Intracellular Signal Transduction Network Research Articles

O-GlcNAcylation mediates H2O2-induced apoptosis through regulation of STAT3 and FOXO1.

The O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is a critical post-translational modification that couples the external stimuli to intracellular signal transduction networks. However, the critical protein targets of O-GlcNAcylation in oxidative stress-induced apoptosis remain to be elucidated. Here, we show that treatment with H2O2 inhibited O-GlcNAcylation, impaired cell viability, increased the cleaved caspase 3 and accelerated apoptosis of neuroblastoma N2a cells. The O-GlcNAc transferase (OGT) inhibitor OSMI-1 or the O-GlcNAcase (OGA) inhibitor Thiamet-G enhanced or inhibited H2O2-induced apoptosis, respectively. The total and phosphorylated protein levels, as well as the promoter activities of signal transducer and activator of transcription factor3 (STAT3) and Forkhead box protein O 1 (FOXO1) were suppressed by OSMI-1. In contrast, overexpressing OGT or treating with Thiamet-G increased the total protein levels of STAT3 and FOXO1. Overexpression of STAT3 or FOXO1 abolished OSMI-1-induced apoptosis. Whereas the anti-apoptotic effect of OGT and Thiamet-G in H2O2-treated cells was abolished by either downregulating the expression or activity of endogenous STAT3 or FOXO1. These results suggest that STAT3 or FOXO1 are the potential targets of O-GlcNAcylation involved in the H2O2-induced apoptosis of N2a cells.

Postnatal skeletal muscle myogenesis governed by signal transduction networks: MAPKs and PI3K–Akt control multiple steps

Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle regeneration provides an in vivo model system of postnatal myogenesis. It comprises multiple steps including muscle stem cell (or satellite cell) quiescence, activation, migration, myogenic determination, myoblast proliferation, myocyte differentiation, myofiber maturation, and hypertrophy. A variety of extracellular signaling and subsequent intracellular signal transduction pathways or networks govern the individual steps of postnatal myogenesis. Among them, MAPK pathways (the ERK, JNK, p38 MAPK, and ERK5 pathways) and PI3K–Akt signaling regulate multiple steps of myogenesis. Ca2+, cytokine, and Wnt signaling also participate in several myogenesis steps. These signaling pathways often control cell cycle regulatory proteins or the muscle-specific MyoD family and the MEF2 family of transcription factors. This article comprehensively reviews molecular mechanisms of the individual steps of postnatal skeletal muscle myogenesis by focusing on signal transduction pathways or networks. Nevertheless, no or only a partial signaling molecules or pathways have been identified in some responses during myogenesis. The elucidation of these unidentified signaling molecules and pathways leads to an extensive understanding of the molecular mechanisms of myogenesis.

Miracle molecule: Salicylic acid

Salicylic acid, a naturally occurring plant hormone acting as an important signaling molecule adds to tolerance against abiotic stresses. It plays a vital role in plant growth, ion uptake and transport. Salicylic acid is also involved in endogenous signaling to trigger plant defense against pathogens. This positive effect of SA could be attributed to an increased CO2 assimilation and photosynthetic rate and increased mineral uptake by the stressed plant under SA treatment. The application of salicylic acid, acetylsalicylic acid or other analogues of SA, to leaves of plants accelerated their leaf area and dry mass production, but plant height and root length remained unaffected. SA reduced the Na uptake of plants and/or increased the uptake of N, P, K, Ca, Mg and the other minerals as compared to control treatment under salt stress. Salicylic acid (SA) plays many roles in plant physiology. Besides pathogenesis-related resistance, SA is involved in the response to abiotic stress. However, the effects of SA on plant resistance to abiotic stress were found contra dictionary, and the actual role of SA in abiotic stress remains unresolved. Generally, deficiency of SA or a very high level of SA increase the plant susceptibility to abiotic stress. The optimal levels for the highest stress tolerance range from 0.1 mm to 0.5 mm for most plants. But the role of SA at a certain level in moderate and severe abiotic stress may be different. This can be attributed to redox regulations in plant cells. In this paper, we discuss the relationship between reactive oxygen species (ROS) and SA, and propose a subsequent intracellular signal transduction network of SA and ROS under abiotic stress. Anti-stress substances besides antioxidant enzymes induced by SA are also summarized.

PLC\u03b32 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer’s disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.

A Bayesian Active Learning Experimental Design for Inferring Signaling Networks.

Machine learning methods for learning network structure are applied to quantitative proteomics experiments and reverse-engineer intracellular signal transduction networks. They provide insight into the rewiring of signaling within the context of a disease or a phenotype. To learn the causal patterns of influence between proteins in the network, the methods require experiments that include targeted interventions that fix the activity of specific proteins. However, the interventions are costly and add experimental complexity. We describe an active learning strategy for selecting optimal interventions. Our approach takes as inputs pathway databases and historic data sets, expresses them in form of prior probability distributions on network structures, and selects interventions that maximize their expected contribution to structure learning. Evaluations on simulated and real data show that the strategy reduces the detection error of validated edges as compared with an unguided choice of interventions and avoids redundant interventions, thereby increasing the effectiveness of the experiment.

Modeling Cell-to-Cell Communication Networks Using Response-Time Distributions.

SUMMARYCell-to-cell communication networks have critical roles in coordinating diverse organismal processes, such as tissue development or immune cell response. However, compared with intracellular signal transduction networks, the function and engineering principles of cell-to-cell communication networks are far less understood. Major complications include: cells are themselves regulated by complex intracellular signaling networks; individual cells are heterogeneous; and output of any one cell can recursively become an additional input signal to other cells. Here, we make use of a framework that treats intracellular signal transduction networks as “black boxes” with characterized input-to-output response relationships. We study simple cell-to-cell communication circuit motifs and find conditions that generate bimodal responses in time, as well as mechanisms for independently controlling synchronization and delay of cell-population responses. We apply our modeling approach to explain otherwise puzzling data on cytokine secretion onset times in T cells. Our approach can be used to predict communication network structure using experimentally accessible input-to-output measurements and without detailed knowledge of intermediate steps.

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Cellular responses to stimuli involve dynamic and localized changes in protein kinases and phosphatases. Here, we report a generalized functional assay for high-throughput profiling of multiple protein phosphatases with subcellular resolution and apply it to analyze coxsackievirus B3 (CVB3) infection counteracted by interferon signaling. Using on-plate cell fractionation optimized for adherent cells, we isolate protein extracts containing active endogenous phosphatases from cell membranes, the cytoplasm, and the nucleus. The extracts contain all major classes of protein phosphatases and catalyze dephosphorylation of plate-bound phosphosubstrates in a microtiter format, with cellular activity quantified at the end point by phosphospecific ELISA. The platform is optimized for six phosphosubstrates (ERK2, JNK1, p38α, MK2, CREB, and STAT1) and measures specific activities from extracts of fewer than 50,000 cells. The assay was exploited to examine viral and antiviral signaling in AC16 cardiomyocytes, which we show can be engineered to serve as susceptible and permissive hosts for CVB3. Phosphatase responses were profiled in these cells by completing a full-factorial experiment for CVB3 infection and type I/II interferon signaling. Over 850 functional measurements revealed several independent, subcellular changes in specific phosphatase activities. During CVB3 infection, we found that type I interferon signaling increases subcellular JNK1 phosphatase activity, inhibiting nuclear JNK1 activity that otherwise promotes viral protein synthesis in the infected host cell. Our assay provides a high-throughput way to capture perturbations in important negative regulators of intracellular signal-transduction networks.

MAKAP-a master scaffold for cardiac remodeling.

Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or "signalosomes" are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPβ serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPβ signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2, and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy.

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils.

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.

Abstract 184: DNA damage response in quiescent fibroblasts elicits a distinct secretory program and confers acquired resistance to cancer therapies

Abstract Cancer treatments including chemotherapy and radiation not only target neoplastic cells thus providing a favorable therapeutic index in early clinical stages, but generate a profound impact on benign cells of the tumor microenvironment (TME). In contrast to cancer cells, the majority of human somatic populations exist in a quiescence stage, which is characterized by reversible exit from the G0/G1 phase of cell cycle, and they can reenter the cell division cycle and continue proliferation under favorable conditions. In this study we sought to determine how quiescent cells respond to DNA-targeting agents originally utilized to kill cancer cells, and to establish the consequence of such alterations to the TME. The results of this study would have broad implications for physiology, cancer biology and targeted therapies. Using genome-wide microarrays, we examined the response of primary human prostate fibroblasts made quiescent by contact inhibition to ionizing radiation, and evaluated the pathological influence through an in vitro system of epithelial-stromal interactions. We found quiescent cells maintained an intact DNA damage response including formation of chromatin foci and although they entered senescence, the proliferative potential was reserved as determined by cell replicative growth surveillance. A secretory expression program based on DNA damage was developed in quiescent cells, termed QASP; this stress-response program was different from that of proliferative cells (DDSP) and the spectrums only partially overlapped. In each case, an intracellular signal transduction network activated by DNA damage repair activities, including but not limited to the mTOR pathway and NF-ΚB complex, emerged and was implicated in mediating a cascade of reactions that culminated in robust secretion, after acute cellular responses. Our data indicate the influence of secreted factors from damaged quiescent fibroblasts on prostate cancer (PCa) cells was comprehensive and significant. In particular, paracrine factors of damaged quiescent fibroblasts supported PCa resistance to cytotoxic therapies. Intriguingly, small molecular inhibitors targeting the mTOR pathway or NF-ΚB complex were able to abrogate secretion of vast majority of quiescent cell-derived effectors thereby minimizing the pathological consequences. To date, a primary barrier to treatment of advanced cancers is the ability of neoplastic cells to acquire resistance to cytotoxicity that results in circumvention of effective targeting. Defining mechanisms underlying drug resistance is critical for designing new therapeutic strategies to selectively bypass or restrain resistance pathways. As such, targeting QASP effectors or more systematically, resistance mechanisms engaged by TME, in conjunction with conventional therapies may improve clinical response to anti-cancer treatments and prolong the patient lifespan. Citation Format: Yu Sun, Ilsa Coleman, Stephen Plymate, Peter Nelson. DNA damage response in quiescent fibroblasts elicits a distinct secretory program and confers acquired resistance to cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 184. doi:10.1158/1538-7445.AM2014-184

Boolean networks with veto functions.

Boolean networks are discrete dynamical systems for modeling regulation and signaling in living cells. We investigate a particular class of Boolean functions with inhibiting inputs exerting a veto (forced zero) on the output. We give analytical expressions for the sensitivity of these functions and provide evidence for their role in natural systems. In an intracellular signal transduction network [Helikar et al., Proc. Natl. Acad. Sci. USA 105, 1913 (2008)], the functions with veto are over-represented by a factor exceeding the over-representation of threshold functions and canalyzing functions in the same system. In Boolean networks for control of the yeast cell cycle [Li et al., Proc. Natl. Acad. Sci. USA 101, 4781 (2004); Davidich et al., PLoS ONE 3, e1672 (2008)], no or minimal changes to the wiring diagrams are necessary to formulate their dynamics in terms of the veto functions introduced here.

Semaphorin signalling during development.

Semaphorins are secreted and membrane-associated proteins that regulate many different developmental processes, including neural circuit assembly, bone formation and angiogenesis. Trans and cis interactions between semaphorins and their multimeric receptors trigger intracellular signal transduction networks that regulate cytoskeletal dynamics and influence cell shape, differentiation, motility and survival. Here and in the accompanying poster we provide an overview of the molecular biology of semaphorin signalling within the context of specific cell and developmental processes, highlighting the mechanisms that act to fine-tune, diversify and spatiotemporally control the effects of semaphorins.

Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation.

The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation.

The scaffold protein muscle A-kinase anchoring protein β orchestrates cardiac myocyte hypertrophic signaling required for the development of heart failure.

Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. Using conditional, cardiac myocyte-specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure.

Light-Mediated Kinetic Control Reveals the Temporal Effect of the Raf/MEK/ERK Pathway in PC12 Cell Neurite Outgrowth

It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.

CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes

BackgroundCIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.ResultsWe now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [3H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.ConclusionsThese results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.

Abstract CN07-01: Modulation of proinflammatory and anti-inflammatory signaling for cancer chemoprevention

Abstract A new horizon in chemoprevention research is the recent discovery of molecular links between inflammation and cancer. The implication of inflammatory tissue damage in pathophysiology of human maligancy is under intense investigation both at the research level and in clinical practice. Numerous studies have been reported with the global ‘omics’ profiling technologies, such as DNA microarray, proteomics, metabolomics, lipidomics, etc., to identify and characterize a series of critical molecules/changes in the inflammatory signaling. It is by gaining this type of mechanistic understanding of a disease that researchers will unlock the keys to discovering new diagnostics and therapeutic strategies for the management of inflammation-associated cancer. Aberrant upregulation of cyclooxygenase-2 (COX-2), a key enzyme in the arachidonic acid cascade, has been frequently observed in various precancerous and malignant tissues. Therefore, the normalization of inappropriately overamplified signaling cascades implicated in chronic inflammation-associated carcinogenesis by use of COX-2 specific inhibitors has been recognized as a rational and pragmatic strategy in molecular target-based chemoprevention. Proinflammatory stimuli trigger the activation of intracellular signal transduction network comprising proline-directed serine/threonine kinases, and their downstream transcription factors, such as NF-κB, resulting in an overexpression of COX-2. Cyclopentenone prostaglandins of J series as products of cyclooxygenase-2 (COX-2)-mediated arachidonic acid cascades have been reported to possess multifaceted cellular functions, including antiinflammatory and cytoprotective effects. A typical example is 15-deoxy-Δ 12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor-γ (PPARγ), that modulates the activities of various intracellular signaling molecules. Because of the electrophilic α,β-unsaturated carbonyl moiety present in its cyclopentenone ring structure, 15d-PGJ2 acts as a Michael addition acceptor and can readily interact with critical cellular nucleophiles, such as cysteine thiol groups of proteins. Many of the biological effects induced by 15d-PGJ2 are mediated by targeting redox-sensitive transcription factors and their regulators, including IκB kinase-NF-κB, AP-1, Nrf2-Keap1, HIF-1α, STAT3and p53 tumor suppressor. The regulation of the aforementioned signaling molecules by 15d-PGJ2 is not likely to be dependent on PPARγ activation, but rather involves direct modification or oxidation of their critical cysteine residues acting as a redox-sensor. Though 15d-PGJ2 exerts cytoprotective effects in normal cells, elevated production of this cyclopentenone prostaglandin has been implicated in tumorigenesis through induction of angiogenesis and metastasis. As one of the terminal products of COX-2-mediated arachidonic acid metabolism, 15d-PGJ2 upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line by activation of Akt and subsequently AP-1. Some malignant cells might exploit this visious cycle of COX-2 up-expression for maintaining invasiveness and taking survival advantage. Cysteine thiols present in various transcription factors and their regulators function as redox sensors in fine-tuning of transcriptional regulation. Thus, oxidation or covalent modification of thiol groups present in the redox-sensitive transcription factors and their regulating molecules can provide a unique strategy for molecular target-based chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN07-01.

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.

Modeling the action of drugs on cellular enzymes by means of optimal control techniques

In this paper we apply Optimal Control techniques, based on the solution of Hamilton–Jacobi–Bellman equations, to simulate the action of a drug on some sub-networks of an intracellular signal transduction network. The cost functional to be minimized is chosen in order to take into account, in the models, different factors, like the toxicity of some reactants, the drug action and the costs of the drug itself. Numerical results are also shown, to test the model.