Jan B. Hoek

Quantification of Short Term Signaling by the Epidermal Growth Factor Receptor

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15–30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

Ethanol, oxidative stress, and cytokine-induced liver cell injury

Both clinical findings and results of experiments with animal models of alcoholic hepatitis have shown the importance of cytokine-mediated cell-cell interactions in the onset of ethanol-induced liver damage. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta (IL-1 beta), and interleukin-6, are released from Kupffer cells or infiltrating neutrophils and macrophages and elicit defensive responses in parenchymal cells, including activation of apoptosis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), generated in response to cytokine-induced stress signals in parenchymal cells and also by activation of Kupffer cells and inflammatory cells, further mobilize cellular defense mechanisms. When these defensive responses are overwhelmed cells may die by necrosis, further stimulating inflammatory responses and infiltration of neutrophils. Chronic ethanol intake (i.e., many years of heavy alcohol use in human patients, several weeks or months in experimental animals) enhances the damaging consequences of these events through a variety of mechanisms. The formation of cytokines in the liver is stimulated by increasing circulating levels of endotoxin and by enhancing the responsiveness of Kupffer cells to such stimuli. In addition, ethanol promotes oxidative stress, both by increased formation of ROS and by depletion of oxidative defenses in the cell. Furthermore, liver cells from ethanol-treated animals are more susceptible to the cytotoxic effects of TNF-alpha and other cytokines than cells from control animals. Mitochondria play a critical role in the apoptotic response, and alterations in mitochondrial function after chronic ethanol treatment may contribute to enhanced cell death by apoptosis or necrosis. How the shift in the balance of cytokine-induced defensive and damage responses in hepatocytes contributes to the liver injury that occurs in alcoholic hepatitis remains poorly characterized and should be a rewarding area for future studies.

Activation of Glycogen Synthase Kinase 3β Disrupts the Binding of Hexokinase II to Mitochondria by Phosphorylating Voltage-Dependent Anion Channel and Potentiates Chemotherapy-Induced Cytotoxicity

Transformed cells are highly glycolytic and overexpress hexokinase II (HXK II). HXK II is capable of binding to the mitochondria through an interaction with the voltage-dependent anion channel (VDAC), an abundant outer mitochondrial membrane protein. The binding of HXK II to mitochondria has been shown to protect against loss of cell viability. Akt activation inhibits apoptosis partly by promoting the binding of HXK II to the mitochondria, but the mechanism through which Akt accomplishes this has not been characterized. The present report shows that Akt mediates the binding of HXK II to the mitochondria by negatively regulating the activity of glycogen synthase kinase 3beta (GSK3beta). On inhibition of Akt, GSK3beta is activated and phosphorylates VDAC. HXK II is unable to bind VDAC phosphorylated by GSK3beta and dissociates from the mitochondria. Inhibition of Akt potentiates chemotherapy-induced cytotoxicity, an effect that is dependent on GSK3beta activation and its attendant ability to disrupt the binding of HXK II to the mitochondria. Moreover, agents that can force the detachment of HXK II from mitochondria in the absence of Akt inhibition or GSK3beta activation promoted a synergistic increase in cell killing when used in conjunction with chemotherapeutic drugs. Such findings indicate that interference with the binding of HXK II to mitochondria may be a practicable modality by which to potentiate the efficacy of conventional chemotherapeutic agents.

Untangling the wires: A strategy to trace functional interactions in signaling and gene networks

Emerging technologies have enabled the acquisition of large genomics and proteomics data sets. However, current methodologies for analysis do not permit interpretation of the data in ways that unravel cellular networking. We propose a quantitative method for determining functional interactions in cellular signaling and gene networks. It can be used to explore cell systems at a mechanistic level or applied within a “modular” framework, which dramatically decreases the number of variables to be assayed. This method is based on a mathematical derivation that demonstrates how the topology and strength of network connections can be retrieved from experimentally measured network responses to successive perturbations of all modules. Importantly, our analysis can reveal functional interactions even when the components of the system are not all known. Under these circumstances, some connections retrieved by the analysis will not be direct but correspond to the interaction routes through unidentified elements. The method is tested and illustrated by using computer-generated responses of a modeled mitogen-activated protein kinase cascade and gene network.

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770–7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. At a concentration of 125 nm, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA). At this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity. By contrast, Bax concentrations between 250 nm and 1 μmcaused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the MPT induced by Bax. Increasing concentrations of calcium caused a greater proportion of the mitochondria to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT. The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space.

Ethanol and signal transduction in the liver.

The liver is a major target for both short‐ and long‐term actions of ethanol. The mechanisms that mediate the response of cells and tissues to chronic intake of ethanol are unknown, but it is likely that both adaptive and deleterious responses are triggered by short‐term interactions of the cell with ethanol. Cellular signaling processes are candidates to mediate the connection between short‐ and long‐term actions of ethanol. Receptor‐coupled signal transduction systems in the plasma membrane of many different cell types are affected by ethanol. In the liver, the signaling processes associated with phospholipases C and D are particularly responsive to ethanol. In this review, we investigate the direct and indirect short‐term effects of ethanol on the signal transduction systems in liver and discuss the possible implications for the responses of the liver to chronic ethanol exposure.—Hoek, J. B.; Thomas, A. P.; Rooney, T. A.: Higashi, K.; Rubin, E. Ethanol and signal transduction in the liver. FASEB J. 6: 2386‐2396; 1992.

Systems-level interactions between insulin-EGF networks amplify mitogenic signaling.

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal‐regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin‐induced increase in the phosphatidylinositol‐3,4,5‐triphosphate (PIP3) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin‐induced amplification of mitogenic signaling is abolished by disrupting PIP3‐mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non‐linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues.

QUANTIFICATION OF INFORMATION TRANSFER VIA CELLULAR SIGNAL TRANSDUCTION PATHWAYS

A conceptual framework is developed for the quantitative analysis of signal transfer through cellular signal transduction pathways and networks. This approach is referred to as signal transfer analysis and is based on formalisms that were first developed for the analysis of metabolic networks. Signal transduction is quantified as the sensitivity, known as the response coefficient of a target (e.g. an ion channel or transcription factor) to a signal (e.g. a hormone, growth factor or neurotransmitter). This response coefficient is defined in terms of the fractional change in the activated target brought about by a small fractional change in the signal. Quantifying the signal transduction in this way makes it possible to prove that for an idealized signaling cascade without feedback loops, the total response equals the product of all the local response coefficients, one for each level of the cascade. We show under which conditions merely having more levels in a cascade can boost the sensitivity of a target to a signal. If a signal propagates to a target through two different routes, these routes contribute independently to the total response, provided there is no feedback from the target. This independence makes the behavior of signaling cascades different from that of metabolic pathways, where different branches are connected through Kirchhoffs law. The relations between the total response and the local kinetics at each level are given for a number of network structures, such as branched signaling pathways and pathways with feedback. The formalism introduced here may provide a general approach to quantify cellular information transfer.

Scaffolding protein Grb2-associated binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops.

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.

Mitochondrial uncoupling: role of uncoupling protein anion carriers and relationship to thermogenesis and weight control "the benefits of losing control".

Uncoupling proteins, a subgroup of the mitochondrial anion transporter superfamily, have beenidentified in prokaryotes, plants, and mammalian cells. Evolutionary conservation of thesemolecules reflects their importance as regulators of two critical mitochondrial functions, i.e.,ATP synthesis and the production of reactive oxygen species (ROS). Although the amino acidsequences of the three mammalian uncoupling proteins, UCP1, UCP2 and UCP3, are verysimilar, each homolog is the product of a unique gene and important differences have beendemonstrated in their tissue-specific expression and regulation. UCP1 and UCP3 appear to bekey regulators of energy expenditure, and hence, nonshivering thermogenesis, either in brownadipose tissue (UCP1) or skeletal muscle (UCP3). UCP2 is expressed more ubiquitously,although generally at low levels, in many tissues. There is conflicting evidence about itsimportance as a regulator of resting metabolic rate. However, evidence suggests that thishomolog might modulate the mitochondrial generation of ROS in some cell types, includingmacrophages and hepatocytes. While the induction of various uncoupling protein homologsprovides adaptive advantages, both to the organism (e.g., thermogenesis) and to individual cells(e.g., reduced ROS), increased uncoupling protein activity also increases cellular vulnerability tonecrosis by compromising the mitochondrial membrane potential. This narrow “risk—benefit”margin necessitates tight control of uncoupling protein activity in order to preserve cellularviability and much remains to be learned about the regulatory mechanisms involved.