Constitutive activation of the Raf-MAPK pathway causes negative feedback inhibition of Ras-PI3K-AKT and cellular arrest through the EphA(2) receptor

The Raf-mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)-AKT pathways are two downstream effectors of the small GTPase Ras. Although both pathways are positively regulated by Ras, the Raf-MAPK and PI3K-AKT pathways have been shown to control opposing functions within the cell, suggesting a need for cross-talk regulation. The PI3K -AKT pathway can inhibit the Raf-MAPK pathway directly during processes such as muscle differentiation. Here we describe the ability of the Raf-MAPK pathway to negatively regulate the PI3K-AKT pathway during cellular arrest. Constitutive activation of Raf or methyl ethyl ketone 1 (MEK1) leads to inhibition of AKT and cellular arrest. Furthermore, we show that activation of Raf-MEK1 signaling causes negative feedback inhibition of Ras through the ephrin receptor EphA(2). EphA(2)-mediated negative feedback inhibition is required for Raf-induced AKT inhibition and cell cycle arrest, therefore establishing the inhibition of the Ras-PI3K-AKT pathway as a necessary event for the Raf-MEK1-regulated cellular arrest.

Antigen-specific T-T interactions regulate CD4 T-cell expansion.

The regulation of CD4 T cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate MHC/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way CD4 T cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

Transcriptional upregulation of SOCS 1 and suppressors of cytokine signaling 3 mRNA in the absence of suppressors of cytokine signaling 2 mRNA after infection with West Nile virus or tick-borne encephalitis virus.

Suppressors of cytokine signaling (SOCS) proteins are a family of proteins that are able to act in a classic negative feedback loop to regulate cytokine signal transduction. The regulation of the immune response by SOCS proteins may contribute to persistent infection or even a fatal outcome. In this study, we have investigated the induction of SOCS 1-3 after peripheral infection with West Nile virus (WNV) or tick-borne encephalitis virus (TBEV) in the murine model. We have shown that the cytokine response after infection of mice with WNV or TBEV induces an upregulation in the brain of mRNA transcripts for SOCS 1 and SOCS 3, but not SOCS 2. We hypothesize that SOCS proteins may play a role in limiting cytokine responses in the brain as a neuroprotective mechanism, which may actually enhance the ability of neuroinvasive viruses such as WNV and TBEV to spread and cause disease.

MicroRNA-155 Promotes Resolution of Hypoxia-Inducible Factor 1{alpha} Activity

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription.

A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a "self-eating" cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.

A novel reciprocal and biphasic relationship between membrane cholesterol and beta-secretase activity in SH-SY5Y cells and in human platelets.

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the A beta peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane beta-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane beta-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH-SY5Y beta-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane beta-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.

N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.

The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression

Suppressors of cytokine signaling (SOCS) are encoded by immediate early genes known to inhibit cytokine responses in a classical feedback loop. SOCS gene expression has been shown to be induced by many cytokines, growth factors, and innate immune stimuli, such as LPS. In this paper, we report that the chemoattractants, IL-8 and fMLP, up-regulate SOCS1 mRNA in human myeloid cells, primary human neutrophils, PBMCs, and dendritic cells. fMLP rapidly up-regulates SOCS1, whereas the induction of SOCS1 upon IL-8 treatment is delayed. IL-8 and fMLP did not signal via Jak/STATs in primary human macrophages, thus implicating the induction of SOCS by other intracellular pathways. As chemoattractant-induced SOCS1 expression in neutrophils may play an important role in regulating the subsequent response to growth promoting cytokines like G-CSF, we investigated the effect of chemoattractant-induced SOCS1 on cytokine signal transduction. We show that pretreatment of primary human neutrophils with fMLP or IL-8 blocks G-CSF-mediated STAT3 activation. This study provides evidence for cross-talk between chemoattractant and cytokine signal transduction pathways involving SOCS proteins, suggesting that these chemotactic factors may desensitize neutrophils to G-CSF via rapid induction of SOCS1 expression.

SOCS2 Can Enhance Interleukin-2 (IL-2) and IL-3 Signalling by Accelerating SOCS3 Degradation

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.

Probabilistic program analysis for parallelizing compilers

Parallelizing compilers have difficulty analysing and optimising complex code. To address this, some analysis may be delayed until run-time, and techniques such as speculative execution used. Furthermore, to enhance performance, a feedback loop may be setup between the compile time and run-time analysis systems, as in iterative compilation. To extend this, it is proposed that the run-time analysis collects information about the values of variables not already determined, and estimates a probability measure for the sampled values. These measures may be used to guide optimisations in further analyses of the program. To address the problem of variables with measures as values, this paper also presents an outline of a novel combination of previous probabilistic denotational semantics models, applied to a simple imperative language.

Researching Vulnerability: What about the Researcher?

Abstract Purpose – The aim of this paper is to explore researcher vulnerability and identify the ways in which research with vulnerable consumers can impact on consumer researchers. Design/methodology/approach – Provides a review of research literature aiming to raise awareness of researcher vulnerability. Findings – Researchers working in the domain of vulnerable consumers need to be aware that feelings of vulnerability may be reflected back to the researcher. Originality/value – Methodological concerns surrounding the research of vulnerable consumers tend to focus on the welfare of respondents; researcher vulnerability has been largely neglected within the consumer research literature. Historically, problems arising in the research process have tended to be disguised so as not to elicit negative feedback. This paper creates an awareness of such aspects of unthought-of ethical and methodological problems.

Bit-Level systolic architectures for high performance IIR filtering

Several novel systolic architectures for implementing densely pipelined bit parallel IIR filter sections are presented. The fundamental problem of latency in the feedback loop is overcome by employing redundant arithmetic in combination with bit-level feedback, allowing a basic first-order section to achieve a wordlength-independent latency of only two clock cycles. This is extended to produce a building block from which higher order sections can be constructed. The architecture is then refined by combining the use of both conventional and redundant arithmetic, resulting in two new structures offering substantial hardware savings over the original design. In contrast to alternative techniques, bit-level pipelinability is achieved with no net cost in hardware. © 1989 Kluwer Academic Publishers.

Programmable high-performance IIR filter chip

The paper presents a state-of-the-art commercial demonstrator chip for infinite impulse response (IIR) filtering. The programmable IIR filter chip contains eight multiplier/accumulators that can be configured in one of five different modes to implement up to a 16th-order IIR filter. The multiply-accumulate block is based on a highly regular systolic array architecture and uses a redundant number system to overcome problems of pipelining in the feedback loop. The chip has been designed using the GEC Plessey Semiconductors CLA 78000 series gate array, operates on 16-bit two's complement data and has a clock speed of 30 MHz. Issues such as overflow detection and design for testability have also been addressed and are described.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.