Cortical injuries are often reported to induce a suppression of the intracortical GABAergic inhibition in the surviving, neighbouring neuronal networks. Since GABAergic transmission provides the main source of inhibition in the mammalian brain, this condition may lead to hyperexcitability and epileptiform activity of cortical networks. However, inhibition plays also a crucial role in limiting the plastic properties of neuronal circuits, and as a consequence, interventions aiming to reestablish a normal level of inhibition might constrain the plastic capacity of the cortical tissue. A promising strategy to minimize the deleterious consequences of a modified inhibitory transmission without preventing the potential beneficial effects on cortical plasticity may be to unravel distinct GABAergic signaling pathways separately mediating these positive and negative events. Here, gathering data from several recent studies, we provide new insights to better face with this “double coin” condition in the attempt to optimize the functional recovery of patients. Cortical injuries are one major cause of death and permanent disabilities worldwide. In the attempt to ameliorate the survival rate and the postlesion rehabilitation of patients, researchers have developed several animal models of cortical injury to reproduce different aspects of this pathological condition.In particular, a great effort has been dedicated in the investigation of the physiological disturbances spreading in the surrounding uninjured tissue and sometimes even in remote brain areas .Even though these lesion-induced functional alterations might notably differ depending on many factors, such as the nature of the insult (cerebrovascular rather than traumatic), the extent of the damage and the cortical structures affected, some pathophysiological events have been systematically reported following many different experimental models of cortical lesion.Interestingly, one of the most frequently observed functional change postlesion is a reduction in the GABA-mediated inhibition which, therefore, seems to be (with some degrees of variability) a general phenomenon taking place as a consequence of a massive neuronal death.Because a deficit in the GABAergic transmission might easily compromise the delicate balance between excitatory and inhibitory neurotransmission  this lesHowever, the inhibitory action of GABA is going far beyond the control of the excitability of neuronal networks. The temporal and spatial precise release of GABA can also guarantee high specific responses of cortical neurons , 7. MoreIn light of these findings, the impaired inhibitory transmission observed postlesion might not be only a deleterious process but, by enhancing the plastic capacity of the cortex, could also promote the functional reorganization of the surrounding uninjured cortical tissue contributing to the functional recovery from the lesion-induced neurological deficits. The injury-induced reduction of inhibition may, therefore, share both detrimental and beneficial effects. Unraveling distinct GABAergic signaling pathways separately mediating these positive and negative events could be extremely helpful in the design of a more effective postlesion rehabilitation therapy. In the attempt to provide new insights to better face with this “double coin” condition, in this paper we will discuss several studies which documented a reduced and/or an altered GABAergic transmission as a consequence of a lesion in the cerebral cortex, and most importantly, we will try to explain how and through which cellular mechanisms the altered GABAergic transmission could influence functions, excitability, and plasticity of cortical networks.A receptors (GABAARs) and GABAB receptors (GABABRs) .The GABA receptors are divided into 2 classes: GABAARs belong to the cys-loop superfamily of ligand-gated ion channels and mediate fast synaptic inhibition in the central nervous system (CNS). GABAARs are heteropentameric structure composed by distinct types of subunit. In the mammalian brain, the majority of synaptic GABAARs are formed by two α, two β and one γ subunit.GABAα, β, and γ subunits have been identified , in the CNS defined combinations of subunits are more frequently found  [Although many different  β3, γ2) . ARs in which the γ subunit has been replaced with the δ subunit are exclusively found extrasynaptically [δ subunit-containing GABAARs are ideally suited to mediate tonic inhibition [Importantly, the combination of these subunits can determine the localization and the functional properties of the receptors. To mention a peculiar example, GABAptically , are actptically , 14. Thahibition . ARs are selectively permeable to Cl− and to a less extent to HCO3− [GABAo HCO3− .− across the membrane  strongly contribute in defining the reverse potential of GABAA-mediated currents (EGABA), and it is, therefore, of fundamental importance to guarantee the inhibitory actions of GABA. This also explains why in immature neuronal networks, where the Cl− intracellular concentration is relatively high, GABA can exert excitatory actions [In the mature CNS, the asymmetrical distribution of Cl actions . BRs are metabotropic, G protein-coupled receptors. They exert their inhibitory action either by decreasing Ca2+ currents or by increasing K+ conductance [BRs are also expressed at the presynaptic site where by reducing the probability of neurotransmitter release seem to offer a negative feedback mechanism to limit synaptic transmission within a certain physiological range [On contrary, GABAductance . GABABRsal range .In the mammalian neocortex, approximately 20%–30% of neurons use GABA as neurotransmitter . In contrast to pyramidal cells, GABAergic neurons are an extremely heterogeneous population of cells. Different criteria have emerged in the attempt to classify interneurons based on their different morphological, physiological, and neurochemical features , but nonA detailed description of the anatomical and functional properties of different interneurons subclasses is beyond the purpose of this paper; however, it is noteworthy to mention one important structural-functional relation emerging from recent studies: interneurons targeting different domains of principal cells seem to subserve specific functional roles . For instance, interneurons preferentially innervating dendrites of principal cells are particularly suited to modulate excitatory postsynaptic potentials (EPSPs) occurring at nearby synapses, thereby limiting the spatiotemporal summation of excitatory inputs and potentially preventing hyperexcitability. Interneurons predominantly sending axons onto the soma and the proximal dendrites (as basket cells) of principal cells are strategically located to control the output of the target neurons, and by operating as a precise clockwork, they can synchronize the firing of large population of principal cells contributing to the generation of cortical oscillatory patterns , 26.Injuries in the cerebral cortex often lead to an abnormal excitability of the surrounding neuronal networks. An increased spontaneous and evoked neuronal firing has been reported following different experimental models of brain injury , 28. In An increased susceptibility to epileptiform discharges have been observed to progressively develop in lesion models of partially isolated cortex –33, in pin vitro studies revealed a postlesion reduction of the GABAergic intracortical inhibition. This reduced inhibitory transmission was, therefore, considered primarily responsible for the lesion-induced hyperexcitability and for the increased propensity to epileptogenesis.In parallel, data from many A reduced strength in the GABAergic intracortical inhibition was reported following a photochemically induced cortical infarct , 37 in aARs in the surround of a cerebral photothrombosis [ARs. Beyond this functional evidence, quantitative receptor autoradiography studies have reported a downregulation of radiolabeled muscimol binding to GABArombosis  as well rombosis . The decAll together, these studies indicate that cortical injuries, independent of their etiology, can similarly lead to a reduced strength of the inhibitory neurotransmission.The lesion-induced suppression of inhibition developed relatively fast, since the effect was already visible one day after the lesion induction , 40, it Unfortunately, due to the few chronic investigations, it is still not clear if these relative long-lasting effects are typical of some lesion models and if they depends on the size and location of the cortical damage. Nonetheless, in the subacute phase postlesion (first week postlesion), the impaired inhibition seems to be a phenomenon systematically observed.Several cellular mechanisms have been proposed to underlie the lesion-induced suppression of inhibition. The degeneration of particular vulnerable interneurons subtypes could constitute one plausible mechanism, especially when brain injuries are followed by extensive secondary brain damage. Some studies, performed in models of ischemic and traumatic brain lesions, indeed reported signs of selective suffering and death of interneurons at the border of the injury , 43.GABAergic interneurons could also survive but enter a functional suppress status. A large body of evidence demonstrates the existence of a series of modulatory (or homeostatic) mechanisms in the CNS trying to maintain the firing rate of neurons within a certain physiological range in face of dynamic changes in synaptic drive –46. The α1 and a slight reduction of the α2 subunit of GABAARs were found in the surround of a photochemically induced cortical lesion in rats [ARs, observed in another study performed with the same lesion model (see above) suggests an overall reduction in the expression of postsynaptic receptors [Consistent with this hypothesis, a reduction in the number of functional GABAergic synapses has been suggested by several studies. In a lesion model of partially isolated cortex, the structural reconstruction of fast spiking interneurons in the vicinity of the “undercut cortex” revealed a significant reduction in their axonal length and a reduced number of large axonal boutons . At the  in rats . Althougeceptors . ARs [Furthermore, since the combination of the subunits determines the cellular localization and the functional properties of the GABAARs , even onGABA.Changes in the physiological properties of GABAergic signaling have been also reported. Intracellular recordings from pyramidal cells in the vicinity of an experimentally induced focal cortical infarct , in the GABA toward more depolarized potentials has been primarily attributed to a downregulation of the specific K+-Cl− cotransporter 2 (KCC2) with a consequent impaired extrusion of Cl−. In support of this hypothesis, some studies performed in traumatic models of axotomized neurons, both in vitro and in vivo, reported a reduction in KCC2 expression at mRNA and protein level [This shift in Ein level , 51. GABA) seem to describe a developmental juvenile status when the GABAergic system is still not fully mature.Interestingly, some of the alterations in the GABA-mediated inhibition  balance is continuously challenged by peripheral stimuli constantly bombarding the CNS.However, accumulating lines of evidence indicate that in sensory cortices, an increase in excitatory conductance is normally counterbalanced by a similar augmentation of inhibitory conductance . FurtherDespite the substantial flexibility, this dynamic equilibrium can be relative easily compromised by different pathological conditions, such as a brain damage. Different studies performed in animal models of ischemic and traumatic brain injuries indeed reported an important shift in the E-I balance in favour of excitation. Morphological and functional analyses of the rat hippocampus performed few months following a global ischemic episode revealed a dramatic loss of GABAergic presynaptic terminals accompanied by an increase in glutamatergic synapses . FurtherSince the recruitment of recurrent inhibitory circuits plays a key role in the maintenance of the E-I balance, the lesion-induced impairment of intracortical inhibition function –39 is moHowever, as already mentioned above, the GABAergic system is extremely heterogeneous, being composed by diverse interneuron cell types with potential specific functional properties . As a coInterestingly, a subclass of dendritic projecting GABAergic neurons expressing somatostatin (SOM neurons) seems to be particularly efficient to counteract increasing levels of cortical excitation. The excitatory synapses impinging on this category of interneurons generate EPSPs which are initially small in amplitude but that progressively increase with the number of subsequent stimuli (facilitating excitatory synapses) eventually leading to the generation of action potentials. This suggests that these cells have the capability to “buffer” a wide range of excitatory inputs before becoming saturated , therebyA selective loss of dendritic-projecting SOM containing interneurons has been also reported in human patients  and in ein vivo electrophysiological recordings from the somatosensory cortex of rats strongly indicate that chandelier cells do not seem particularly suited to encode incoming ascending information, but they seem indeed strongly involved in preventing hyperexcitability of cortical networks [Another interneuron subtype which seems to strongly contribute in dampening excessive cortical excitability is constituted by chandelier cells. This category of interneurons, by selectively forming GABAergic synapses onto the axon initial segment of principal cells, can strongly control the generation of action potentials in pyramidal neurons, and therefore, they might have the capability to prevent excessive firing . In linenetworks . Furthernetworks , 61.Parvalbumin-containing (PV) basket cells constitute another important class of interneurons strongly participating in the cortical recurrent inhibitory circuits. In distinction to the above-described subtypes of interneurons, PV basket cells seem to strongly participate in the cortical information processing. The fast spiking phenotype –64, the Together, these electrophysiological and anatomical properties define the fundamental role of PV basket cells in synchronizing action potential discharges of large numbers of principal cells promoting the emergence of network oscillations in the gamma frequency band (30–80 Hz) , 70, 71.Oscillatory activity in the gamma range has been reported to play a crucial role in the perception and processing of sensory stimuli , in focuNowadays, it is still unknown whether the observed reduction of inhibitory transmission following cortical injuries is the result of a lesion-induced effect on a specific subpopulation of inhibitory cells or if all classes of interneurons are equally affected. Potentially, a lesion-induced reduction in the activity of SOM neurons or chandelier cells might critically compromise the E-I balance especially during high level of excitation, while a lesion-induced change in the activity of PV basket cells could have a profound impact on the cortical information processing. The recent availability of transgenic mice expressing fluorescent proteins (such as the green fluorescent protein GFP) in defined classes of interneurons –77 offerThe reduced strength of the inhibitory transmission, often observed following cortical injuries, does not seem to be simply the result of a lesion-induced degeneration or reduced activity of GABAergic interneurons. The situation is far more complicated, since functional modifications of the GABAergic signaling have been reported after cortical lesions as well. GABA [An important phenomenon, described following different cortical lesion models, which could potentially compromise the E-I balance, is the positive shift in EGABA , 48–51. GABA at values above the resting membrane potential (mV) could lead to the straightforward conclusion of an increase in the neuronal excitability due to a GABA-mediated depolarization of the postsynaptic membrane. Generally, GABA is known to exert its inhibitory action by leading to a hyperpolarization of the postsynaptic neuron, thereby driving the membrane potential away from the threshold for the generation of spikes (spike threshold). The positive shift of EHowever, depolarization is not always synonymous of excitation . A-mediated depolarizing responses can still exert an inhibitory action (on conditions that EGABA remains more negative than the spike threshold) by increasing the membrane conductance of the postsynaptic neurons, and thereby shunting excitatory inputs “just” generated at nearby synapses. This inhibitory mechanism, known as “shunting inhibition”, has been shown to operate even under physiological conditions at many cortical and hippocampal synapses, where EGABA was found between the resting mV and the spike threshold [GABAhreshold –81. GABA might convert many hyperpolarizing GABAergic synapses into shunting ones. From this evidence, one can predict that the pathological positive shift in EARs exhibit a higher ionic conductance (or outward rectification) [Nonetheless to estimate the consequences of such a phenomenon on the E-I balance is not an easy task. Shunting inhibition can have in some instances a stronger inhibitory effect than hyperpolarization. This is because at depolarized membrane potentials GABAication) , 82. Morication) . For theication) . However, on the other side, the efficacy of shunting inhibition is strictly dependent on how the excitatory and inhibitory inputs are spatially and temporally related on the membrane of the postsynaptic cell . TemporaGABA at these synapses could, therefore, critically favour hyperexcitability of cortical networks.In contrast, IPSPs temporally and spatially isolated from EPSPs need to be hyperpolarizing to provide an inhibitory action; otherwise, they will generate depolarizing waves propagating toward the soma of the cell which will sum to depolarizing EPSPs . A pathoGABA is due to an impaired Cl− extrusion, as suggested by Jin and colleagues, repetitive synaptic GABAARs activation, normally occurring in vivo, could promote a transient additional intracellular Cl− accumulation which will depolarize EGABA further until the action of GABA will be fully excitatory [GABA could facilitate recurrent excitation between pyramidal cells potentially leading to the generation of epileptic discharges. Finally, if the depolarized Ecitatory . This hyGABA on the excitability of neuronal networks. Nonetheless, if EGABA is equally affected at all synapses, a general shift in favour of excitation should be expected.All these considerations explain why it is difficult to predict the consequences of the reported lesion-induced depolarized EARs containing the subunit α5 and δ in the peri-infarct cortex [A receptors containing these subunits are normally located extrasynaptically [Despite the large number of studies strongly indicating a postlesion impairment in GABA-mediated inhibition, a recent study performed in a phototrombotic model of stroke in the motor cortex of mice, revealed a lesion-induced enhancement of tonic inhibition due to an increased activity of GABAt cortex . GABAA rptically , 87, wheptically . The autInteresting, in a study performed in the hippocampus of guinea pigs tonic inhibition was found to be most prominently expressed at interneurons . AssuminA cortical lesion may, therefore, produce a shift from a phasic to a tonic GABAergic transmission with profound consequences on neuronal network functions .GABA by promoting intracellular Cl− accumulation .Tonic inhibition lacks the spatial and temporal precision of synaptic transmission, and by producing a “long-term” reduction in the resistance of the postsynaptic neurons, it could prevent an appropriate neurotransmission, potentially constraining plastic processes to occur. Moreover, the enhanced tonic inhibition might also contribute to the above-mentioned depolarizing shift in EThe functional consequences of neocortical injuries are often not limited to the neuronal circuits surrounding the primary lesion but can be observed in remote projection cortical areas as well as in some subcortical structures . These rIn different clinical studies , 30 and in vitro extracellular recordings performed in photothrombotic and ischemic unilateral cortical lesion models [In parallel, n models –39 reveaIt is, therefore, conceivable that the reduced inhibitory tone may be responsible for the described abnormal activation of the hemisphere contralateral to the lesion. As a consequence of these findings, the hypothesis emerged that a lesion-induced dysinhibition of the contralateral cortex could potentially contribute to the functional recovery postinjury by compensating or at least partially taking over the function of the damaged brain area. In this regard, longitudinal studies, comparing the extent of the hyperexcitability of the unaffected cortex with the degree of recovery from neurological deficits, suggested that the abnormal activity of the contralateral hemisphere during the acute/subacute phase postlesion could indicate a sort of bihemispheric cooperation which might be indispensible for performing even simple tasks involving the affected side of the body. However, the contribution of the contralateral cortex, in the recovery of function, seems to diminish over time, since the better final outcomes are observed when the brain regions, normally executing a function, are reintegrated into the active network . It is, therefore, plausible that shortly after a focal cortical injury, the dysinhibition of anatomically connected remote areas might constitute a compensatory mechanism to temporary relieve the neurological deficits before a consistent functional reorganization will gradually guaranty a permanent, at least partial, functional recovery. However, it is also not possible to rule out a potential involvement of these hyperexcitable remote neuronal networks in promoting the generation of epileptic events after a brain injury.Since the brain areas most likely affected by a cortical damage are the one anatomically connected to the lesion site, the dense corticothalamic thalamocortical connections strongly suggest a likely lesion-induced physiological alteration at the level of the thalamus. One study performed in a phototrombotic model of cortical infarct in the somatosensory cortex of rats indeed reported a strong reduction in the excitability of interneurons located in the reticular thalamic nucleus . The retIt is, therefore, important, when searching for the cellular mechanisms responsible for epilepsy after cortical injuries, to do not underestimate potential alterations in the physiological properties of thalamic neurons.From the considerations drawn so far, it seems pretty evident that the pathological alterations of GABAergic inhibition following a cortical lesion can lead to severe negative consequences on the excitability and function of neuronal circuits. One might, therefore, conclude that a simple pharmacological enhancement of the GABAegic synaptic transmission could be the best approach to restore a normal brain activity after a lesion. However, the reduced inhibition could be also viewed as an evolutionary conserved mechanism initiated in front of a dramatic alteration of cortical activity, as in the case of cortical injuries, with potential beneficial effects.In line with this assumption, different studies indicated that the level of intracortical inhibition is important to define the plastic properties of neuronal circuits.During the development of the mammal CNS, the slowly increasing strength of inhibitory transmission is suggested to modulate cortical plasticity by crossing two thresholds: crossing the first threshold defines the onset of a period, known as critical period, characterized by high experience-dependent plasticity of neuronal networks, while passing the second threshold closes the time window of high plasticity and open a period of restricted plasticity which is protracting throughout the life of an animal , 98. The relation between the level of intracortical inhibition and the critical period for plasticity has been extensively studied in the visual cortex of rodents, where the critical period is normally determined by the successful induction of ocular dominance (OD) plasticity . In thisBased on these observations, if the reduced strength of inhibition observed after a cortical lesion matches a level similar to that found during the critical period, the remodeling capacity of the surrounding cortical networks could be strongly enhanced.In order to take advantage of the described findings, it is fundamental to elucidate the cellular and physiological mechanisms mediating the influence of the strength of inhibition on cortical plasticity.As early as in 1987, Artola and Singer proposed that strong inhibitory synapses, by reducing EPSPs, could prevent the activation of NMDARs, indispensible for many forms of synaptic plasticity .Shortly afterwards, Kirkwood and Bear also suggested that a mature inhibitory circuitry in layer 4 of sensory cortices might act as a kind of filter by limiting the activity pattern able to gain access from subcortical structures to the supragranular layers of the cortex .This observation indicates that inhibition may control plasticity of neuronal networks by selectively permitting or preventing plasticity at excitatory synapses. A reduced/immature GABAergic transmission might, therefore, act as a permissive substrate allowing sensory experience to remodel structure and functions of cortical networks. However, a too drastic impairment of synaptic GABAergic transmission might be deleterious by preventing accurate cortical information processing and by promoting epileptic activity. On this concern, Feldman proposed the existence of an ideal level of inhibition, on the one hand low enough to permit the potentiation or the depression of excitatory connections but on the other hand sufficient to guarantee an appropriate temporal encoding of relevant inputs .Reducing the strength of inhibition or adjusting an impaired inhibition postlesion to an ideal level could, therefore, constitute a promising tool to restore and/or enhanced experience-dependent plastic processes in the adult CNS. Among the different interneuron subtypes, PV basket cells have been suggested to contribute, more than others, in the expression of the critical period for cortical plasticity , 104. Different functional properties of PV basket cells can indeed support their role in modulating plastic processes. Their fast somatic inhibition could filter the action potentials able to access the dendritic arbor by back propagation, thereby allowing postsynaptic spikes to meet presynaptic inputs within specific temporal windows appropriate for synaptic plasticity induction . FurtherBeside permitting or preventing structural and functional modifications of excitatory connections, inhibitory networks can themselves undergo plastic processes. Firstly, the activity of cortical interneurons is highly sensitive to global changes in the activity of cortical circuits. A reduced cortical activity leads normally to an impaired GABAergic innervation , 108 andin vitro studies could demonstrate the efficacy of different stimulation protocols in the induction of long-term modifications at cortical GABAergic synapses [Not only homeostatic but also Hebbian plastic mechanisms have been observed at inhibitory synapses. Several synapses –114. in vivo studies performed in different sensory systems provided evidence of inhibitory-plasticity-dependent changes in cortical maps. Moreover, some ARs blocker indicating that the suppression of responses to irrelevant inputs was likely due to a potentiation of GABAergic synapses [In these studies, a shift in cortical maps was obtained by exposing the animals to an abnormal sensory experience for a define period of time. This condition produced a receptive field shift away from deprived/inappropriate inputs towards new behavioral relevant inputs. Interestingly, this receptive field plasticity could be reversed by the application of a GABAsynapses , 116.Intracortical GABA-mediated inhibition also strongly contributes in shaping the receptive fields of cortical neurons.ARs antagonist produced an enlargement of the receptive field's size of single neurons [This important function of GABA was first appreciated in a series of electrophysiological studies mainly performed in primary sensory systems of mammals. In these studies, the application of a GABA neurons , 118 as  neurons , 119 and neurons . Remarkably, increased and/or altered receptive fields were observed following cortical lesions in the surrounding brain areas –123. The lesion-induced reduction of inhibition might enlarge the receptive fields by bringing suprathreshold and thereby unmasking previously silent (subthreshold) inputs .per se a mechanism of functional reorganization, but most importantly, as already outlined above, this process may allow new functional excitatory inputs to enter in competition with others and to undergo potentiation or depression following Hebbian-based learning rules. Converting silent connections into functional ones is In this way, a reduced level of inhibition could strongly contribute in the plasticity of cortical maps.The above-mentioned studies provide several lines of evidence for an important role of inhibition in influencing the plasticity of cortical networks. Here, we briefly discuss how the plasticity of neuronal networks surrounding a cortical lesion could mediate the recovery of function and how the lesion-induced reduction in inhibition could contribute to this process. First of all, to achieve a functional recovery after a cortical lesion the information previously processed by the injured cortex needs somehow to be rerepresented by the remaining cortical areas.In the neocortex, due to the dense and exuberant cortical connectivity , some ofThe initial depression of GABAergic inputs, by unmasking subthreshold excitatory connections, plays therefore a crucial role in the initiation of cortical map plasticity.This is of fundamental importance since cortical map plasticity is largely responsible for the “long-term” functional recovery postlesion. Subsequently, to guarantee a stable rewiring of neuronal circuits, experience-dependent plastic processes will likely lead to the reinforcement of some of these new functional inputs, which turn out to be behaviorally relevant, and eventually lead to the suppression of inputs which became irrelevant after the lesion .The reinforcement of the relevant inputs most likely involves long-term potentiation (LTP) of excitatory connections –128, whiFinally, structural modifications might stabilize the new connectivity patterns.This process can therefore induce at least a partial functional recovery postlesion, because it promotes the cortical area surrounding the damage to gradually take over the functions before belonging to the death cortical tissue.As we intensively discussed, the altered GABA-mediated inhibition often observed following cortical injuries can have both detrimental consequences by modifying excitability and functions of cortical networks as well as beneficial effects by promoting cortical plasticity. Intuitively, in order to optimize the functional recovery of patients suffering from a cortical injury, a therapy should aim to minimize the deleterious consequences of a modified inhibitory transmission without preventing the potential beneficial effects on cortical plasticity. This scope can be achieved if we could distinguish that the positive and negative effects of the altered GABAergic transmission differ somehow in the cellular mechanisms of their induction, in the GABAergic networks that they affect and/or in the temporal window postlesion of their expression.Although much more needs to be done to give a final answer to these questions, a consistent amount of information can already be found in many studies investigating the role of GABA-mediated inhibition on cortical function and plasticity. ARs, produced a significant improvement in the motor recovery of the animals [For instance, in the above-mentioned study of Clarkson and colleagues it has been proposed that the stroke-induced increase in the tonic GABAergic transmission is one of the constraining factors for cortical plasticity. The authors were indeed able to demonstrate that dampening the excessive tonic inhibition, by selectively antagonize the function of extrasynaptic GABA animals . ARs subunits exclusively or predominately contained in extrasynaptic GABAARs [The availability of antagonists for specific GABA GABAARs  makes thThe identification of specific networks of GABAergic neurons primarily involved in the reorganization of cortical circuits postlesion could also promote the development of a better targeted therapy to improve the functional recovery of patients. On this regard, although it is still not possible to attribute an exclusive function to each subtype of cortical interneurons, compelling evidence indicates that some classes of GABAergic cells might be more relevant than others in mediating cortical plastic processes.In particular, through the lines of this paper different points stress the importance of the fast-spiking PV basket cells in the regulation of cortical network functions as well as in the modulation of experience-dependent plastic processes. A drastic impairment in the function of these interneurons should be, therefore, avoided although a moderate reduction in their activity might facilitate cortical plastic processes. On contrary, other subtypes of interneurons seem to contribute to a lesser extent in the induction of cortical plasticity and to be more closely involved in controlling the excitability of cortical networks. On the basis of recent findings, these subpopulations might include dendritic projecting SOM interneurons and chandelier cells , 59, 104α1 subunit-containing GABAARs [α2-containing GABAARs [Interestingly, PV basket cells form predominately perisomatic synapses enriched in  GABAARs , while o GABAARs .The development of pharmacological agents showing specific-subunit sensitivity might, therefore, provide a strategic tool able to modulate the function of a particular class of interneurons and might be more effective in reducing postlesion cortical hyperexcitability without constraining cortical plasticity. Since experience-dependent changes in synaptic plasticity likely contribute to the functional rewiring of cortical networks, a physical rehabilitation accompanying a pharmacological approach will remain essential. Finally, the identification of an optimal therapeutical time window for pharmacological and rehabilitative interventions could also be extremely helpful. In this regard, results from clinical studies indicate that pharmacological therapies following cortical injuries showed a moderate efficacy and only if administered very early after the lesion (few hours postlesion) . This miNow, it seems that the attention is shifting toward the development of a therapy aiming to amplify endogenous mechanisms of repair . This miAs extensively described in this paper this time window postlesion seems to be characterized by a profound alterations in the GABAergic transmission which might strongly influence cellular mechanism of neuroplasticity. A therapeutical approach able to precisely target the GABAergic signaling involved in the modulation of neuronal plastic processes may, therefore, constitute a powerful instrument to improve the rehabilitation of patients suffering from traumatic brain injuries and stroke.


The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells. Ataxia telangiectasia mutated (ATM) and the related kinase ATM- and Rad3-related (ATR) are principal signal transducers that mediate DNA damage signalling. While ATM is recruited to DNA double strand breaks (DSBs) by the Mre11, Rad50 and Nbs1 complex, ATR and its constitutive interacting partner ATRIP bind to replication protein A (RPA)-coated single-stranded DNA (ssDNA). ATR can then be further activated by direct interactions with DNA topoisomerase 2-binding protein 1 (TopBP1), which is recruited to ssDNA/double-stranded DNA junctions by the Rad9-Rad1-Hus1 (9-1-1) complex. Claspin-mediated phosphorylation of Chk1 kinase at serines 317 and 345 by ATR regulates Chk1 activity  repair. ATR is activated after ionizing radiation (IR), and this may be associated with the DNA end resection of DSBs that induces RPA-coated DNA prior to the formation of Rad51 filaments during HDR ,4. BecauBecause ataxia telangiectasia patients, who typically express no ATM protein, are the most radiosensitive humans that have been identified , it has Here, we used a reverse chemical genetics approach to study ATR function. Selective and reversible ATR kinase inhibitors allowed us to investigate the consequences of transient ATR kinase inhibition in cells after IR. Surprisingly, ATR inhibition caused significantly more potent radiosensitization than ATM inhibition. Transient ATR inhibition in synchronized cells revealed a novel role of ATR in G1 phase and identified a short time interval after IR where ATR activity is critical for the repair of IR-induced damage and cell survival. ATR colocalized with RPA foci and was activated in irradiated G1 phase cells in the absence of RPA2 phosphorylation. Thus, ATR activation does not require extensive DNA end resection as previously postulated, indicating a potential mechanism of ATR activation in G1 phase cells in the absence of HDR.ATM kinase inhibitor KU55933  was used at final concentrations of 10 μM. ATR kinase inhibitors ETP-46464 and Vertex compound 45 were synthesized at the Medicinal Chemistry Shared Resource of the Ohio State University Comprehensive Cancer Center . ETP-46464 and Vertex compound 45 were used at a final concentration of 10 μM. Chk1 kinase inhibitor UCN-01  and CDK4/6 kinase inhibitor PD0332991  were used at a final concentration of 100 nM. ATM, ATR, Chk1 and CDK4/6 kinase inhibitors were reconstituted in dimethyl sulfoxide. Apurinic/apyrimidinic endonuclease 1 (APE1) inhibitor NSC332395  (gift fr137Cs] irradiator (J.L. Shepherd & Associates) at a dose rate of 0.711 Gy/min.Dr. Jiri Lukas (University of Copenhagen) and Dr. Stephen Jackson (University of Cambridge) provided U2OS cells stably expressing green fluorescent protein (GFP)-tagged ATR or p53-binding protein 1 (53BP1). Dr. Jill Siegfried (University of Pittsburgh Cancer Institute) provided the lung cancer cells 201 T and 239 T . U2OS, CAntisera against ATM phospho-S1981 , generic ATM , Chk1 phospho-S345 , generic Chk1 , glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , ATR , RPA2 , RPA2 phospho-S4/8 , geminin , cyclin A , cyclin D1 , CDC25C phospho-S216  and generic CDC25C  were used in immunoblotting and/or cell staining. Whole cell extracts were prepared in lysis buffer: 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1% Tween-20, 0.5% NP40 and 1× protease inhibitor mixture (Roche Applied Science). Cleared cell extracts were resolved in either 3–8% Tris-Acetate or 4–12% Bis-Tris gels (Invitrogen). For cell staining, cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% Triton X100 in phosphate buffered saline and saturated with bovine serum albumin. Cells were then incubated with primary antibodies for 2 h at room temperature, washed and incubated with secondary antibody for 1 h at 4°C in the dark. Dilution of primary antibodies was 1:400 for anti-RPA2, 1:600 for anti-cyclin A, 1:200 for anti-geminin and 1:200 for anti-cyclin D1. Dilution of secondary antibodies was 1:300 for anti-mouse-AlexaFluor488 and anti-mouse-AlexaFluor555 (both from Invitrogen) and 1:1000 for anti-rabbit-Cy3 (Jackson Immunoresearch).t-test was done for a two-tailed distribution of two samples with unequal variances. The *, ** and *** denote P-values smaller than 0.1, 0.05 or 0.01, respectively. To evaluate colocalization of GFP-ATR and RPA, foci were examined in a single Z-stack.Fixed cells for foci analysis were imaged on an inverted confocal microscope  controlled by NIS Elements software. Z-stacks were taken to cover the entire thickness of the cell in a step size of 0.4 µm. The excitation wavelengths for GFP, 4′, 6-diamidino-2-phenylindole (DAPI) and RFP were 488, 405 and 543 nm, respectively. The signal was collected through a 1.40 60× oil immersion objective. For live cell imaging, cells were mounted on an environmentally controlled live cell chamber and monitored for 24 h on a Nikon A1 microscope with a 60× 1.49 NA total internal reflection fluorescence (TIRF) objective. Foci were counted in at least three independent experiments after reassembling Z-stacks. Error bars denote standard deviations. A Student’s Cells were seeded in 60 mm petri dishes and treated with inhibitor(s) and increasing doses of IR. Drug treatments were removed 4 h post IR or 75 min post IR in the case of 1 h treatment. Vehicle dimethyl sulfoxide served as control. After 10–12 days, depending on the cell line, colonies were stained with Giemsa solution. A colony was defined as a cluster of ≥50 cells, presumably having formed from a single cell. Three independent experiments were performed, each in triplicate. Error bars denote standard deviations derived from triplicates.ETP-46464  and Vertin vitro . Surprisingly, we observed that a 4 h inhibition of ATR kinase activity with ETP-46464, from −15 min to +4 h post IR, caused significantly more IR-induced cell killing than ATM kinase inhibition with KU55933 over the same interval in U2OS, H460, 201 T, 239 T and Calu6 cells . Similar results were obtained when ATR kinase activity was inhibited using Vertex compound 45, confirming the specificity of the radiosensitization by ATR inhibition . Concurrent inhibition of both ATR and ATM kinase activity radiosensitized cells still further, suggesting that the ATR and ATM kinase-dependent mechanisms of radioprotection are at least partially independent during this 4 h interval.Selective small molecule ATM kinase inhibitors have been shown to radiosensitize cells in vitro . We undeBecause increased cell killing could be due to unrepaired DNA damage, we quantitated IR-induced DNA damage foci in cells treated with or without ATR kinase inhibitor. Transient ATR kinase inhibition caused an increase in the number of cells containing >50 RPA foci assayed 24 h after 2 or 10 Gy IR E, suggesSupplementary Figure S2A and B). Significant aspects of ATR kinase signalling are mediated by the downstream Chk1 kinase . To investigate the contribution of ATR kinase-dependent Chk1 signalling to radioprotection, we undertook clonogenic survival assays using ETP-46464 and UCN-01. Chk1 kinase inhibition for 1 h did not increase IR-induced cell killing in U2OS, H460, 201 T, 239 T or Calu6  and exposed G1, S and G2/M phase populations to 2 Gy IR. Synchronized cells were treated with ETP-46464 from +15 min to +75 min post IR, at which point whole cell lysates were prepared. Strong IR-induced ATR kinase-dependent phosphorylation of Chk1 was observed in S, G2/M and G1 phase cells showing that ATR kinase is functional in G1 phase , showing that IR-induced GFP-ATR foci form in all phases of the cell cycle. GFP-tagged ATR has been shown to form foci in U2OS cells in S and G2 phase after IR ,23. We eSupplementary Information). This is important because it excludes the possibility that the observed GFP-ATR foci in these Cdt1-RFP positive cells result from IR-induced damage in mitotic cells or appeared at the onset of S phase.Cdt1 promotes the assembly of the pre-replication complex that is required for the loading of the DNA replication machinery. Cdt1 accumulates during G1 phase and is degraded in early S phase by ubiquitin-mediated proteolysis ,26. We uSupplementary Figure S4B). Thus, ATR kinase activity is required for the formation or stability of IR-induced ATR foci, indicating that phosphorylation of a yet to be identified target within 1 h after IR is an important step in the DNA damage signalling.To determine whether the formation of IR-induced ATR foci is ATR kinase-dependent, we examined GFP-ATR foci in cells 75 min post IR. ATR kinase inhibition from +15 to +75 min post IR strongly inhibited the formation of ATR foci in both synchronized G1 phase cells B and C aATR recruitment to RPA-coated ssDNA via its binding partner ATRIP is the initial step required for DNA damage-induced ATR kinase activation ,28. PrevSupplementary Figure S5). Furthermore, IR-induced RPA foci were evident in all U2OS cells regardless of positive or negative staining for geminin, cyclin A or cyclin D1 . First, we tested whether ATR kinase activity in G1 phase was transcription-dependent by inhibiting RNA polymerases II and III with a high dose of α-amanitin (50 µg/ml) in synchronized U2OS cells prior to IR. No significant reduction in IR-induced Chk1 phosphorylation was detected 1 h after 2 Gy IR when transcription was inhibited B. Next, We discovered that ATR inhibition during a short interval after IR is sufficient to not only significantly radiosensitize cells, but also has long-term effects on the DNA damage signalling. Remarkably, Chk1 and RPA2 phosphorylation are not recovered when ATR kinase is transiently inhibited for 1 h after IR A despiteSupplementary Data are available at NAR Online.NIH Grants ; ETP-46464 and Vertex compound 45 were synthesized by the Medicinal Chemistry Shared Resource of the Ohio State University Comprehensive Cancer Center , the National Cancer Institute Grant [P30 CA16058] (in part). Funding for open access charge: [RO1 CA148644].Frieda G. and Saul F. Shapira BRCA Cancer Research Program (in part) and Conflict of interest statement. None declared.


Angelica sinensis (Oliv.) Diels , showed strong inhibition of human placental GST. Its 50% inhibition concentration (IC50) was 0.3 μM, which was greater than a known GSTP1-1 inhibitor, ethacrynic acid (EA), using the established high-throughput screening model. Kinetic analysis and computational docking were used to examine the mechanism of GST inhibition by CF. Computational docking found that CF could be fully docked into the gorge of GSTP1-1. The further exploration of the mechanisms showed that CF was a reversible noncompetitive inhibitor with respect to GSH and CDNB, and it has much less cytotoxicity. Apoptosis and the expression of P-gp mRNA were evaluated in the MDR positive B-MD-C1 (ADR+/+) cell line to investigate the MDR reversal effect of CF. Moreover, CF showed strong apoptogenic activity and could markedly decrease the overexpressed P-gp. The results demonstrated that CF could inhibit GST activity in a concentration-dependent manner and showed a potential MDR reversal effect for antitumour adjuvant therapy.Glutathione S-transferase (GST) is the key enzyme in multidrug resistance (MDR) of tumour. Inhibition of the expression or activity of GST has emerged as a promising therapeutic strategy for the reversal of MDR. Coniferyl ferulate (CF), isolated from the root of  Chemotherapeutics provide the most effective treatment modality for metastatic cancer. However, resistance to anticancer chemotherapy remains a serious obstacle in cancer treatment. Primary and acquired resistance of tumour cells to anticancer drugs are major causes of the limited efficacy of chemotherapy , 2. TumoGlutathione S-transferases  are a superfamily of Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds including chemotherapeutic agents . GSTs ar ABCB1/MDR1) and acts as an ATP-dependent active efflux pump for chemotherapeutic agents. P-gp-mediated MDR appears to be a major feature in drug resistance   was obtained with different CF or EA concentrations  and different GST concentrations of 0.0075–0.18 U/mL to distinguish between reversible and irreversible inhibition. A Hanes plot of v versus [S] was performed at GSH concentrations from 0.07 to 2.24 mM or CDNB concentrations from 0.5 to 44.1 μM, with CF at concentrations of 0, 0.25, and 0.5 μM.Enzyme kinetic experiments were performed to elucidate the interaction of human GSTs with CF in detail. Firstly, the initial rate of the enzyme was analysed by measuring the formation of catalysate at 340 nm. The reaction was carried out at 37°C for 5 min after preincubating GST with various concentrations of CF for 5 min. Furthermore, a plot of Computational docking was performed with FlexX software in SBVS . The protein data bank (PDB) file 2GSS and the cocrystal format of the inhibitor, EA, in the active site of GST (human GSTP1-1) were optimised and used. EA was selected as a reference ligand structure which was a fixed conformation docked into the active site of the enzyme. The flexible docking conformations of EA were then created with 30 docking conformation options. Each conformation was energy-minimised using a molecular mechanics program. All ligands were predicted based on the active sites being within a 6.5 Å radius from the bound ligand. Water and metals not involved in binding were removed from the protein. The docking scores in GSTP1-1 were employed and elucidated in detail.3 cells per well) and incubated in the presence of different concentrations of positive control EA (10 μM–200 μM) and CF (4.12 μM–3 mM) for 48 h. The cytotoxic activity and dose response of EA and CF to these cell lines were determined using the MTT assay. Normalised data were plotted graphically as percentages of viable cells. Each study was performed in triplicate and repeated three times.The wild-type human endometrial cancer B-MD-C1 cells or the multidrug-resistant B-MD-C1 (ADR+/+) cells were plated in quadruplicate (6 × 107/mL) were incubated with CF at various concentrations or positive control EA (40 μm) for 48 h, and then the medium was replaced with fresh medium in the presence of Adriamycin (5 μg/mL). The B-MD-C1 (ADR+/+) cells only treated with Adriamycin were used as a control. After incubation for 24 h at 37°C, the cells were collected and fixed in 70% cold ethanol (−20°C) overnight. The cell suspensions were washed twice with phosphate-buffered saline (PBS) and resuspended in phosphate-buffered saline containing 1% foetal calf serum. RNA in the fixed cells was digested with RNase A (0.5 mg/mL) at 37°C for 1 h. Finally, the cells were centrifuged at 1000 revolutions/min for 5 min. The cells were resuspended with binding buffer and stained with propidium iodide (2.5 μg/mL) at room temperature for 15 min. The samples were measured by flow cytometry with CellQuest software .Flow cytometry was used to evaluate the effect of CF on apoptosis of the multidrug-resistant B-MD-C1 (ADR+/+) cells treated with Adriamycin. Briefly, B-MD-C1 (ADR+/+) cells . The amplification reaction was carried out with 2 μL of cDNA product for 35 cycles with each cycle consisting of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, followed by a final 5 min elongation at 72°C. The final RT-PCR products were visualised by ultraviolet illumination after electrophoresis through 1.5% agarose gel, with 0.5 mg/mL ethidium bromide at 50 V at 2 h, and scanned using Kodak gel analysis software. RNA amounts were normalised against the β-actin mRNA levels. EA (40 μM) was also used as the positive control in this assay.MDR1 gene expression levels were evaluated by RT-PCR assay on the B-MD-C1 and B-MD-C1 (ADR+/+) cells described above. Total cellular RNA was isolated using the TRIzol reagent (Life Technologies). First-strand cDNA synthesis was performed using a kit (Life Technologies). The primers used for the analysis of MDR1 were sense 5′-TCGTAGGAGTATCCGTGGAT-3′ and antisense 5′-CATTGGCGAGCCTGGTAG-3′ (455 bp); t-test. The results were considered to be statistically significant when P < 0.05.Data were expressed as the means ± SD and analysed statistically using Student's 50 value of 4.89 μM (see 50) of CF was approximately 0.3 μM. Moreover, the inhibitory kinetics and the associated binding mode were investigated because they may provide an exploitable mechanism for developing potent drugs with desirable properties. The kinetic analysis of the mechanism of inhibition to GST by CF was investigated by varying either GSH or CDNB concentration. The results showed that the inhibition of GST by CF might be reversible  with respect to GSH and CDNB. Therefore, CF was likely to act as a reversible noncompetitive inhibitor of GST.As shown in 9 μM see . Notablyversible .  FigureIn order to gain an insight into the structural basis by which CF exerts its inhibitory activity on GST, the active site of GST was analysed using the GST-EA complex, and molecular docking studies were performed on CF using the PDB file 2GSS as a reference structure. The computational docking method was used with FlexX software to dock CF into the active site of GST. This method demonstrated that CF might insert into the enzyme molecule active cavity but not bind to the active site. A compound with a high-scoring conformation would display a high inhibitory activity on GST, and the computational docking scoring conformations were consistent with the results of the high-throughput screening. CF's hydroxyl oxygen atoms in the phenyl ring formed a hydrogen bond with active site amino acid TYR7. Methoxy oxygen atoms in the same phenyl ring also formed a hydrogen bond with active site amino acid TYR106. In addition, hydroxyl, hydrogen atoms in another phenyl ring formed a hydrogen bond with the carbonyl oxygen atom of active site amino acid VAL35. The conformation score was −11.0. The reference compound, EA, has a side chain carbonyl oxygen atom that forms a hydrogen bond with active site amino acid TYR108. The carboxyl oxygen atom forms a hydrogen bond with active site amino acid LEU52. The conformation score for the ethacrynic acid interaction is −13.3 .μM was above 45%). Our data showed that CF hardly had any cytotoxic effect on the wild-type B-MD-C1 and the B-MD-C1 (ADR+/+) cells when its dose was less than 3 mM  had no obvious influence on the proliferation of B-MD-C1 and B-MD-C1 (ADR+/+) cells (the inhibition rate of 3 mM was below 30%), while EA showed more potent inhibition of cell growth and induction of cell death than CF in these two cells  cells by flow cytometry. After the cells were exposed to 5  control . B-MD-C1P < 0.01, RT-PCR analysis of the expression level of P-gp in the studied groups further revealed the effect of CF on reversing MDR in MDR-positive cells , lane 1.Conventional cancer chemotherapy is seriously limited by MDR commonly exhibited by tumour cells. MDR has been recognised as an important type of resistance that can be due to various mechanisms. These various mechanisms might explain why treatment regimens that combine multiple agents with different targets are less effective than expected , 27, 28.Of these mechanisms, one of the most frequently encountered mechanisms for the acquisition of MDR by tumour cells is the induction and activation of efflux transporter proteins. P-gp is commonly accepted as one of the best characterized transporters responsible for the multidrug resistance phenotype exhibited by cancer cells. Most of the researchers pay more attention to find molecules that can directly block the activity of P-gp, which is a common step and a well-accepted strategy to reverse MDR phenotype . HoweverCancer cells can also acquire resistance by overexpressing GSTs that may increase detoxification and circumvent the cytotoxic action of antitumour drugs. In particular, a number of chemotherapy agents currently used in cancer therapy are known to be substrates of GSTs , and it Angelica sinensis (Oliv.) Diels, which has been used in traditional Chinese medicine (TCM) for more than 2000 years  and nuclear magnetic resonance spectroscopy [1H-NMR spectrum (500 MHz) of CF in CDCl3 and 13C-NMR spectrum (125 MHz) of CF in CDCl3] had been provided as the supporting information.Click here for additional data file.


In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS).Avian infectious bronchitis (IB) is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV). Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection.in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection  Gamma coronavirus of the coronavirus genus and replicates primarily in the upper respiratory tract, kidney, and oviduct of chickens  -1-propanesulfonate; DTT: dithiothreitol; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IBV: Infectious bronchitis coronavirus; IEF: isoelectric focusing; IPG: immobilized pH gradient; MALDI-TOF-TOF/MS: matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; PMF: peptide mass fingerprinting; RT-PCR: reverse transcriptase-polymerase chain reaction;SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPF: specific pathogen free; TFA: trifluoroacetic acid; 2-DE: two-dimensional gel electrophoresis.The authors declare that they have no competing interests.SL designed the study. SL and ZC drafted the manuscript. ZC, ZH and YS carried out virus infection and test for the presence of IBV. ZC and HG carried out the 2-DE experiments, image analysis, excised the protein spots, data analysis and interpretation, and confirmed the differential expression by real-time RT-PCR and Western blotting analysis. SL wrote the manuscript. XK revised the manuscript. All authors read and approved the final manuscript.Additional_file_1.doc containing the MALDI-TOF spectrum and MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chicken embryo tracheal tissues.Click here for fileAdditional_file_2.doc containing the PMF spectrum and Mascot database search results of differentially expressed protein spots in IBV-infected chicken embryo tracheal tissues.Click here for fileAdditional_file_3.doc containing the MALDI-TOF spectrum and MALDI-TOF-TOF spectrum of differentially expressed protein spots in IBV-infected chicken embryo kidney tissues.Click here for fileAdditional_file_4.doc containing the PMF spectrum and Mascot database search results of differentially expressed protein spots in IBV-infected chicken embryo kidney tissues.Click here for file


Reduced muscle mass is a hallmark of metabolic diseases like diabetes and cancer. The mammalian (mechanistic) target of rapamycin complex 1/S6 kinase 1 (mTORC1/S6K1) pathway is critical to the regulation of muscle protein synthesis and mass but its mechanism of action is not completely understood.Using L6 myotubes, we characterized the regulation of programmed cell death 4 (PDCD4), a recently described substrate of S6K1. The abundance, but not Ser67 phosphorylation, of PDCD4 was sensitive to amino acid and serum deprivation: values in starved cells were 4.5X of control (P < 0.001). Refeeding had opposite effects. Growth factors, compared to amino acids, appeared more critical in regulating PDCD4 abundance. Furthermore, inhibition of mTORC1 or the proteasome prevented the refeeding-associated decrease in PDCD4 abundance. Amino acid and serum deprivation significantly increased PDCD4 binding to eIF4A (P < 0.05); this was reversed during refeeding. PDCD4 depletion by RNA interference had no significant effect on phenylalanine incorporation into myotube mixed proteins in control cells but further suppressed (30%) this measure in nutrient-deprived cells (P < 0.0005). This was not observed in myoblasts. In starved myotubes, PDCD4 depletion further reduced the association of eIF4G with eIF4E.Our data suggest that in myotubes, PDCD4 abundance is sensitive to nutritional manipulation in an mTORC1 and proteasome depended manner. Furthermore, the role of PDCD4 in regulating protein synthesis appears dependent on the developmental state of the cell. The mammalian (mechanistic) target of rapamycin complex 1/ribosomal protein S6 kinase 1 (mTORC1/S6K1) signalling is a critical regulator of skeletal muscle mass and metabolism, and mechanisms that regulate it are studied as possible targets for the treatment/prevention of loss of muscle mass in diverse muscle atrophying conditions . HoweverProgrammed cell death 4 (PDCD4),  , H731 99, and inMuch of what is known about PDCD4 is from cancer studies where PDCD4 is proposed to function as a cell cycle inhibitor/tumor suppressor. Loss of this protein is associated with invasion, progression or increased aggression of numerous, but not all , cancersIn the present work, using L6 myotubes, our specific objectives were to: 1) examine the requirement for mTORC1/S6K1 and the ubiquitin proteolytic system in regulating PDCD4; 2) examine the contribution of amino acids vs. growth factors in mediating the effect of nutrition on PDCD4; and 3) determine whether nutritional status affects the interaction of PDCD4 with components of eIF4F. Because others have suggested that signalling pathways that regulate protein metabolism may be regulated differently in myotubes versus myoblasts  and becaGiven the identification of PDCD4 as a substrate of mTORC1/S6K1 signalling, and the fact this kinase pathway is regulated by nutrients, we examined the effect of nutrient deprivation on the regulation PDCD4 in L6 myotubes. Neither 12 h of serum and amino acid deprivation nor refeeding in a complete medium had any significant effect on PDCD4 Ser67 (the residue targeted by S6K1) phosphorylation . Amino acid-free medium (RPMI 1640) was obtained from US Biological . PDCD4 siRNA oligonucleotides, phosphatase and protease inhibitor cocktails were purchased from Sigma-Aldrich . Alpha Modification of Eagle’s Medium (AMEM) was obtained from Wisent .Fetal Bovine Serum (FBS), Horse Serum (HS), Lipofectamine RNAiAntibodies to eIF4A, eIF4G, phosphorylated (Thr389) S6K1, and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse) were purchased from Cell Signaling Technology . Antibody against PDCD4 was from Cell Signaling Technology or Santa Cruz Biotechnology . Antibodies against phosphorylated  PDCD4 were from Sigma-Aldrich or Aviva Systems Biology .L6 myoblasts were cultured in 12-well plates in growth medium  until they reached ~80% confluency. Cells were then shifted into differentiation medium (DM: AMEM as above except that 2% HS replaced 10% FBS). Experiments were carried out on day 4–5 of differentiation. For starvation experiments, myotubes were grown in differentiation medium or starved in amino-acid free, serum-free medium (amino acid-free RPMI 1640) for 12 h. They were then refed in DM for 1 or 3 h. To examine the roles of amino acids and growth factors in regulating PDCD4 abundance, in some experiments refeeding was done in incubation media of varied composition. To examine the requirement for mTORC1 or the ubiquitin dependent proteolytic system on the regulation of PDCD4, additional refeeding experiments were carried out in the presence of inhibitors of these pathways  or equivalent volumes of DMSO. At the end of the experiments, cells were harvested in a lysis buffer  sodium dodecyl sulphate (SDS), 1 mM DTT, supplemented with protease (10 μl/mL) and phosphatase (10 μl/mL) inhibitor cocktails).Max (Life Technologies) as previously described [Myotubes on day 4 of differentiation were transfected with 30 nM siRNA oligonucleotides designed against PDCD4 or with a proprietary scrambled oligonucleotide ), using Lipofectamine RNAiescribed . We usedescribed ,40.Proteins were resolved on 7.5, 10, or 15% SDS-PAGE (depending on the antigens being studied), transferred onto polyvinylidene difluoride (PVDF) membranes, which were then immunoblotted for the indicated antigens, as previously described . ImmunobTo immunoprecipitate eIF4A or PDCD4, myotubes were cultured in 10-cm plates. Following appropriate treatments, cells were rinsed in ice-cold PBS and then lysed in 500 μl of ice-cold lysis buffer (40 mm HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol 2 phosphate, 0.5 mM orthovanadate) supplemented with 0.03% CHAPS, 1 mM DTT, 0.5 mM NaV, 1 mM benzamidine, 6.25 mM N-ethyl-maleimide (NEM) and protease inhibitor cocktail (10 μL/mL). One hundred micrograms of myotube proteins were combined with either anti eIF4A or anti-PDCD4 (Santa Cruz biotechnology) antibodies and the mix rotated overnight at 4°C. The following day and in order to precipitate the antigen-antibody complex, 50 μL of re-suspended BioMag protein G–bound beads  were added to each of the immunoprecipitation tubes and the suspension rocked gently at 4°C for 1 h. The beads were collected on a magnetic stand (Life Technologies) and washed 3 times with 0.1 M sodium phosphate buffer. After the last wash, beads were re-suspended in 1X SDS-PAGE sample buffer and boiled at 95°C for 2 minutes. Following a brief centrifugation, eluates were collected, separated on 10% SDS-PAGE, and blotted for PDCD4 and eIF4A.Data are presented as means ± SEM. Treatment means were compared using a one-way analysis of variance (ANOVA) and differences among individual means assessed using the Bonferroni multiple comparison test or, as in Figures The authors declare that they have no competing interest.OAJA conceived, designed, conducted experiments, wrote and reviewed the manuscript; DK, NI and NM conducted experiments and reviewed the manuscript. All authors read and approved the final version of the manuscript.PDCD4 phosphorylation (Ser457) in L6 myotubes.Click here for fileMyofibrillar protein synthesis in L6 myotubes.Click here for file


The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2•−) production in human neutrophils and to probe the concept of ligand-specific receptor conformations  in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2•− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2•− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2•− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2•− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2•− production after β2AR-stimulation although cAMP-increasing substances inhibited O2•− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2•− production in a cAMP-independent manner.In neutrophils, activation of the β After phagocytosis of invading agents neutrophils are able to destruct them, the respiratory burst NADPH oxidase being a major player Classic models of G-protein-coupled receptor (GPCR) activation postulate the existence of a single active (R*) and an inactive (R) state s-coupled receptor, the histamine H2 receptor, in two native cell systems, human eosinophils and neutrophils 2R agonists as well as H2R antagonists do not match by comparing their effects on eosinophils and neutrophils as well as by comparing these parameters with data obtained in a recombinant test system. Moreover, even in the same cell type, differences were observed when ligands were characterized determining two different parameters. Each ligand triggers unique effects depending on the test system and parameters measured which is of importance for further drug development.Recently, we have reported on the functional selectivity of another G2AR on human neutrophils with a series of structurally diverse β2AR ligands and thereby, to probe the concept of ligand-specific receptor conformations on one of the most important and best-characterized GPCRs in a physiologically relevant native human cell system. Two distinct parameters were chosen for the characterization of the β2AR on neutrophils. The first parameter was measurement of the cAMP content in neutrophils. The second parameter was monitoring of the β2AR-mediated effects on fMLP-stimulated O2•− production. We also examined the effects of various pharmacological tools including protein kinase inhibitors, AC inhibitors and activators and various cAMP analogues on fMLP-induced O2•− production in order to obtain further insights into the mechanisms underlying inhibition of NADPH oxidase.The aim of the present study was the characterization of the βS*,S*)-3-(Isopropylamino)-1-butan-2-ol) ((±)--ICI 118551) (ICI) and (±)-bisoprolol (BIS) were obtained from Tocris Bioscience . (−)-Isoproterenol (ISO), (−)-adrenaline (ADR), (±)-salbutamol (SAL), (±)-dobutamine (DOB), (±)-metoprolol (MET), (±)-alprenolol (ALP), (±)-atenolol (ATE) and forskolin (FSK) were from Sigma-Aldrich . (−)-Ephedrine (EPH) was from Mallinckrodt  and (±)-dichlorisoproterenol (DCI) from Aldrich . Chemical structures of ligands are depicted in 2+ and Mg2+ (pH 6.5–7.0) was purchased from PAN Biotech  and Biocoll separating solution from Biochrom . Trypan blue solution, ferricytochrome c, cytochalasin B, fMLP and IBMX were from Sigma-Aldrich. Solvents for extraction and HPLC analysis were purchased as follows: HPLC-gradient grade water and methanol from J. T. Baker , ammonium acetate from Sigma-Aldrich and acetic acid from Riedel-de Haen . Tenofovir was obtained from the National Institutes of Health . N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate (DB-cAMP)  was purchased from Sigma-Aldrich. cAMP (>99.9%), adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) (>99.94%), N6-monobutyryladenosine-3′,5′-cyclic monophosphate (6-MB-cAMP) (>99.56%), 2′-O-monobutyryladenosine-3′,5′-cyclic monophosphate (2′-O-MB-cAMP) (>99.69%), adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS) (>99.96%), and 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) (>99.97%) were obtained from BIOLOG Life Science Institute . Structures of cAMP analogs are shown in (6 cells/ml for the O2•− assay or 1×107 cells/ml for the determination of cAMP) and stored on ice until use. Experiments were performed within 4 h after completion of isolation because at later time points, viability of cells declined substantially as assessed by trypan blue dye uptake and declined responsiveness to receptor ligands (data not shown).This study and the consent procedure were approved by the Ethics Committee of the Medical School of Hannover. Written consent was obtained by all volunteers. The completed and signed consent forms are kept on file in the secretary of the Institute of Pharmacology of the Medical School of Hannover. Human neutrophils were isolated from venous blood of healthy volunteers of either sex (1.6 mg EDTA/ml blood as anticoagulant) or from buffy coat obtained from the Institute for Transfusion Medicine . Buffy coat preparations were also obtained from individual donors. All isolation steps were carried out at room temperature. Firstly, 7 ml of venous blood or 5 ml of buffy coat were diluted to 35 ml with 1×DPBS and carefully layered onto 15 ml of Biocoll separating solution (density 1.077 g/ml) in a 50 ml-Falcon tube. Following centrifugation , the upper three layers were removed. The residual pellet (∼2 ml), which contained erythrocytes and granulocytes, was resuspended in 18 ml of Millipore water and incubated for 1 min under gentle agitation in order to achieve selective lysis of erythrocytes. Afterwards, isotonicity was restored by adding 2.2 ml of 10×DPBS, and centrifugation at 300×g for 5 min followed. The lysis step was repeated once to remove residual erythrocytes. The cell pellet was re-suspended in 5 ml of 1×DPBS and sedimented by centrifugation at 300×g for 5 min. The resulting cell preparation consisted of viable neutrophils (>98%), as assessed by the trypan blue exclusion test. Finally, neutrophils were suspended in 1×PBS  5 neutrophils in 1×DPBS. After pre-incubation of the reaction mixtures for 3 min at 37°C, reactions were initiated by addition of fMLP (1 µM). Reference samples contained all components listed above except for fMLP. O2•− formation was continuously measured by monitoring the reduction of ferricytochrome c at 550 nm for 30 min at 37°C, using a Synergy 4 microplate reader . The difference in absorbance at 550 nm between 0 min (addition of fMLP) and 30 min was used for subsequent data analysis, in order to assess agonistic activity of examined ligands. With the exception of DOB, all examined test compounds did neither reduce ferricytochrome c nor stimulate O2•− production per se nor acted as radical scavenger as assessed by the lack of effect on phorbol ester-stimulated O2•− production (data not shown). As at DOB concentrations higher than 500 nM, ferricytochrome c reduction took place, the maximum concentration of DOB used in the O2•− assays was 500 nM.Reactions were carried out in 96-well plates in triplicate. Standard reaction mixtures  contained 1 mM CaCl2 , IBMX  and the respective ligand at different concentrations in 1 x DPBS were pre-incubated for 5 min at 37°C. Isolated neutrophils suspended in 1×DPBS were pre-incubated separately for 10 min at 37°C. Following the addition of 50 µl of neutrophils (5×105 cells/reaction vessel) to reaction mixture, samples were incubated for 10 min at 37°C. Afterwards, samples were incubated for 10 min at 95°C in order to denature cell proteins and then cooled to 4°C. One hundred µl of ice-cold internal standard  in eluent A  were added. The suspension was centrifuged at 20.800×g at 4°C for 5 min in order to remove denatured proteins. The cAMP concentration of the supernatant was determined by reversed phase HPLC coupled to mass spectrometry (HPLC-MS/MS).Reactions were conducted in triplicate in 1.5 ml Eppendorf reaction vessels in a total volume of 100 µl. Fifty µl of the reaction mixture containing CaClIn this study, cAMP levels were determined by HPLC-MS/MS which is characterized by extremely high sensitivity and specificity 2AR-GsαS, was performed as described previously 2•−, cAMP and GTPase assays were analyzed with the Prism 5.01 software . The means ± S.E.M. were always determined by the analysis of at least three independent experiments, performed in triplicate, if not indicated otherwise.Chromatograms, obtained by the HPLC-MS/MS analysis, were analyzed with the Analyst Software 1.5.1 (AB Sciex). Steady-state GTPase activity assay, using membrane preparations of Sf9 insect cells, expressing fusion protein βEmax) of ISO in each assay was set to 1.00 and the efficacies of other ligands were referred to this value. The pKB values for β2AR antagonists were calculated according to Cheng and Prusoff KB = - log(ICantagonist50/(1+(cISO/x))); ICantagonist50 – IC50 value of an antagonist, determined in antagonist mode, cISO – used concentration of ISO, ×– IC50 (O2•− assay) or EC50 value (cAMP assay and GTPase assay) for ISO, determined in agonist mode .Data for receptor ligands were analyzed using one-way ANOVA, followed by Bonferroni’s multiple comparison test, in order to compare p2AR agonists with efficacies varying from very weak partial to full agonism 2AR agonists were measured as inhibition of fMLP-stimulated O2•− production  instead of fMLP, the examined β2AR agonists had no effect on O2•− production at all (data not shown).A trivial explanation for the differing effects of βKB values were determined for β2AR antagonists 2AR antagonist potency is also apparent in a native test system, namely in neutrophils.According to conventional models of GPCR activation, potency of an antagonist for a given receptor is constant irrespective of the tissue or recombinant system selected for the characterization, the agonist used for the stimulation of GPCR and downstream signaling event monitored KB values for ICI, MET, ALP, BIS and ATE were determined in the cAMP and O2•− assay by applying a submaximally effective concentration of ISO and increasing concentrations of β2AR antagonists. Concentration-response curves for antagonists are shown in 2AR-GsαS). The statistical analysis of the obtained data revealed no difference of pKB values between the two different parameters in neutrophils, BIS being an exception. However, in comparison with data on neutrophils, the potencies of all antagonists were significantly reduced on the recombinantly expressed β2AR-GsαS. KB values of antagonists for the various parameters analyzed. It is evident that the correlations for antagonists are much better than the corresponding correlations for agonists shown in The p2AR-signaling is very complex, depending on ligand and test system 2AR-mediated inhibition of fMLP-stimulated O2•− production is thought to be due to cAMP production and PKA activation 2AR stimulation is a crucial event for inhibition of fMLP-stimulated O2•− production . Even the cAMP antagonist Rp-cAMPS which competes with cAMP for the binding to the regulatory subunits of PKA 2•− production in human neutrophils . The activator of the cAMP effector protein Epac, 8-pCPT-2′-O-Me-cAMP 2•− production, arguing against an involvement of Epac in NADPH oxidase regulation. We also observed that the PKA inhibitors H89, KT5720 and Rp-cAMPS showed no reversing effect on the inhibition of O2•− production caused by DB-cAMP  and an inactive (R) state has now been superseded by a more complex model involving multiple active receptor conformations that lead to ligand-specific receptor activation, also referred to as functional selectivity or biased agonism t system . If only2AR antagonists, effects were similar in the cAMP and O2•− assay on neutrophils  in neutrophils are independent of each other. The lack of effect of DCI and EPH on cAMP accumulation despite inhibitory effects of these ligands on O2•− production supports the concept of cAMP-independent inhibition of NADPH oxidase. Moreover, SAL is more effective at inhibiting NADPH oxidase than at increasing cAMP. Our failure to detect stimulatory effects of DCI and EPH on cAMP accumulation and ineffective stimulation of cAMP accumulation by SAL are not due to cAMP degradation since we included a phosphodiesterase inhibitor into the cAMP assay. Moreover, we applied a highly sensitive and specific MS method to detect cAMP, avoiding notorious cross-reactivity problems of antibodies widely applied in cyclic nucleotide detection 2AR although, in principle, cAMP can inhibit O2•− production .Stimulation of cAMP accumulation and reduction of Othe case , and morant β2AR . Thus, t2AR-dependent impedance response to ISO is the result of activation of multiple signaling pathways, including Gs and Gi coupling, Gβγ-dependent signaling, cAMP production, extracellular signal-regulated kinase (ERK) 1/2 activation as well as Ca2+ mobilization. Therefore, when stimulation of the β2AR does not activate the PKA-dependent pathway in human neutrophils or this pathway does not interfere with the NADPH oxidase signaling, modulation of the e.g. ERK1/2-pathway could be the explanation for the inhibition of the NADPH oxidase. Interestingly, in other studies on human neutrophils, a correlation between activation of cAMP/PKA signaling pathway and inhibition of ERK phosphorylation was observed, resulting in reduced fMLP-induced O2•− production 2AR was reported to activate ERK signaling pathway via interaction with Gi, Src and/or arrestin proteins in addition to Gs proteins in other systems 2AR-signalling strongly depends on the cell system used 2AR activation, ERK activation and fMLP-stimulated O2•− production in future studies as well. This could provide an explanation for the observed biased effects of some examined β2AR ligands on neutrophils.Stallaert and coworkers 2AR; e.g. we tested the p38 inhibitor SB203580, the JNK inhibitors SP600125 and SP600123, the ERK inhibitor PD980598, the PI3 kinase inhibitor LY294002 and the protein kinase C inhibitor Goe 6978. Unfortunately, these compounds per se inhibited fMLP-stimulated O2•− production (data not shown) so that separate effects of these compounds on ISO actions could not be properly dissected. An alternative approach will be the examination of the effects of β2AR ligands on protein phosphorylation in neutrophils, using screening approaches encompassing numerous protein kinases as starting point. Lastly, it will also be very important to explore the possibility that the new signaling molecules cyclic CMP (cCMP) and cyclic UMP (cUMP) 2AR-mediated signal transduction.In preliminary studies we examined a number of pharmacological inhibitors to explore alternative signaling pathways of the β2AR agonists as anti-inflammatory agents 2AR ligands on cAMP accumulation and O2•− production in human neutrophils. However, there are many native test systems described, which would afford an expansion of our knowledge about functional selectivity of β2AR ligands. One example could be the parallel measurement of cAMP concentration and contractility of cardiomyocytes or endothelial cells as functional parameter 2AR ligands. All in all, there is a need to assess biased signaling through the β2AR in a wide spectrum of native test systems in order to improve the desired therapeutic effect of developed compounds on the one and to minimize side effects on the other. Furthermore, β2AR agonists that have been used for many years in the therapy of humans, e.g. as bronchodilators in patients with asthma or chronic obstructive pulmonary disease, should be reassessed using various native test systems, as there is a potential to improve already existing therapies, particularly by minimizing unwanted effects.Data from the literature suggest a feasible use of βAs a general approach to study functional selectivity in native cells, it is necessary to construct a systematic data matrix in which multiple ligands are examined at multiple concentrations (enabling determination of precise potencies and efficacies) and for multiple parameters. Previous studies with native human cells may have largely overlooked functional selectivity at GPCRs because there was no appreciation of the necessity to generate such a systematic data collection in order to understand cell biology. It is evident that availability of human cells is an issue for comprehensive pharmacological studies. The human neutrophil, despite its inherent problems, i.e. short survival time after isolation, variable responsiveness and poor accessibility to genetic manipulation, is a suitable model system to test functional selectivity for several reasons. Specifically, neutrophils can be obtained in large quantities, express multiple receptors, display numerous cell functions that can be assessed quantitatively and are pathophysiologically relevant for inflammation.Figure S12AR agonists and antagonists examined in this study.Structures of the β(PPTX)Click here for additional data file.Figure S2Structures of cAMP and cAMP analogs examined in this study.(PPTX)Click here for additional data file.


To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals’ knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H Reactive oxygen species (ROS) function as important physiological regulators of intracellular signaling pathways . High ROThe mitogen-activated protein kinase (MAPK) signaling pathways allow cells to interpret a wide range of external signals and respond by generating a plethora of different biological effects. Members of the MAPK family, including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, are activated by ROS. The activation of these kinases usually regulates the expression of a variety of genes involved in survival, proliferation or cell death, depending on the stimulus and the cell-type studied ,3,7.2O2-induced cell death [2O2 dose and duration of the stress signal [The ribosomal protein S6 kinase 1 (S6K1) is a common downstream target of signaling by hormones and nutrients. S6K1 is a substrate of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). This complex is a Ser/Thr kinase that regulates S6K1 activation through its phosphorylation at Thr389 (T389). Activated S6K1 regulates the phosphorylation of other substrates such as the ribosomal protein S6 to promote protein synthesis, cell growth and cell proliferation . In recell death , others ll death . In somell death ,13, wherll death ,14,15. Ts signal .S6K1 activation is measured by the increase of its phosphorylation at T389 and/or by the phosphorylation increase of its substrate, the ribosomal protein S6, at S235/S236. Thus, antibodies against these phosphorylated residues are a valuable tool for analyzing S6K1 activation. The specificity of these antibodies is crucial to interpretation of the data. S6K1 is member of a family of serine /threonine kinases named AGC. Other members of this family, such as the mitogen- and stress-activated kinases (MSK) and the p90 ribosomal S6 kinases (RSK), show a high degree of homology, in particular a serine residue within the hydrophobic motif of the RSK and MSK proteins . Previou2O2 and that the anti-P-T389-S6K1 antibody detected the phosphorylation of RSK and MSK proteins by H2O2 in a p38- and ERK-dependent manner.In response to oxidative stress, MAPK signaling pathways are activated; contradictory data have been reported for the S6K1 signaling pathway. We asked whether under these conditions the anti-P-T389-S6K1 antibody detected RSK and MSK proteins and could be a motif to misinterpret these findings. In the present study, we showed that S6K1 is not involved in the fast response to incubation with H2O2) (Panreac); U0126 and SB203580 ; anti-mTOR, anti-P-T389-S6K1 (1A5), anti-P-S380-RSK, anti-P-S376-MSK, anti-P-S235/236-S6, anti-S6 (54D2) and anti-PT180/Y182-p38 antibodies ; anti-MSK, anti-S6K1 (C-18) and anti-RSK1 (C-21) antibodies ; Alexa Fluor 488, Alexa Fluor 546, TO-PRO3 (Molecular Probes); anti-P- H2AX antibody, Immobilon-P PVDF transfer membrane (Millipore Corporation); siRNA used: mTOR (CCCUGCCUUUGUCAUGCCUdTdT), S6K1 (GGGGGCUAUGGAAAGGUUUdTdT), RSK1 (GCUAUACCGUCGUGA-GAUCdTdT), RSK2 (GGAGGAGAUUAACCCACAAdTdT), MSK1 (GGAACUGG-AGCUUAUGGAAdTdT), MSK2 (UUGCACAUGAUCUCGGCCGdTdT) and non-targeting control (UAGCGACUAAACACAUCAAdTdT).Insulin, wortmannin, rapamycin and anti-P-ERK1/2 antibody (Sigma-Aldrich); hydrogen peroxide solution  . All celPreviously treated cells were lysed in CHAPS lysis buffer  for 1 h at 4°C and equal amounts of proteins were separated by electrophoresis. To analyze simultaneously large and small proteins in the same gel, we used Tris-Acetate PAGE systems . After rMCF-7 cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT). The cells were blocked and permeabilized with 10% fetal bovine serum and 0.1% Triton X-100 in PBS for 2h. The primary antibodies, anti-PT180/Y182-p38 (1:200), anti-P-S380-RSK (1:50), anti-P-ERK1/2 (1:200) and anti-P- H2AX (γH2AX) (1:500), were incubated at 4°C overnight; and the secondary antibodies, at RT for 2h. Nuclei were stained with TO-PRO-3 and the cells were examined by laser confocal microscopy.2, 1 mM DTT). The kinase assay was performed as previously described [Lysates from MCF7 cells were immunoprecipitated with anti-RSK or anti-MSK antibodies. Lysis and immunoprecipitation were carried out in a buffer containing 40 mM Hepes, pH 7.5, 120 mM NaCl, 50 mM NaF, 0.3% CHAPS and the above protease inhibitors. Immunoprecipitates were washed three times with lysis buffer and once with kinase buffer . MCF7 cells were incubated with H2O2 for 30 min. We used these human cells because they have been extensively used to study the response to oxidative stress. As a positive control of S6K1 activation, MCF7 cells were stimulated with insulin after overnight serum deprivation. As shown in 2O2, we detected an increase in a band with similar mobility to S6K1 p85 isoform that we named p85 protein. No increase was seen in p70 isoform phosphorylation. With both treatments, an increase in the phosphorylation of the ribosomal S6 protein was observed. These treatments did not modify endogenous levels of S6K1 or S6 proteins  and specific phosphorylation of endogenous p85 protein was seen on incubation with various concentrations of H2O2 . Maximum2O2 in the presence of known inhibitors of S6K1 activation  and MSK (MSK1/MSK2). The mix of siRNA against RSK or MSK isoforms decreased the p85 detection with anti-P-T389-S6K1 antibody . This de2O2. To show this point, kinase assays were performed using purified kinases and GST-S6 fusion protein as substrate. As it is shown in 2O2.The previous data suggest that the kinase activity of RSK and/or MSK is regulated by H2O2 through Akt-independent mechanisms [2O2  through the analysis of S6K1 phosphorylation at T389 using anti-P-T389 antibodies. In some cases inhibition of mTORC1 activity was reported [2O2 concentration and duration of the stress signal [The mTOR signaling pathway has an essential role in the regulation of mammalian growth and development. Hormones such as insulin and nutrients such as amino acids mediate their cellular effects through this pathway . Severalreported ,14,15, wreported ,13,22. Ts signal .2O2 concentrations and/or during long time periods produces DNA damage and p53 activation. Thus, in these conditions, inhibition of the mTOR signaling pathway would be expected [2O2 concentrations or during shorter time periods, we showed that mTOR signaling was not involved and explained previous observations by the use of the anti-P-T389-S6K1 antibody from Cell Signaling. We showed that this antibody recognized a phosphorylated protein of 85 kDa in response to H2O2. Knockdown experiments together with the use of specific inhibitors let us show that this phosphorylated protein of 85 kDa was not regulated by mTOR and was not S6K1. A similar situation had been previously reported in signaling by amino acids, for which authors showed that the phosphorylated p85 protein recognized by the anti-P-T389-S6K1 antibody was phosphorylated RSK and MSK proteins [Stress conditions that produce DNA damage activate cell repair mechanisms where p53 activation is involved ,23,24. Dexpected ,14,15. Fproteins . To avoiproteins ,17, resu2O2. Moreover, we reported the phosphorylation and activation of their substrates RSK and MSK. Interestingly, in human cells phosphorylation of both RSK and MSK proteins was sensitive to ERK and p38 activities, indicating that both kinases were necessary to phosphorylate RSK and MSK proteins completely in response to H2O2. In contrast, in mouse cells, phosphorylation of MSK by H2O2 seemed exclusively dependent on p38 since, in the absence of p38α, MSK phosphorylation was completely abolished. In this context, regulation was reported of mTORC1/p70 S6K1 by p38 in Drosophila melanogaster cells [2O2. As expected, insulin did not activate the MAPK signaling pathways. Instead, oxidative stress activated phosphorylation of p38/ ERK/MSK in WT MEFs and of RSK in p38α-deficient MEFs. Anti-P-T389-S6K1 antibody detected the phosphorylated p85 protein and its regulation correlated with phosphorylated RSK and MSK proteins. All these observations suggest that, at least in mouse and in human cells, the fast response to oxidative stress caused by low concentrations of H2O2 is mediated by MAPK signaling pathways and not by the mTORC1/p70 S6K1 signaling pathway. In summary, we believe that this report helps to explain previous controversial results and to clarify the cellular signaling activated in response to oxidative stress.Members of the MAPK family such as ERK, JNK or p38 are phosphorylated and activated in response to oxidative stress ,7,20. Weer cells  and in Mer cells . Using t