,本工作以成年雄性Balh／c小鼠为研究对象,通过腹腔注射ISO,采用蛋白免疫印迹杂交方法观察ISO对小鼠心肌丝裂素活化蛋白激酶(mito-gen-activted protein kinase,MAPK)、核因子—κB(NFκB)和Janus激酶／信号转导因子和转录激活因子(JAK／STAT)途径的激活效应。结果发现,ISO腹腔注射后可早期(5min)激活心肌MAPK(ERK1／2和p38);ISO对心肌NFKB的激活效应表现为双相性,激活高峰分别为5和120min;ISO腹腔注射60min后可显著促进STAT3的酪氨酸磷酸化,6h时基本恢复到基础水平。上述结果提示,ISO对多种细胞内信号转导途径均具有激活效应,但表现出明显的时相差异。探明这些信号转导途径的时空整合规律,将有助于深化对心肌重塑发生机制的认识。     

SOCS regulation of the JAK/STAT signalling pathway

The suppressor of cytokine signalling (SOCS) proteins were, as their name suggests, first described as inhibitors of cytokine signalling. While their actions clearly now extend to other intracellular pathways, they remain key negative regulators of cytokine and growth factor signalling. In this review we focus on the mechanics of SOCS action and the complexities of the mouse models that have underpinned our current understanding of SOCS biology.     

Insulin inhibits growth hormone signaling via the growth hormone receptor/JAK2/STAT5B pathway

Insulin is important for maintaining the responsiveness of the liver to growth hormone (GH). Insulin deficiency results in a decrease in liver GH receptor (GHR) expression, which can be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether continuous insulin stimulation, similar to what occurs in hyperinsulinemic states, results in GH resistance. Our present studies suggest that insulin treatment of hepatoma cells results in a time-dependent inhibition of acute GH-induced phosphorylation of STAT5B. Whereas total protein levels of JAK2 were not reduced after insulin pretreatment for 16 h, GH-induced JAK2 phosphorylation was inhibited. There was a concomitant decrease in GH binding and a reduction in immunoreactive GHR levels following pretreatment with insulin for 8-24 h. In summary, continuous insulin treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine phosphorylation of STAT5B. These findings suggest that hepatic GH resistance may develop when a patient exhibits chronic hyperinsulinemia, a condition often observed in patients with obesity and in the early stage of Type 2 diabetes.     

JAK/STAT3-dependent activation of the RalGDS/Ral pathway in M1 mouse myeloid leukemia cells.

The Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral guanine nucleotide dissociation stimulator (RalGDS), a putative effector protein of Ras. Recently, it was proven that Ral regulates c-Src activity and subsequent phosphorylation of its substrate, STAT3. Here, we show that STAT3 inversely regulates activation of Ral through induction of expression of RalGDS. To identify new leukemia inhibitory factor-induced genes, we have performed representational difference analysis using M1 mouse myeloid leukemia cells and cloned RalGDS. The expression of RalGDS and subsequent activation of RalA were clearly suppressed by a dominant negative form of STAT3 and a JAK inhibitor, JAB/SOCS1/SSI-1, indicating that RalGDS/RalA signaling requires the activation of the JAK/STAT3 pathway. An experiment using a Ras inhibitor demonstrated that full activation of RalA also requires activation of Ras. These results suggest a novel cross-talk between JAK/STAT3 and the Ras/RalGDS/Ral signaling pathways through gp130.     

The Polyhomeotic protein induces hyperplastic tissue overgrowth through the activation of the JAK/STAT pathway

Epigenetic mechanisms controlling cellular proliferation are essential to animal development. Moreover, altered levels of expression of the epigenetic regulator proteins are associated with the development and progression of human diseases like cancer. We have studied the effects of high levels of Polyhomeotic (PH) protein, a member of the Polycomb Group (PcG), during the proliferation of the imaginal discs in Drosophila. Over expression of PH protein causes induction of proliferation, accompanied with induction of JNK-dependent apoptosis. As a result, massive hyperplastic overgrowth is produced and the corresponding differentiated tissues show phenotypes related with mis-regulation of homeotic gene expression. We have found that high levels of PH up-regulate the JAK/STAT pathway through the de-repression of Unpaired (UPD), the extracellular ligand of the Drosophila JAK/STAT signalling cascade. Moreover, inactivation of the JAK/STAT pathway in the presence of a large amount of PH protein greatly reduces the tissue overgrowth, demonstrating a functional role of JAK/STAT in PH-induced hyperplasia. Finally, we have observed that decapentaplegic and d-myc, two growth genes and putative targets of the JAK/STAT pathway, are also over expressed in the PH-induced tumors. We propose that during normal development, the PcG proteins act to maintain inactive the JAK/STAT pathway. Upon cellular stress, changes in the levels of PcG proteins expression are induced and JAK/STAT is activated leading to tumor development. Our results show a functional relationship between the PcG gene expression and the JAK/STAT pathway, both of which are found to be perturbed in tumorigenesis.     

Nestin-expressing cells in the pancreatic islets of Langerhans

The pancreatic islets of Langerhans produce several peptide hormones, predominantly the metabolically active hormones insulin and glucagon, which are critical for maintaining normal fuel homeostasis. Some evidence exists that pancreatic endocrine cells turn over at a slow rate and can regenerate in certain conditions. This could be due to the presence of pluripotent cells residing in the pancreas. Recently the intermediate filament protein nestin has been identified to be a marker for a multipotent stem cell in the central nervous system. Given the similarity between the pancreatic islets and neuronal cells, we hypothesized that stem cells expressing nestin might be present in the pancreas. Here we present evidence that a subset of cells in the pancreatic islets express the stem cell marker nestin. These cells might serve as precursors of differentiated pancreatic endocrine cells.     

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.     

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating the JAK/STAT signaling pathway

This article aimed to reveal the mechanism of long noncoding RNA (lncRNA) urothelial cancer-associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via mediating the activation of the JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into the Sham group (without any treatments), KA group, normal control (NC; injection with empty vector) + KA group, and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in the hippocampus was determined by quantitative real time polymerase chain reaction (qRT-PCR). Surviving neurons were counted by Nissl staining, and expression levels of glial cells glial fibrillary acidic protein (GFAP), p-JAK1, and p-STAT3 and glutamate/aspartate transporter (GLAST) were analyzed by immunofluorescence and Western blot analysis. A rat model of TLE was established by intraperitoneal injection of KA. qRT-PCR and fluorescence analyses showed that UCA1 inhibited astrocyte activation in the hippocampus of epileptic rats. Meanwhile, the Morris water maze analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might have a protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of the JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and JAK/STAT/GLAST expression in TLE rats and improved the adverse reactions caused by epilepsy.