During the last decade, we have gained much insight into the mechanisms that open and close a sensitive period of plasticity in the visual cortex. This brings the hope that novel treatments can be developed for brain injuries requiring renewed plasticity potential and neurodevelopmental brain disorders caused by defective synaptic plasticity. One of the central mechanisms responsible for opening the sensitive period is the maturation of inhibitory innervation. Many molecular and cellular events have been identified that drive this developmental process, including signaling through BDNF and IGF-1, transcriptional control by OTX2, maturation of the extracellular matrix, and GABA-regulated inhibitory synapse formation. The mechanisms through which the development of inhibitory innervation triggers and potentially closes the sensitive period may involve plasticity of inhibitory inputs or permissive regulation of excitatory synapse plasticity. Here, we discuss the current state of knowledge in the field and open questions to be addressed. Many things can be learned more easily during childhood than in adulthood, including speaking a new language, playing an instrument, or performing a sport. This is the consequence of how our brain develops. It seems to make sense to learn these skills in a rather permanent way when we are young so that we can take advantage of them when we are adults. This is not only true for learning skills or facts but reflects a general property of brain development where periods of enhanced experience-dependent plasticity in different cortical and subcortical brain regions are essential for achieving functional and reliable connectivity between brain areas. During the last decade, it has become clear that specific molecular and cellular mechanisms are in place that regulate the onset and offset of these sensitive periods , indicatIn order to be able to treat such disorders in the future and to develop approaches to increase adult plasticity for treating brain damage, extensive research on the mechanisms opening and closing sensitive periods has been performed. Plasticity in the visual cortex has been the most used model to address this. The primary visual cortex (V1) receives inputs conveying information from both eyes, and neurons in V1 have a preference for inputs from one of the eyes . This soThe initial evidence supporting the involvement of the inhibitory system in sensitive period onset was the discovery that in mice in which GABA synthesis is reduced due to the deletion of the glutamic acid decarboxylase GAD65, no sensitive period plasticity can be induced until the moment that they are treated with the GABA(A) receptor agonist benzodiazepine . InteresIn order to understand the mechanism through which inhibition may initiate the sensitive period, it is important to know which interneuron subtypes are involved. Most evidence points towards the involvement of fast-spiking Parvalbumin (PV) expressing basket cells. These interneurons innervate excitatory neurons , each otGad-1 gene  in individual neurons in the visual cortex, which results in reduced synapse formation and axon branching [The formation of inhibitory inputs by PV basket cells onto the somata of excitatory neurons is strongly influenced by neuronal activity around the start of the sensitive period. This development of perisomatic inhibitory innervation can be replicated in cortical slice cultures. Using cultures from transgenic mice expressing GFP in PV interneurons it was found that innervation by PV interneurons was only reduced when activity was inhibited by TTX between p18 and p24, but not when this was done at later stages . Similarranching . OverexpSuch a positive feedback loop seems to fit nicely with other findings. For example, as explained above, in mice deficient for GAD65, the sensitive period does not start until inhibitory inputs are strengthened with benzodiazepines . HoweverEvidence for increased inhibitory inputs being central to the initiation of the sensitive period of OD plasticity is very convincing . It is lHowever, the interpretation of how the ECM limits adult cortical plasticity may have to be adjusted based on novel evidence . While tWhat other evidence is there to support the notion that mature levels of inhibitory inputs restrict adult cortical plasticity? Recent studies have shown that when adult rats are monocularly deprived when housed in an enriched environment, OD plasticity can be induced more effectively than in normally reared rats . HistoloAlternatively, the maturation of inhibitory input does not only alter the levels of inhibition  but also causes qualitative changes in inhibitory synaptic transmission. Several recent findings seem to support this notion. One study has shown that when inhibitory neurons are isolated from 12–16-day-old embryo's and transplanted into newborn mice, a second sensitive period occurs around the time that the transplanted interneurons reached an equivalent of approximately 4 weeks postnatally, exactly when the sensitive period normally occurs in mice . It did While evidence for maturation of inhibitory innervation regulating sensitive period onset is very solid, the mechanism of how inhibition regulates plasticity remains unclear. There are two likely explanations, which are not mutually exclusive. The first is that maturation of inhibition moderates the level of plasticity in excitatory synapses. It is known from slice physiology that reduced GABAergic inhibition facilitates synaptic plasticity . PV baskGad-1 gene (encoding the rate limiting glutamate decarboxylase GAD67) resulting in expression of GFP in most interneurons [Suppression of deprived-eye responses by inhibition can in principle be achieved through increased excitation of interneurons responding to the deprived-eye, or through strengthening of their inputs onto excitatory neurons. A few studies have been published in recent years addressing these possibilities. We will first discuss three studies which have investigated how the responses of interneurons change during OD plasticity. Two of these employed two photon imaging of calcium responses of individual neurons in mice expressing a fluorescent protein in inhibitory neurons. The first study employed mice in which the GFP coding sequence was knocked into the rneurons . This strneurons , using arneurons , 56. Therneurons . Again, rneurons . UnfortuGad-1 allele [Which interneuron subsets were assessed in the different studies? The two-photon studies employed different mouse lines in which GFP variants were expressed in interneurons. Although in the two mouse lines, there is good co-expression of GFP and GABA  miniature inhibitory postsynaptic currents (mIPSCs) in layer 4 star pyramidal neurons of the binocular visual cortex, without a change in mIPSC frequency . After tAlthough it is unknown if the synaptic changes induced by MD correlate with OD plasticity, it is likely that the increased inhibition reduces the responses of the deprived-eye. To prove this, it would be necessary to study the inhibitory synaptic strength in vivo and correlate it with OD. This experiment has not been done yet, but one study looked at the inhibitory contribution to the change in OD of pyramidal cells in vivo . The OD After MD, the removal of inhibition caused a reversal of the eye preference in the spike response. Such a reversal can only happen if the inhibitory input is biased in the same direction as the excitatory input, and opposite to the pyramidal cell's spiking output. The difference in the effect of removal of inhibition with and without MD can be interpreted as an increase in inhibition from deprived-eye stimulation , 64. It In the adult animal, no change in the spike biases was observed after removal of inhibition, even after MD. This means that in the adult animal the spiking bias is more similar to the excitatory input bias than in the juvenile animal. But even in adult animals, OD of the population response can be changed by altering inhibition by pharmacology, for instance in undeprived cats  and in mMore evidence suggesting that inhibition can alter the expression of the OD shift after MD comes from experiments assessing the influence of callosal connections on sensitive period plasticity. After a week of MD, the OD shift in the binocular visual cortex contralateral to the deprived-eye is removed by acutely silencing the visual cortex on the opposite side . SurprisThis change in inhibitory synaptic strength, the reported increase in mini IPSC amplitude  and the Another interesting possible mechanism for the increase in synaptic strength of specifically deprived-eye dominated inhibitory synapses, is the possible reduction of the endocannabinoid receptor dependent form of long-term depression of inhibitory synapses (iLTD)  by MD. IRecently, it was discovered that MD does not reduce responses to the deprived-eye in all neurons in visual cortex, but that a selection of cells with little input from the open eye often show more response to the deprived-eye than in the normal situation . This caIt has long been known that removal of visual input to the adult visual cortex leads to a decrease in levels of GABA, its synthesizing enzymes GAD65 and GAD67 and the beta2/3 subunit of the GABA(A) receptor –76. Ten As discussed earlier, the state of the inhibitory system is an important factor in the opening of the sensitive period. This correlation between the state of inhibition and plasticity may be through changes in the plasticity of the response or connections of inhibitory neurons themselves, as suggested for instance by the experiments on iLTD . AlternaA more indirect way through which inhibition may alter plasticity is through controlling brain oscillations. In particular fast-spiking PV basket cells are important for the generation of gamma rhythms in the brain , 89–91. During the last decade convincing evidence has accumulated showing that the development of inhibitory synapses formed by PV interneurons defines the onset of the sensitive period of OD plasticity in V1. However, we are still in the dark about the mechanisms by which inhibitory innervation permits or instructs these experience dependent changes in neuronal responsiveness. A crucial question that remains unanswered is whether PV basket cells permit cortical plasticity by influencing neuronal synchrony and spike timing-dependent plasticity, or instruct plasticity by specifically suppressing deprived-eye responses. Experiments to address these issues will be technically difficult. Studying whether spike timing-dependent plasticity is altered through the changing influence of PV basket cell inputs during development would require in vivo experiments in which electric stimulation of individual pyramidal neurons is time-locked to visually evoked responses, a challenging undertaking. To test whether plasticity of inhibitory inputs influence the expression of OD or its plasticity, experiments involving the inactivation of interneuron plasticity or brief suppression of specific interneuron subsets during the assessment of OD will be required.There is also an almost complete lack of information on the dynamics of structural plasticity of excitatory inputs onto inhibitory neurons and vice versa during OD plasticity. Knowledge is currently limited to the finding that there is an increase in growth and retraction of inhibitory dendritic branch tips upon MD in early adulthood . This suAlthough until now evidence points towards PV basket cells being important for initiating the sensitive period, they are most likely not the only subset of interneurons that is involved in cortical plasticity. We will therefore need to dissect which types of interneurons are involved in any of the mechanisms described above. This will also require better insight into the exact wiring diagrams of excitatory and inhibitory neurons within and between different cortical layers. The production of mice expressing the Crerecombinase in specific interneurons subsets will be of tremendous help to achieve this.A recent study has shown that there are important differences between the development of GABAergic innervation in layer 4 and layers 2/3 . It was


Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs. Odor recognition is not just a simple summation of responses to the components of an odorant mixture. It has long been known to involve the synthesis of complex, poorly understood interactions variously described by terms like inhibition, suppression, hypoadditivity, masking, and synergy versus eugenol, can directly interfere with ORN activation in a manner indicative of, or at least strongly suggestive of, competitive inhibition at the binding pocket of the olfactory receptor (OR) versus octanol, can inhibit mammalian ORNs in a phosphoinositide 3-kinase (PI3K)-dependent manner Evidence for inhibitory odorant interactions within complex mixtures has been found in many different species, arguing that inhibitory or opponent signaling is a fundamental principle of odor coding Understanding the overall contribution and mechanistic nature of inhibitory input to olfactory coding first requires addressing some basic questions that have largely gone unanswered. Are there dedicated ‘inhibitory’ odorants and how many are there? Can ‘inhibitory’ odorants be assigned to a particular subset of odorants? Is there relationship between the inhibitory and conventional excitatory odorants for a given OR? Here we address these questions in the context of PI3K-dependent inhibition. We show that there is a large repertoire of odorants from diverse molecular classes that can activate PI3K in a manner physiologically relevant to olfactory transduction, including those occurring in a behaviorally-salient natural odor mixture. We further show that the PI3K-dependent inhibitory odorants overlap with conventional excitatory odorants, but do not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs.All experiments were performed on adult female Sprague-Dawley rats. All procedures were carried out in accordance with protocols approved by the University of Florida IACUC, protocol # 201105796. Rats were euthanized by inhalation of carbon dioxide and decapitated. All experiments were performed at room temperature (22–25°C).2 and 5% CO2 that contained (in mM): 120 NaCl, 25 NaHCO3, 5 KCl, 1.25 Na2HPO4, 1 MgSO4, 1 CaCl2, 10 glucose, 305 mOsm, pH7.4. After enzymatic digestion at 37°C, the tissue was gently washed with normal oxygenated ACSF and accurately triturated with a large bore fire polished glass transfer pipette. The resulting suspension was filtered through a 40 µm cell strainer (Fisher Scientific) and stored at 4°C until needed. An aliquot of the suspension was mixed with 10 µM Fluo-3/AM (AnaSpec) containing 0.04% Pluronic F127 and placed on a glass coverslip coated with concanavalin A (Sigma-Aldrich) in a recording chamber . The chamber was transferred to the stage of an inverted microscope  equipped with a 10×/0.5NA Fluar objective. To increase the sample size in each experimental session the field of view was further expanded using a 0.63× reducer video tube (Diagnostic Instruments) between the microscope and the camera. Depending on the quality of the preparation, 300–600 ORNs sensitive to IBMX/forskolin stimulation could be analyzed at once. The cells were illuminated at 500 nm  and the emitted light was collected at 530 nm  by a 12-bit cooled CCD camera . Both the illumination system  and image acquisition were controlled by Imaging Workbench 6 software (INDEC BioSystems).Experiments were performed as described previously 0 where F0 is the baseline fluorescence before odorant application. Response was detected and measured when the change in fluorescence intensity exceeded two standard deviations above the noise median level. For quantitative comparison, the peak amplitudes of the responses of different cells were normalized to the saturated responses elicited by application of a mixture of 100 µM IBMX (a phosphodiesterase inhibitor) and 10 µM forskolin (a selective agonist of adenylate cyclases) to robustly activate the cyclic nucleotide signaling pathway. Application of IBMX/forskolin was also used to identify functional ORNs. We elected to use this stimulation instead of applying saline with high KCl since often acutely dissociated cells lack dendrites and cilia yet are still able to react to the membrane depolarization induced by high KCl. The resulting values are referred to as relative units. In each experiment, groups of nine odorants were tested and then the results of all experiments representing survey of 42 odorants were pooled for further analysis. Special attention was paid to assure we were imaging individual cells when several ORNs in a cluster were co-activated.Each cell was assigned a region of interest (ROI) and changes in fluorescence intensity within each ROI were analyzed and expressed as the peak fractional change in fluorescent light intensity F/F3-isobutyl-1-methylxanthine (IBMX) and 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxylabd-14-en-11-one (forskolin) were from Sigma-Aldrich. Odorants were delivered as aqueous solutions prepared in freshly oxygenated ACSF. Single odorants were of highest purity obtained from Sigma-Aldrich, Acros Chemicals or Alpha Aesar (purchased through Fisher Scientific) and were prepared as 0.5 M stock solution in anhydrous DMSO. Cycloheptanecarbaldehyde was purchased from Wako Pure Chemical Industries (Japan). All odorant stocks were kept frozen at -20°C and the final aqueous solutions were prepared on the day of experiment. ACSF supplemented with 0.1% DMSO, the odorant carrier, served as the control solution. PI3K beta and gamma isoform-specific blockers TGX221 and AS252525  were supplied as 3.3 mM solution in pure ethanol.All data are expressed as mean±SEM. Statistical and clustering analyses were performed using build-in functions in SigmaPlot 10  or XLSTAT 12 .To address the question of whether there are dedicated PI3K-dependent inhibitory odorants, we began by assembling a panel of 42 conventional odorants, all of which are known to elicit olfactory responses in rodents. These odorants represented a relatively broad range of chemical features, such as carbon chain length and functional group, with sufficient diversity to approximate the odor space of rats. Odorants were analyzed using an optimized set of 38 chemical descriptors generated by CHEMMINE Tools for that cell.We screened the odorants at a single concentration (50 µM) on a large group of rat ORNs sensitive to IBMX/forskolin stimulation (IF+ ORNs) for the ability to evoke PI3K-dependent inhibition . The calst response) vs 0.36±0.07  . In a number of cases (3.6%), PI3K blockade decreased the amplitude of the response  to each odorant before and after PI3K blockade against the chemical class of the odorant. The odorants are sparsely distributed along the incidence scale  and therWhile there was no particular class that was responsible for evoking PI3K-dependent inhibition, odorants within chemical classes with few chemical features , as well as each individual odorant (at 50 µM), was applied to a different population of 3,680 IF+ ORNs, using the protocol from the paired-pulse screen. The ORNs showed strong conventional excitatory and PI3K-dependent inhibitory responses to mix8 . 162 4.. In compWe next examined the relationship between the conventional excitatory and PI3K-dependent inhibitory responses to the tomato volatiles. As with the 9 random odorants, ORN tuning to PI3K-dependent inhibitory odorants was narrower than to conventional excitatory odorants, and each odorant acted on a different type of ORN. Cluster analysis of the tomato odorant-evoked responses of just the 36 cells that showed PI3K-dependent inhibition reveals 15 conventional excitatory response profiles in comparison to just 10 PI3K-dependent inhibitory profiles. When odorants were correlated with the neuronal activity to build similarity matrices, comparison did not reveal a significant cross-correlation between activation patterns before and after PI3K blockade  0.381, p-value (two-tailed) 0.093). Additionally, PCA analysis of the data set shows that the PI3K-dependent inhibitory tomato odorants are sparsely distributed in the neural space, suggesting that each acts on a distinct ORN type . OverallPreviously, we demonstrated that activation of rat ORNs could be inhibited in a PI3K-dependent manner by studying a few inhibitory odorant pairs in detail 3, the product of PI3K activation, negatively regulates the olfactory cyclic nucleotide-gated (CNG) channel It is important to have implicated PI3K signaling in a physiological context relative to olfactory transduction. PI3K signaling regulates multiple cellular processes such as proliferation, apoptosis, and intracellular trafficking Interestingly, the incidence or bias of the PI3K-dependent inhibitory odorants did not correlate strongly with that of odorants shown in the present study and elsewhere to be conventional excitatory odorants. For example, the incidence of the responses to aliphatic aldehydes, alcohols, and acids, among the most potent excitatory odorants for rodents Depending on the extent of this partitioning it could have practical consequences for defining the molecular receptive range (MRR) of an ORN. The MRR is intended to represent the full set of odorants that interacts with a given receptor cell or its OR, and typically is determined by identifying odorants that evoke conventional excitatory (cyclic nucleotide-dependent) responses Each rodent ORN expresses only one of approximately 1,000 functional ORs Our data do not directly address whether excitatory and inhibitory odorants target the same OR. However, finding that the PI3K-dependent inhibitory responses and the conventional excitatory responses of individual ORNs are tuned to different numbers of odorants and not the same odorants bears on the question. Given that each rodent ORN expresses only one of approximately 1,000 functional ORs Overall, finding that a large repertoire of different molecular classes of odorants, including those found in a behaviorally-salient complex odor mixture, can activate PI3K, and do so in a physiologically relevant context to olfactory transduction, argues that PI3K-dependent inhibition can provide a broad basis for opponent coding in mammalian olfaction. The extent to which inhibitory input contributes to the combinatorial code that is generally assumed to be the basis for odor coding and discrimination in many different animals


Covalent modification of nuclear receptors by the Small Ubiquitin-like Modifier (SUMO) is dynamically regulated by competing conjugation/deconjugation steps that modulate their overall transcriptional activity. SUMO conjugation of progesterone receptors (PRs) at the N-terminal lysine (K) 388 residue of PR-B is hormone-dependent and suppresses PR-dependent transcription. Mutation of the SUMOylation motif promotes transcriptional synergy.2). This occurs in part by raising PR sensitivity to ligands. The C-terminal ligand binding domain of PR is required for the transcriptional stimulatory effects of N-terminal deSUMOylation, but neither a functional PR dimerization interface, nor a DNA binding domain exhibiting PR specificity, are required.The present studies address mechanisms underlying this transcriptional synergy by using SUMOylation deficient PR mutants and PR specifically deSUMOylated by Sentrin-specific proteases (SENPs). We show that deSUMOylation of a small pool of receptors by catalytically competent SENPs globally modulates the cooperativity-driven transcriptional synergy between PR observed on exogenous promoters containing at least two progesterone-response elements (PREWe conclude that direct and reversible SUMOylation of a minor PR protein subpopulation tightly controls the overall transcriptional activity of the receptors at complex synthetic promoters. Transcriptional synergism controlled by SENP-dependent PR deSUMOylation is dissociable from MAPK-catalyzed receptor phosphorylation, from SRC-1 coactivation and from recruitment of histone deacetylases to promoters. This will provide more information for targeting PR as a part of hormonal therapy of breast cancer. Taken together, these data demonstrate that the SUMOylation/deSUMOylation pathway is an interesting target for therapeutic treatment of breast cancer. Its effects are mediated by progesterone receptors (PRs), members of the steroid hormone receptor superfamily of ligand-dependent transcription factors. PRs exist as two major, functionally different  isoformsactivity . Becauseactivity ,18-20, sin vitro translated AR and GR, but not ERα or ERβ, are SUMOylated [Additional complexity arises from the structure of DNA to which PRs bind. Cooperativity among receptors bound at compound promoters consisting of two or more PREs results in synergism defined as a "more-than-additive" transcriptional effect . IniguezMOylated .SUMO conjugation of PR-B at K388  is hormone dependent and occurs via PIAS1  or PIAS3In the present study we analyze the functional effects of SENP-induced PR deSUMOylation in detail. Our results indicate that on a compound promoter, SENP1 enhances transcription in a dose-dependent manner, but this requires full-length PR. However enhanced transcription is independent of PR DNA binding specificity or the PR S294 phosphorylation site. By deSUMOylating PR, SENP increases PR sensitivity to hormone. The histone deacetylase inhibitor Trichostatin A (TSA) has a marked biphasic effect. At high concentrations, which promote global histone hyperacetylation and modify many proteins , TSA strFigure 2) or more palindromic PREs but not a single PRE. To assess the generality of this, we used the MMTV-LTR, which contains 1 palindromic PRE and 3 PRE half-sites. In contrast to GRs that prefer the palindrome, the half-sites are preferentially used by PRs [2-Luc  led to a decrement in transcription likely due to transcription factor "squelching" [2-LucWe previously showed  that SUMd by PRs , possibld by PRs . To examd by PRs , togethec Figure  or MMTV-c Figure  reporterelching" . Wild-tyin vivo [in vivo PR deSUMOylation, wild-type PR-B (1 μg DNA) and GFP-SUMO1 (0.1 μg DNA) were cotransfected into HeLa cells together with SENP1 or SENP2 expression vectors (0.1 μg DNA), and unliganded or liganded PR-B SUMOylation states were assessed by immunoblotting  encoding SENP1, SENP1m and SENP2 on PREs Figure  or stabl000 ng ens Figure . It is os Figure . Indeed,2-Luc  where SENP1 had no effect despite strong transcription with wild-type PR-B, confirming that the PREs of MMTV-LTR are not PR SUMOylation sensitive  NT-B, a constitutively active PR N-terminal construct [2-Luc. NT-B is strongly active in the absence of ligand  linked to luciferase. The PR-B specificity mutant was treated with R5020 (since it contains a PR LBD); ER was treated with 17β-estradiol. The receptor-encoding constructs (100 ng DNA) were transfected into HeLa cells without or with hormones together with increasing (20-1000 ng) SENP1 concentrations. The PR-B specificity mutant exhibited weak ligand-dependent transcriptional activity, which was dramatically enhanced by SENP1-mediated deSUMOylation in a dose-dependent manner. This suggests that unlike the PR LBD, neither the PR DBD nor its DNA binding site influence SUMOylation of the PR N-terminus. The DBD dimer interface of steroid receptors stabilizes binding to palindromic HREs. Interestingly, disruption of the dimer interface markedly increases transcriptional activity of receptors bound to multiple PREs  a full-length PR-B Spec "specificity" mutant in which the PR DBD was replaced by the DBD of ER Figure , and 2) -15 to 10-8 M. Transcription levels on PRE2-Luc were plotted as a percent of maximal induction by 10-8 M R5020 above "no hormone" controls. Curve fitting was performed by Prism Graph as described under "Experimental Procedures"  of the SUMOylation deficient K388R mutant whose intrinsic R5020-binding affinity exceeded that of wild-type PR ~2-fold. This indicates that deSUMOylated PR are exquisitely sensitive to very low hormone concentrations; also explaining enhancement of the agonist properties of RU486  or high (500 nM) TSA concentrations. At low TSA concentrations  TSA concentrations together with the deSUMOylase SENP1. HeLa cells were transfected with wild-type PR-B Figure , left orVarious regulators of SUMO-dependent transcriptional repression have been proposed, which include chromatin-associated proteins , histoneLike GR, AR are SUMOylated at two N-terminal Lys residues and mutation of one (K385) enhances cooperativity on palindromic but not direct-repeat HREs. Callevaert et al.  posit thThe SENPs deconjugate SUMO-modified proteins and are critical for maintaining physiological ratios of SUMOylated to deSUMOylated substrates. Studies in knockout mice demonstrate that a fine balance of SUMOylation/deSUMOylation is required for normal embryonic development . This baSENP1 stimulates the transcriptional activity of ARs and two different mechanisms have been proposed. Cheng et al.  suggest Both PR SUMOylation and PR phosphorylation are enhanced with similar kinetics by progestin binding to the receptors . HoweverIn contrast to the stimulatory effects of SENP1 on PR activity Figure , the effAcetylation of steroid receptors results in either transcriptional activation or repression, depending on alterations in DNA binding affinities, coregulator recruitment, or hormone responsiveness ,70-72. AIt has been suggested that SUMOylation represses transcription by recruiting repressors, including HDAC to SUMOylated substrates . HoweverPRs are major markers in breast cancer. Their presence indicates that a tumor is hormone-dependent and a candidate for endocrine therapies. The role of progesterone in activating these transcription factors is complex, however. After binding PR, progestin agonists and antagonists can have either transcriptional activating or suppressive effects modulated in part by enhancing or suppressing PR SUMOylation ,31,33,352-Luc, PRE2-Luc and MMTV-Luc reporter plasmids were described previously [The expression plasmids pSG5 hPR, encoding human PR-B and HEGO, encoding human ER, cloned into pSG5 were a gift of P. Chambon . Cloning of pSG5 hPR1 K388R, pSG5 hPR1 S294/344/345A, pSG5 NT-B, pSG5 hPR1 R606A (PR-B DNA dimerization mutant was a gift of B. Jacobsen), pCMV5-MEKK1 and pSG5 DBD-LBD were described previously ,17,33. W5 cells per 60 mm dish, 1 day prior to transfection. Cells were transfected by calcium phosphate co-precipitation [Renilla luciferase was added as an internal control at 20 ng/dish. Twenty four hours later, cells expressing LBD-containing constructs were washed and incubated 24 hrs with the synthetic progestin R5020  at final concentrations indicated in the figures. Control cells received ethanol only. Cells were collected in 150 μl lysis buffer (Promega), and 50 μl were analyzed on a dual luminometer [HeLa cells were plated in minimum Eagle's medium containing 5% FBS  at a density of 1.2 × 10pitation  with coninometer . ResultsN-ethylmaleimide, and cell extracts were prepared in 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 15 mM dithiothreitol, a protease inhibitor mixture , and 20 mM N-ethylmaleimide. The expressed proteins were resolved on SDS-PAGE, and conjugated protein was detected by immunoblotting with PgR1294.Whole cell extracts were prepared from HeLa cells transiently transfected with PR expression vectors as described . Cells wt test, and the differences were considered statistically significant at a P value of 0.05.Prism GraphPad software version 4 . was used to determine least-squares best fit of the experimental data to the theoretical dose-response curve. All values represent at least three independent experiments and are expressed as the means ± SD. Data sets were analyzed with GraphPad Prism 4 Statistical significance was determined by two-tailed unpaired student's N-ethylmaleimide; NR: Nuclear receptors; NT-B: N-terminal region of PR-B; PIAS: Protein inhibitor of activated transducer and activator of transcription; PR: Progesterone receptors; PRE: Progesterone response element; SENP: SUMO specific protease; SRC: Steroid receptor coactivator; SUMO: Small ubiquitin like modifier; TSA: Trichostatin AAF: Activation function; AR: Androgen receptors; BUS: PR-B-upstream segment; DBD: DNA binding domain; ER: Estrogen receptors; GFP: Green fluorescent protein; GR: Glucocorticoid receptors; HDAC: Histone deacetylase; LBD: Ligand binding domain; MMTV: Mouse mammary tumor virus; MR: Mineralocorticoid receptors; NEM: The authors declare that they have no competing interests.HA-H was involved in all of the experimental and theoretical work. HA-H and KBH participated in the design of the experiments. HA-H and KBH wrote the manuscript. All authors have read and approved the final manuscript.Figure S1. A) DeSUMOylation of PR by SENP1 depends on its catalytic activity. HeLa cells were transiently transfected with expression vectors encoding wild type PR-B together with a GFP-SUMO-1 expression vector (+), and wild type or mutant (m) SENP1. Cells were treated 24 hrs without (-) or with (+) 10 nM R5020. Western blot analysis was performed on cell extracts probed with the anti-PR1294 monoclonal antibody or anti -actin control. B) SENP1 enhances PR-B activity in T47D breast cancer cells. PR-negative T47D-Y breast cancer cells stably expressing PR-B were transfected with the PRE2-Luc reporter plasmid in the presence of pSV40-Renilla as internal control along with increasing amount (20-1000 ng) of SENP1 expression vector, or an empty vector control (-). Cells were treated without (-) or with (+) 10 nM R5020 for 24 hrs before being assayed for luciferase activity. C) SENP1 enhances transcription by the partial agonist RU486. HeLa cells were transfected with 2 g of PRE2-luciferase reporters together with 50 ng of a PR-B expression vector and Renilla-Luc as an internal control in the presence or absence of 100 ng SENP1 or SENP1m expression vectors. The cells were treated for 24 hrs with the agonist R5020 (10 nM), partial agonist RU486 (100 nM), or the pure antagonist ZK98299 (100 nM) then harvested and lysed. The extracts were assayed for luciferase activities as in Figure Figure S2. The PR DBD dimerization interface is necessary for effective synergy control. HeLa cells were transfected with 2 g of PRE2-luciferase reporters together with 50 ng of a wild type PR -B, the PR-B K388R SUMOylation deficient, or a PR-B DBD dimerization mutant (PR-B DX) expression vector and Renilla-Luc as an internal control in the presence or absence of 100 ng SENP1 expression vectors. The cells were treated for 24 hrs with the agonist R5020 (10 nM), then harvested and lysed. The extracts were assayed for luciferase activities as in Figure Figure S3. A) The stimulatory effect of MEKK1 on PR-B transcriptional activity is LBD and hormone independent. HeLa cells were transfected with 2 g of PRE2-luciferase reporters together with 500 ng of NTB-DBD, a constitutively active PR N-terminal expression vector in the presence of pSV40-Renilla as internal control along with increasing amount (5-200 ng) of constitutively active MEKK1 expression vector, or an empty vector control (-). The extracts were assayed for luciferase activities as in Figure B) Concentration dependent effect of MEKK1 on PR SUMOylation. HeLa cells were transiently transfected with expression vectors encoding wild type PR-B together with a GFP-SUMO-1 expression vector (+) in the absence (-) or presence of increasing amount of MEKK1 expression vector. Cells were treated 24 hrs without (-) or with (+) 10 nM R5020. Western blot analysis was performed on cell extracts probed with the anti-PR1294 monoclonal antibody or anti -actin control.Click here for file


Rheumatoid arthritis (RA) is a destructive arthropathy with systemic manifestations, characterized by chronic synovial inflammation. Under the influence of the pro-inflammatory milieu synovial fibroblasts (SFs), the main effector cells in disease pathogenesis become activated and hyperplastic while releasing a number of signals that include pro-inflammatory factors and tissue remodeling enzymes. Activated RA SFs in mouse or human arthritic joints express significant quantities of autotaxin (ATX), a lysophospholipase D responsible for the majority of lysophosphatidic acid (LPA) production in the serum and inflamed sites. Conditional genetic ablation of ATX from SFs resulted in attenuation of disease symptoms in animal models, an effect attributed to diminished LPA signaling in the synovium, shown to activate SF effector functions. Here we show that administration of 1-bromo-3(S)-hydroxy-4-butyl-phosphonate (BrP-LPA), a metabolically stabilized analog of LPA and a dual function inhibitor of ATX and pan-antagonist of LPA receptors, attenuates collagen induced arthritis (CIA) development, thus validating the ATX/LPA axis as a novel therapeutic target in RA. Rheumatoid arthritis (RA) is a destructive arthropathy with a high prevalence, imposing a substantial socioeconomic burden RA pathogenesis is characterized by the symmetric, chronic inflammation of the joints, eventually leading to the destruction of cartilage and bone Autotaxin  is a secreted glycoprotein widely present in biological fluids, including synovial fluid ATX was originally isolated as an autocrine motility factor from the supernatant of melanoma cells Soon after the discovery of the potential roles of ATX and LPA signaling in carcinogenesis, many ATX inhibitors and LPAR antagonists were developed S)-hydroxy-4-butyl-phosphonate (BrP-LPA), is an α-halo-substituted phosphonate, a metabolically stabilized analog of LPA that acts as a dual function pan-antagonist of LPA receptors and inhibitor of the lysophospholipase D activity of ATX ras and ATX by 40% and to decrease their chemotaxis by 23% in vitro and to cause tumor regression in vivoin vitro and to inhibit tumor growth and angiogenesis in an engineered three-dimensional non-small cell lung cancer xenograft model in vitro and to reduce liver tumor weight and volume in vivo1-bromo-3 The ligand properties of BrP-LPA were previously evaluated with Cain vitro and ex vivo utilizing natural LPC substrates. The full pharmacokinetic profile of BrP-LPA in vivo was also determined showing that BrP-LPA rises to its highest level of 8 μΜ (Cmax) one hour (tmax) post administration, accompanied by a parallel decrease in ATX activity levels (48% reduction) and LPA levels (82% reduction). Most importantly, intraperitoneal injections of BrP-LPA were able to prevent the arthritic symptoms in groups of mice undergoing the development of collagen induced arthritis (CIA), establishing the ATX/LPA axis as a novel therapeutic target in RA.In this report, we first characterized the potency of BrP-LPA on the inhibition of ATX All mice were bred at the animal facilities of the Alexander Fleming Biomedical Sciences Research Center, under specific pathogen-free conditions. Mice were housed at 20–22°C, 55±5% humidity, and a 12-h light-dark cycle; water and food were given ad libitum. Mice were bred and maintained in their respective genetic backgrounds for more than ten generations. All experimentation was approved by the Institutional Animal Ethical Committee of Biomedical Sciences Research Centre Alexander Fleming , as well as by the Veterinary Service and Fishery Department of the local governmental prefecture. All efforts were made to minimize suffering during injections, and all surgery was performed in humanly sacrificed mice.CIA in DBA/1 mice was induced by immunization with collagen type II emulsified in complete Freund's adjuvant essentially as previously described q) background. 0: no detectable pathology; 1: hyperplasia of the synovial membrane and presence of polymorphonuclear infiltrates – mild tendonitis may be present; 2: pannus and fibrous tissue formation and focal subchondrial bone erosion: 3: cartilage destruction and bone erosion in multiple foci; 4: extensive cartilage destruction and bone erosion- bone outline structure lost. Presented results refer to an average score of all four joints.Paraffin-embedded joint tissue samples were sectioned and stained with hematoxylin and eosin as previously described The various LPA and LPC species  and their internal standards  were procured from Avanti Polar Lipids Inc. . Recombinant ATX(β) enzyme was obtained from R&D , SinoBiological Inc  and Echelon . HPLC solvents , fatty acid–free BSA, chicken collagen type II, choline chloride, 4-aminoantipyrene (4-AAP) and horseradish peroxidase were obtained from Sigma . Choline oxidase was from MP Biomedical , TOOS reagent was from TCI Europe , Amplex Red was from Invitrogen  and FS-3 was from Echelon.3/CH3OH  followed by 2 ml of PBS-saturated ice-cold CHCl3/CH3OH . Each extraction step was followed by a 60 s vortex and a 1 min centrifugation step in a bench-top clinical centrifuge in a cold room at 4°C at 3,000 rpm. The lower chloroform organic phases from both neutral extraction steps were pooled and kept for LPC measurement. The remained aqueous phase was chilled on ice for 10 min and extracted twice with ice-cold acidified with acetic acid CHCl3/CH3OH  as above. The lower organic phases were pooled, neutralized to pH 6–7 (checked with pH paper as above) on ice and kept for LPA, BrP-LPA measurements. The kept neutral lower organic phase of neutral extraction and the neutralized acidified lower organic phase were evaporated to dryness. The resulting residues were resuspended in 0.15 ml of isopropanol and after brief vortex mixing the solution was transferred to the auto sampler vial. Recovery of LPA species was between 60% and 95%. Recovery of BrP-LPA was 70% and the recovery of LPC species was between 80% and 100%. The Relative Standard Deviation (RSD) was less than 7% from the plasma fortified with three different concentrations  of LPAs,  of BrP-LPA and  of LPCs. Analysis was performed using a Phenomenex  Luna Silica column  using a binary gradient program consisting of isopropanol:hexane:100 mM NH4CO2H(aq) 58:40:2 (mobile phase A) and isopropanol:hexane:100 mM NH4CO2H(aq) 50:40:10 (mobile phase B). The mobile phase was delivered at a flow rate of 0.3 ml/min and the total run time was 50 min/sample. The analysis was performed with electrospray ionization in negative SIM (Single Ion Monitoring) mode for LPA species and BrP-LPA and in positive mode for LPC species (data dependent MS/MS). The resolution was 100 K providing high accuracy for lysophospholipid measurements. Data were collected and processed with the Xcalibur software (Thermo Scientific). The precursor ion mass was used for the quantification while lipid identity was confirmed by precursor ion fragmentation using collision induced dissociation (CID). The peak area ratios  versus the molar ratios  were plotted and fitted to a linear regression. The linear dynamic range of LPA species and BrP-LPA was between 0.01 μM and 10 μΜ.BrP-LPA, LPA species  and LPC species  were measured in plasma with HPLC-ESI/MS/MS using RSLCnano system (Ultimate 3000) coupled in-line with an LTQ Orbitrap XL mass spectrometer . BrP-LPA and lipid  extraction from plasma was performed on ice according to the Folch method with minor modifications 2O2). The latter in the presence of horseradish peroxidase reacts with TOOS and 4-aminoantipyrene (4-AAP) to form a pink quinoneimine dye measured at 555 nm ATX/LysoPLD activity was assessed based on the amount of choline released from LPC with the use of TOOS (N-ethyl-N-(2-hydoroxy-3-sulfopropyl)-3-methylaniline) reagent. In this assay ATX-mediated LPC-liberated choline is oxidised by choline oxidase to betaine and hydrogen peroxide  or recombinant ATX protein (2 nM) were incubated with LPC in the presence of 100 mmol/l Tris–HCl/pH 9.0, 500 mmol/l NaCl, 5 mmol/l MgCl−1) and choline oxidase (2 UnitmL−1). Absorbance was measured at 555 nm and data were analyzed using the statistical software package Prism 4.0 . For each sample and control the absorbance was plotted against time and the slope (da/min) for the linear (steady-state) portion of each reaction profile was calculated. Absorbance units were converted to nmol of choline/min/ml by comparison with a choline standard curve or by use of the extinction coefficient for the quinoneimine dye according to the following equation:2); and 1/2: Factor based on the fact that 1 mol of hydrogen peroxide produces 1/2 mol of quinoneimine dye.The liberated choline was detected by adding 100 μl of a color mix containing 4-AAP and TOOS (0.3 mM), horseradish peroxidase  set to make fluorescence measurements every 1 min for a total period of 30 min. Autotaxin activity was quantified by measuring the rate of increase in fluorescence at 528 nm with excitation at 485 nm. At the end of the measurement, each data point was plotted on a graph of fluorescence versus time. A linear regression was fitted in the linear portion of the graph (3–20 min).Inhibition of ATX activity by BrP-LPA was determined using the cleavage of the ATX-specific fluorogenic substrate FS-3 . Assays were conducted in a final volume of 100 μl in the presence of 140 mM NaCl, 5 mM KCl, 1 mM MgClBrP-LPA inhibition was also tested with the more sensitive reagent Amplex Red based on the Amplex™ Red PLD assay kit  originally designed for the measurement of the PLD activity m) was obtained by fitting the substrate dependence of V (ATX activity) to a rectangular hyperbola using the non-linear regression analysis of the statistical software package Prism 4.0 . Km and Vmax were evaluated from the rectangular hyperbola curve with the use of GraphPad software.The biochemistry of lyso-PLD was assessed by following the kinetics data using a wide panel of different ATX substrates , three different recombinant ATX proteins  and three different ATX activity assays . The ATX activity was measured at different substrate concentrations  in order to examine whether ATX follows Michaelis-Menten kinetics. The Michaelis constant  and appropriate dilutions were made in water. The inhibition of ATX by BrP-LPA was measured employing 2 nM recombinant ATX, 50 μΜ LPC  and 0.01 to 100 μmol/L of BrP-LPA . The in vitro inhibition of ATX by BrP-LPA was evaluated with three different assays . To ensure that BrP-LPA did not interfere with the colouring agent or hydrogen peroxide that is generated during the colour forming reaction in Amplex Red and TOOS activity assay, samples with BrP-LPA were incubated with only choline and the colouring reagents were added subsequently. BrP-LPA was also tested for its ability to inhibit mouse plasma ATX activity. Diluted mouse plasma was used as the source of ATX and the analysis of inhibition was made with TOOS as the in vitro inhibition assay.For i) and IC50 values were determined using the mean values obtained from two independent experiments. The relative remaining ATX activity or LPA levels in the presence of the BrP-LPA was expressed as a percentage of the corresponding control value . The IC50 value was determined graphically by plotting the log concentration of BrP-LPA versus relative remaining ATX activity or LPA levels of each test sample using the sigmoid dose–response fitting method (Prism® software). The IC50 (concentration of BrP-LPA sufficient to inhibit ATX activity or diminish LPA levels by 50%) was calculated by the equation f =  y0+a/(1+exp(-(x-x0)/b)) given by SigmaPlot 11.0 .The inhibition model  was evaluated by graphical analysis with Lineweaver–Burk double reciprocal plots (1/[velocity] versus 1/[substrate]) and Cornish-Bowden plot ([substrate]/[velocity] versus [Inhibitor]). These plots were generated by the measurement of ATX activity in the presence of different BrP-LPA concentrations (0–10 μΜ) and increased amounts of 16:0 LPC . The inhibition constants  was determined at two or more substrate concentrations and over a range of inhibitor concentrations (I). In a plot of 1/v against I, data for each substrate concentration fall on straight lines that intersect at I = −Ki and 1/v = 1/Vmax (competitive inhibition) and at I = −Ki and 1/v = [1−(Ki/Ki')]/Vmax (mixed type inhibition).Kg for 10 min to pellet the cells, which has been reported to remove most platelets 50 values using ATX activity and LPA levels were determined graphically as described above.Venous blood from wild type mouse was collected in EDTA tubes. Blood was incubated with BrP-LPA at different final concentrations (0–10 μM) in 96-well collection plates for 2 h at 37°C. The reaction was stopped by centrifugation at 1000 max), their time of occurrence (Tmax) and half-life (t1/2) were both obtained directly from the acquired data.BrP-LPA was administered intraperitoneally (IP) at 10 mg/kg in water to 6–8 week old female mice. Control mice (n = 3) received only the vehicle (water). Blood samples were collected from each animal  via a cardiac puncture at time points up to 16 hours post administration. Plasma was prepared by centrifugation of whole blood at 1000 g for 15 min at 4°C, and was stored frozen (−80°C) in siliconized tubes. Plasma samples were then analysed for plasma ATX activity and BrP-LPA/LPA/LPC levels with the TOOS activity assay and HPLC-MS/MS respectively. Maximum plasma concentrations , p<0.01 (**) and p<0.001 (***) were considered significant. For CIA clinical scores the Mann-Whitney rank sum test was used to determine the level of significance between means of groups.Statistical significance of all values, always in pair-wise comparisons with control values, was assessed with a paired Student's In addition to its antagonistic effect on LPA receptors, BrP-LPA was shown to inhibit the lysophospholipase D activity of ATX (94% inhibition at 10 μM) o) of LPC 16:0, 18:0 and 18:1 by ATX was found to depend hyperbolically on the LPC concentration and to follow Michaelis-Menten kinetics , followed by 16:0 LPC (Km = 60 μM) and 18:0 LPC (Km = 82 μM)  hydrolysis was found linear within the first 20 minutes (data not shown), so all subsequent analyses were performed at this time point. The hydrolysis rate (Vkinetics . The Km = 82 μM) . Similar= 82 μM) . Moreove= 82 μM) . In compbstrates , confirmm values. A clear dose response effect on ATX inhibition was observed for BrP-LPA for all LPC species  tested . Moreover, the mode of inhibition was determined from the inhibitory effect of BrP-LPA on Km and Vmax values with 16:0 LPC as a substrate. BrP-LPA was found to inhibit ATX lysoPLD activity in a competitive manner from Cornish-Bowden  tested . Similar) tested . IC50 anh-Bowden  and Lineh-Bowden  plots, atrations .ex vivo. Increasing amounts of BrP-LPA were added to mouse plasma ex vivo and ATX activity was measured with the TOOS assay utilizing different exogenous natural LPC substrates at final concentrations of 50 μM. In agreement with the in vitro ATX activity inhibition studies, BrP-LPA also inhibited plasma ATX/lysoPLD activity  followed by 20:4 LPA (IC50 = 559 nM), 16:0 LPA (IC50 = 1340 nM) and 18:1 LPA (IC50 = 1520 nM).In the same experiments no significant changes were observed in the endogenous LPC levels upon the addition of BrP-LPA . This isA levels . The effA levels , while Bin vivo, we next administered BrP-LPA (10 mg/kg) or vehicle alone as a single intra-peritoneal bolus injection to groups of mice. The levels of BrP-LPA, ATX activity, LPA and LPC were then determined in plasma 0.5, 1, 2, 4, 8 and 16 h post injection. The pharmacokinetic (PK) analysis of BrP-LPA shows that it rose to its highest level of 8 μΜ (Cmax) at 1 h (tmax) post-administration and declined rapidly   . The phaat 16 h)  as well at 16 h)  levels 1at 16 h)  while LPat 16 h) . BrP-LPAat 16 h) , possibleriments .ATX levels have been found to be elevated in the synovial fluids of RA patients, as well as in joint tissues and sera of both RA patients and arthritic models ATX induction in the synovium and other inflammatory sites is likely to stimulate local LPA production and its autocrine and paracrine effects. This hypothesis was tested in a rat air pouch model, where potent ATX inhibition provided >95% reduction in air pouch LPA, indicating ATX as the major source of LPA at inflammatory sites To examine the therapeutic potential of targeting the ATX/LPA axis in RA, BrP-LPA was injected intraperitoneally twice a week , indicating the absence of systemic side effects. Moreover, BrP-LPA administration did not interfere with the levels of anti-collagen antibodies . It has in vivo in an orthotopic breast cancer xenograft model Similar therapeutic results were also obtained when the BrP-LPA injection dosing regimen was increased to three times per week as well as to daily dosing (data not shown); however, the lower, biweekly dosage scheme was generally employed as it has been previously reported to be effective It was recently reported that pharmacological antagonism of LPA receptors 1 and 3 with ki16425 The exact mode of action of BrP-LPA, moderate ATX inhibition and/or LPA receptor antagonism or both (or other undetermined off site effects), in the context of disease pathogenesis remains to be elucidated. Nonetheless, the future commercialization of novel, potent specific inhibitors for ATX and antagonists for LPA receptors Figure S1Analysis of ATX kinetics and in vitro characterization of BrP-LPA inhibition with different recombinant ATX proteins and different ATX activity assays.A–B. The hydrolysis of LPC  by R&D recombinant ATX protein measured with TOOS reagent and C–D. The hydrolysis of LPC  measured with Amplex Red reagent and Echelon recombinant ATX follows Michaelis Menten kinetics. E. [FS-3] dependence of the steady state FS-3 hydrolysis rate by Echelon recombinant ATX, measured with FS-3 activity assay. The solid line represents the best fit to a rectangular hyperbola. F–H. Dose-response curves in the presence of various BrP-LPA concentrations (0.01–100 μM), show the percent residual ATX activity (% ATX activity) of Echelon recombinant protein, measured with  Amplex Red assay by choline release from different LPC species (50 μΜ); (F) 14:0 LPC; (G) 18:0 LPC and with H. FS-3 activity assay by the use of fluorescent lipid substrate FS-3. The presented values are the means (±std) of two independent experiments. The log concentration of BrP-LPA is used for the sigmoidal dose response curves.(TIF)Click here for additional data file.


Hepatic gluconeogenesis tightly controls blood glucose levels in healthy individuals, yet disorders of fatty acids (FAs) oxidation are characterized by hypoglycemia. We studied the ability of free-FAs to directly inhibit gluconeogenesis, as a novel mechanism that elucidates the hypoglycemic effect of FAs oxidation defects.Primary rat hepatocytes were pre-treated with FAs prior to gluconeogenic stimuli with glucagon or dexamethasone and cAMP.Pre-treatment with 1 mM FAs  for 1 hour prior to gluconeogenic induction, significantly decreases the induced expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6pase) as well as the induced glucose production by the cells. The inhibitory effect of FAs upon gluconeogenesis is abolished when pre-treatment is elongated to 18 hours, allowing clearance of FAs into triglycerides by the cells. Replacement of palmitate with the non-metabolic fatty acid 2-bromopalmitate inhibits esterification of FAs into triglycerides. Accordingly, the increased exposure to unesterified-FAs allows their inhibitory effect to be extended even when pre-treatment is elongated to 18 hours. Similar changes were caused by FAs to the induction of peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) expression, indicating this transcriptional coactivator as the mediating link of the effect. This inhibitory effect of FAs upon gluconeogenic induction is shown to involve reduced activation of cAMP response element-binding (CREB) transcription factor.The present results demonstrate that free-FAs directly inhibit the induced gluconeogenic response in hepatocytes. Hence, high levels of free-FAs may attenuate hepatic gluconeogenesis, and liver glucose output. The liver is a major site of systemic metabolic regulation. Pathways of synthesis and degradation of lipids and carbohydrates are controlled by complex interactions in the hepatocytes . InductiThe main cellular site for oxidation of FAs is the mitochondria , where tThe pathogenic mechanisms leading to hypoglycemia in FAs oxidation defects have only partially been elucidated . The obsThe current study shades new light on the mechanistic basis of limited gluconeogenic capacity among patients with defects in FAs oxidation. It is demonstrated, for the first time, that FAs directly attenuate the induction of gluconeogenic programme in isolated primary hepatocytes and that removal of FAs restores the gluconeogenic response. Our findings indicate that free-FAs may be involved in inhibition of GNG induction, mediating hypoglycemia in FAs oxidation disorders.Rapid uptake of FAs by cultured primary hepatocytes is indicated by TLC separation of cellular lipid extracts. Eminent increase in cellular FAs amounts is noticed after 1 hour exposure to FAs Figure . Longer As seen also by FACS analysis after Nile-Red staining Figure , TGs accReplacement of palmitate acid with the non-metabolic FA 2-bromopalmitate (FAs-Br) markedly prevented the TGs accumulation observed after 18 hours  mRNA expression level Figure , left. PSimilar effects of free-FAs on PEPCK expression levels are seen when gluconeogenesis is induced by dexamethasone and cAMP Figure , left.In addition, pre-treatment with FAs for 1 hour prior to glucagon and cAMP abolishes the induction of glucose-6-phosphatase (G6pase) expression in primary hepatocytes Figure , right. As seen in Figure Since both PEPCK and G6pase are rate-limiting enzymes in the gluconeogenic pathway, the inhibition of their induction represent the ability of free-FAs to inhibit induction of GNG in hepatocytes. The inhibition of GNG by free-FAs was observed as pre-treatment with free-FAs for 1 hour, but not for 18 hours, significantly inhibited glucose production by hepatocytes, when induced by glucagon and cAMP Figure  or by deThe similar effect of FAs upon PEPCK and G6pase expression imply an upstream target for the inhibitory effect, and therefore the effect of free-FAs on PGC1α induction was examined.As shown in Figure As shown by the evaluation of p-CREB/CREB protein ratio in cellular extracts, CREB activation is induced in response to gluconeogenic stimuli of the cells with glucagon and cAMP Figure  as well Hence, inhibition of CREB activation may precede the observed effects of free-FAs on gluconeogenic response in primary hepatocytes.in vitro model of primary hepatocytes, this study provided the first evidence that free-FAs can directly attenuate the activation of gluconeogenic pathway by decreasing CREB activation. The results demonstrate that induction of glucose production and gluconeogenic enzymes is strongly inhibited by free-FAs in hepatocytes, and that the mediating link of this inhibitory effect might be inhibition of PGC1α. The inhibitory effect of free-FAs is observed when GNG is induced by different stimuli in hepatocytes, and relieved when they are esterified into TGs. Due to rapid esterification of FAs by the cells, the time window for detection of this effect is found to be short and critical. However, the duration of the effect of free-FAs is extended when their esterification is prevented by using non-metabolic FAs. Together, this implies a mechanism by which GNG is suppressed physiologically when FAs-metabolism takes over during long term fasting in vivo. Furthermore, exceptionally high levels of free-FAs found in the milieu of the β-oxidation deficient liver can directly attenuate GNG and therefore facilitate hypoglycemia among patients.Using an Although a role for FAs in modulation of GNG was previously suggested, it remained controversial whether FAs affect hepatic glucose production and how . Some fiin vitro with freshly added 1 mM PMSF and protease inhibitor cocktail . Lysates were transferred through 23 G needles, incubated on ice for 30 minutes, vigorously vortexed and centrifuged for 15 min at 12000 × g (4°C). Supernatants were collected and used for Western blot analysis.Determination of total protein concentrations was carried out by the DC Protein Assay Reagents . Protein expression was analyzed by standard Western blot techniques. In brief, protein samples were denatured in electrophoresis buffer , at 95°C for 5 minutes and equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto nitrocellulose membranes, and equal loading was confirmed by ponceau red staining. The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk, or 5% BSA  for 1 hour at room temperature. Immunodetection was done by using anti-CREB antibody  or anti-p-CREB antibody , diluted 1 : 1000 in TBS-T with 5% milk or 5% BSA, accordingly, at 4°C overnight. After a subsequent washing step, peroxidase-conjugated anti-rabbit immunoglobulin  was used as a secondary antibody (diluted 1 : 3500 in TBS-T with 2.5% milk at room temperature for 1 hour). Visualization of immunoreactive bands was performed by using ECL detection reagents, and the signal was detected by short exposure to x-ray film. Bands were quantified by scanning densitometry and expressed as arbitrary units. Expression levels of p-CREB in each sample were normalized to their respective CREB levels.The results are the means ± S.D of experiments performed in triplicate. Each experiment was repeated in cells of at least two independent cell isolation procedures. Analysis was performed by ANOVA using JMP Software . Comparisons between groups were performed using Tukey’s honestly significant differences (HSD) test. Differences with a value of P < 0.05 were considered to be statistically significant.FAs: Fatty acids; GNG: Gluconeogenesis; BSA: Bovine serum albumin; TLC: Thin-layer chromatography; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; TGs: Triglycerides; PEPCK: Phosphoenolpyruvate carboxykinase; G6pase: Glucose-6-phosphatase; CPT1: Carnitine palmitoyltransferase 1; GPAT: Glycerol-3-phosphate acyltransferase; PGC1α: Peroxisome-proliferator-activated receptor-γ coactivator 1α; CREB: cAMP response element-binding.The authors declare that there are no competing interests.OT and ZM conceived the experimental design and performed the proofreading of manuscript. NBH performed the experiments and the statistical analysis and wrote the manuscript. SA conducted the FACS analysis. All authors discussed analyses and interpretation, read and approved the final manuscript.


On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs.Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm Infrared radiation is invisible electromagnetic radiation, the wavelength of which is longer than that of visible light. According to differences in wavelength, the International Commission on Illumination (CIE) recommends dividing infrared radiation into the following three bands: near-infrared radiation (IR-A: 0.7∼1.4 µm), middle-infrared radiation (IR-B: 1.4∼3 µm), and far-infrared (FIR) radiation (IR-C: 3∼1000 µm). FIR therapy has the potential to improve endothelial function and reduce the frequency of some vascular-related diseases The overwhelming disadvantage of an AVF in HD patients is its propensity for venous stenosis. A histological evaluation revealed that endothelial and fibromuscular hyperplasia is the leading cause of venous stenosis Many studies revealed that the biological activities of irradiation with FIR are highly associated with the endothelial nitric oxide (NO) synthetase (eNOS)/NO pathway. In rat models, the beneficial effects of FIR therapy on skin blood flow were suggested to be related to the L-arginine/NO pathway In this study, we evaluated the biological effect of FIR on VEGF-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that a non-thermal effect of FIR induced translocation of the transcription factor, promyelocytic leukemia zinc finger (PLZF) protein, to nuclei, and ultimately inhibited VEGF-induced proliferation in HUVECs via the phosphoinositide 3-kinase/Akt signaling pathway.VEGF and other chemicals of reagent grade were obtained from Sigma . The primary antibodies for PI3K p85, Akt, phospho-Akt (Ser 473), ERK1/2, phospho-ERK1/2 (Thr 202/Tyr 204), eNOS, and phospho-eNOS (Ser 1177) were purchased from Cell Signaling . Primary antibodies for PLZF and GAPDH were respectively purchased from Calbiochem  and AbFrontier .The HUVEC line was purchased from the Bioresource Collection and Research Center , and cultured in 90% medium 199 with 25 U/ml heparin and 30 µg/ml endothelial cell growth supplement (ECGS) adjusted to contain 1.5 g/L sodium bicarbonate with 10% fetal bovine serum. They were grown until the monolayer became confluent. The medium for culturing cells was then changed to serum-free medium, and cells were incubated overnight before the experiment. Cell culture reagents were obtained from Sigma-Aldrich .2 atmosphere. The FIR intensity was adjusted by changing the distance between the FIR emitter and culture chamber. The effective FIR intensity and wavelength received by HUVECs in culture dishes were detected and calculated by China National Infrared & Industrial Electrothermal Products Quality Supervision & Test Center . The effective primary FIR emission that the culture cells received ranged 3∼5 µm. Additionally, as a result of FIR exposure, there was secondary emission from the Petri dish lids with a peak wavelength of approximately 10 µm, calculated according to Wein's law.A ceramic FIR generator, a WS TY301 FIR emitter , was used to provide FIR exposure. This FIR emitter generates electromagnetic waves with wavelengths in the range of 3∼25 µm. HUVECs were cultured in a 6-cm Petri dish with 3 ml medium for FIR exposure. During FIR exposure, an experimental group and a negative control covered with aluminum foil were set up in a culture chamber of a LiveCell™ system  at 37°C with a 5% CO4 cells/well) were cultured in a 96-well microtiter plate in a final volume of 100 µl/well of culture medium. After FIR exposure, cells were incubated at 37°C overnight. Cell proliferation was analyzed using a Quick Cell Proliferation Assay kit according to the instructions provided by the manufacturer , and results are presented as the absorbance of each sample at 440 nm.HUVECs  (at a 1∶5000 dilution) was used as the second antibody to detect primary antibodies by enhanced chemiluminescence . Data of protein bands on Western blots were also quantitated with QuantiScan software .g for 10 min), and then injected into a collection chamber containing 5% VCl3. This strong reducing environment converted both nitrate and nitrite to nitric oxide (NO). A constant stream of helium gas carried the NO into an NO analyzer , where the NO reacted with ozone, resulting in the emission of light. The light emitted was proportional to the quantity of NO formed; standard amounts of nitrate were used for calibration.Nitrate/nitrite in the culture medium was measured as described in a previous report 3 cells per 400 µl culture medium. Before the experimental treatment, slides were preincubated with 10 µM DCF-DA at 37°C for 30 min in the dark, and then subjected to experiments. Fluorescence was observed by fluoromicroscopy with excitation and emission wavelengths of 485 and 530 nm, respectively.Intracellular ROS production was detected with a fluorescent microscope using the fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCF-DA) . Cells were cultured overnight on 4-well chamber slides  at a concentration of 5×10Nitrotyrosine formation was measured using Nitrotyrosine ELISA kits , following the manufacturer's instructions. A standard curve was used to determine the absolute concentration. Values were standardized to micrograms of protein for each sample.HUVECs were lysed at 4°C in lysis buffer . PTEN was collected by using immunoprecipitation kits  with specific antibodies and protein-G-agarose, following the manufacturer's instructions. Precipitates were washed with lysate buffer, and the activity of PTEN was analyzed by using PTEN Malachite Green Assay Kit . Absorbance was detected at 600 nm. Released phosphate was determined relative to a standard curve.Total RNA was extracted from HUVECs using the TRIzol method according to the protocol recommended by the manufacturer , and used to synthesize single-stranded complementary (c)DNA with a High-Capacity cDNA Reverse Transcription Kit . PI3K p85 subunit messenger (m)RNAs were quantified using TaqMan Gene Expression Master Mix (Applied Biosystems) with specific primers in an ABI 7300 Real-Time PCR System (Applied Biosystems). TaqMan Gene Expression Assay kits containing specific primers for PI3K p85-α (cat. no. Hs00933163_m1), PI3K p85-β (cat. no. Hs00178181_m1), and GAPDH (cat. no. Hs99999905_m1) were obtained from Applied Biosystems. Specific primers for GAPDH were used to normalize the amount of sample added. Relative amounts of PI3K p85 mRNA were quantitated using the comparative Ct method. All quantifications were performed on triplicate samples of three separate experiments.g to obtain nuclear pellets. The nuclear pellets were resuspended in cold buffer B , vigorously agitated, and then centrifuged.HUVECs were suspended in cold buffer A  for 15 min, lysed by adding 10% NP-40, and then centrifuged at 5000×PLZF siRNA (cat. no. sc-37149) and control siRNA (cat. no. sc-44234) were purchased from Santa Cruz Biotechnology. Cells were grown to 70% confluence, and PLZF siRNA and control siRNA were transfected using the TurboFect reagent according to the manufacturer's instructions . The final concentration of PLZF siRNA for transfection was 100 nM.t-test was used in all statistical tests. Distributions of continuous variables in groups were expressed as the mean±S.D. A value of p of <0.05 was considered to indicate statistical significance.A Student's 2 of FIR for 30 min and then cultured overnight. A cell proliferation analysis revealed that both VEGF and FIR exposure significantly increased cell proliferation in HUVECs , but was not influenced by FIR exposure . Both apTo understand the molecular mechanisms underlying FIR-induced eNOS activation, we focused on PI3K-dependent activation of Akt, a known eNOS kinase. As shown in The influence of FIR on PI3K signaling pathways was investigated by monitoring phosphatase and tensin homolog (PTEN) protein activity and PI3K expression. PTEN is a phosphatase which inhibits Akt signaling pathways. However, there was no influence of FIR on PTEN activity in HUVECs . A westePI3K-p85 is known to be positively regulated by PLZF 2 for 30 min achieved the maximum inhibitory effect of FIR on VEGF-induced proliferation in HUVECs. A higher FIR intensity or longer exposure time was unable to increase the inhibitory effect of FIR. FIR exposure also reduced VEGF-induced ERK1/2 phosphorylation, and increased the phosphorylation of eNOS. Applications of the NOS inhibitor and ROS scavengers showed that the inhibition effect of FIR was associated with eNOS-mediated NO and VEGF-induced ROS. Although FIR exposure usually accompanies thermal transmission, we found thermal pretreatments slightly increased VEGF-induced proliferation in HUVECs. The thermal effect of 38 or 39°C did not influence phosphorylation levels of ERK1/2 or eNOS. Compared to the influence of FIR on the eNOS signaling pathway, these results show that the biological effects of FIR in HUVECs do not result from a thermal effect. Moreover, we found that FIR exposure induced the nuclear translocation of PLZF which increased PI3K expression. This PI3K increase activated Akt which phosphorylated eNOS to generate NO in HUVECs. Pretreatment at 38 or 39°C did not induce PLZF translocation. Therefore, a non-thermal effect of FIR inhibits VEGF-induced proliferation via PLZF activation.Based on our results, the FIR exposure at 0.13 mW/cm2H2 zinc finger domains and a POZ/BTB protein-binding domain PLZF is a sequence-specific DNA-binding transcriptional factor. It comprises 673 amino acids and contains nine Kruppel-like CNO is a free radical species that diffuses and concentrates in the hydrophobic core of low-density lipoproteins to serve as a potent antioxidant FIR therapy was found to improve inadequate access flow and survival of an AVF in HD patients, although the mechanism of its non-thermal effects is still unknown Because thermal transmission always accompanies FIR emission, FIR therapy often possesses a thermal effect. In this study, we evaluated the thermal effect at temperatures (37∼39°C) generated by FIR exposure. Unlike the biological effect of FIR, the thermal effect slightly increased VEGF-induced proliferation in HUVECs. The influence of the thermal effect differed from the biological effect of FIR on PLZF translocation and PI3K/Akt signaling pathways. Actually, the increase in FIR intensity or exposure time gradually reduced the inhibitory effect of FIR. This may have resulted from the thermal effect caused by FIR exposure because the increase in FIR intensity or exposure time significantly raised the temperature of the culture medium. Thus, we suggest that the temperature increase should be limited in FIR therapy on AVFs in HD patients to achieve maximum therapeutic effects.In conclusion, the present study reveals that FIR exposure induced the nuclear translocation of PLZF which upregulated PI3K to activate Akt, and then activated eNOS to induce NO generation. This NO generation combined with VEGF-induced ROS generation resulted in inhibition of VEGF-induced proliferation in HUVECs. Through a PLZF-mediated pathway, FIR therapy is a potential therapeutic modality to maintain vascular endothelial health and function.


In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.In this study, Tyr The membrane-bound GC (guanylate cyclase) family consists of five (human) and seven (rodent) members . Among t2+ to stabilize GC activity orthophosphate for 4 h. HeLa cells expressing Myc-GC-B were homogenized on ice with RIPA buffer , and the homogenates were centrifuged for 10 min at 21000 g at 4°C. The resulting supernatants were transferred to a tube containing mouse anti-myc antibody (5 μg/ml) and mixed for 4 h at 4°C. Protein G-Sepharose was then added to each tube and further mixed for 2 h at 4°C. The Protein-G-Sepharose-anti-myc antibody complex was washed five times with RIPA buffer, and the final pellet was mixed with 2× SDS/PAGE sample buffer (Bio-Rad) containing 5% (v/v) 2-mercaptoethanol. Samples were boiled for 3 min, aliquots of the homogenate were separated by SDS/PAGE (6% polyacrylamide gel), and then blotted on to a nitrocellulose membrane. Phosphorylated WT Myc-GC-B and Y808E bands were detected by autoradiography, and WT Myc-GC-B and Y808E proteins were detected using anti-myc antibody and the ECL system (GE).Myc-GC-B or Y808E mutant cDNA was transfected into HeLa cells grown in 6-well plates, and the cells were cultured in the growth medium overnight. After 24 h culture in DMEM containing 0.5% FBS, cells were labelled with serum- and phosphate-free DMEM containing 0.1 mCi/ml [http://www.cbs.dtu.dk/services/NetPhos/), and selected residues having scores greater than 0.800 for mutation. Based on this search, I identified 14 potential serine/threonine phosphorylation sites and two potential tyrosine phosphorylation sites which had not previously been examined . To further analyse the significance of Tyr808 for GC-B activity, I substituted this residue with amino acids having different chemical characteristics. The effect of residue volume was examined by substituting Tyr808 with smaller  or larger (tryptophan) residues , I found that cells expressing the Y808F mutant produced more than 30-fold higher levels of cGMP than cells expressing WT GC-B upon stimulation with 0.1 μM CNP B, despitectively ). The im WT GC-B . Upon st WT GC-B G. These 808 mutations affected the suppression of GC activity by well-characterized GC-B inhibitors, such as S1P, PMA and Na3VO4. As demonstrated above, S1P-dependent suppression of GC-B activity was unaffected by the Y808F mutation, nor was it affected by the Y808W mutation  in the presence of Mn ligands . To testractions . Consist523 and Ser526 to alanine or glutamine greatly reduced GC-B activity [P< 0.01) of 32Pi incorporation level into WT GC-B  into all six potential phosphorylation sites in the juxtamembrane portion of both WT GC-B (WT GC-B-6D) and the Y808E mutant (Y808E-6D), and measured cGMP production in cells expressing these constructs, in the absence or presence of CNP B. Consis808 plays a critical role in the regulation of the enzymatic activity of GC-B. Mutations of Tyr808 resulted in a wide range of effects on cyclase activity, depending on the nature of the substituting residue  or charged (glutamic acid) amino acid, probably because these mutations alter the orientation of GC domain to KHD, allowing GTP to access the catalytic site with less steric hindrance  domain, which is found in soluble GCs and bacterial haem-binding protein kinases [808 is located within the helical linker region. Mutations of Tyr808 may alter the dimerization state of GC-B and thereby control its catalytic activity, which fits our hypothesis shown in Based on NCBI  Conserved Domain Database , Tyr808  kinases ,34 (Figu808-equivalent tyrosine residue with alanine or phenylalanine, and of obtaining constitutively active cyclases by replacing that residue with serine or glutamic acid. Interestingly, mutations in Glu837, Arg838 and Thr839 of GC-E (shown in bold italics in 808 in GC-B, have been linked to dominant cone–rod dystrophy [All membrane-bound GCs, as well as adenylyl cyclases 2 and 4, have tyrosine at the position corresponding to residue 808 of GC-B B. Therefystrophy –38. Thes808 or its neighbouring residues. Current models suggest that membrane-bound GCs are present in an auto-inhibited conformation in unstimulated cells, and that binding of natriuretic peptides to their extracellular domains releases this inhibitory constraint. Mechanisms that underlie GC auto-inhibition have remained elusive, primarily because three-dimensional structures of the intracellular domains of membrane-bound GCs have not yet been solved. My results suggest that Tyr808 contributes to the auto-inhibition of GC-B, and raises the possibility that binding of CNP to the receptor induces a conformational change that displaces this residue from its inhibitory orientation.Due to its location between the KHD and the GC domain, scant attention has been paid to the potential biochemical significance of Tyr


Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKα phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK  increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPKα, β, and γ by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis.In this study, we investigated inhibition of the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm,  Bombyx moriDrosophila melanogaster, is a PTTH receptor B. mori and Manduca sexta. Initial studies showed that cAMP and Ca2+ are intracellular second messengers involved in PTTH-stimulated ecdysteroidogenesis in both M. sextaB. moriM. sextaB. mori prothoracic glands B. mori prothoracic glands by PTTH Ecdysteroids are the insect steroid hormones that are synthesized and secreted by the prothoracic glands upon stimulation by the brain neurohormone, prothoracicotropic hormone (PTTH) Drosophila, single genes encoding homologues of the α, β, and γ subunits of mammalian AMPK were identified Drosophila AMPK is highly similar to mammalian AMPK, as it is formed via a heterotrimeric complex, is activated by AMP, and has many of the same targets, including acetyl-CoA carboxylase (ACC) Drosophila through RNAi knockdown leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity In eukaryotic cells, adenosine 5′-monophosphate-activated protein kinase (AMPK) is a key regulatory enzyme of cellular energy homeostasis and is involved in regulating a diverse range of metabolic pathways B. mori prothoracic glands. Our results showed that PTTH inhibited the phosphorylation of AMPK both in vitro and in vivo. Moreover, PI3K appeared to be partially involved in PTTH-inhibited AMPK phosphorylation. The AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), prevented PTTH-inhibited AMPK phosphorylation and greatly inhibited PTTH-stimulated ecdysteroidogenesis, suggesting that AMPK signaling is indeed involved in PTTH-stimulated ecdysteroidogenesis of prothoracic glands in B. mori.In the present study, we investigated the involvement of AMPK signaling in PTTH-stimulated ecdysteroidogenesis in Larvae of the F1 racial hybrid, Guofu × Nongfong, were reared on fresh mulberry leaves at 25°C under a 12-h light: 12-h dark photoperiod. Newly-ecdysed last instar larvae were collected and used for each experiment.B. mori PTTH (PTTH) was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as described previously in vivo injections. Each incubation (50 µl) contained about 0.15 ng PTTH. Grace’s insect cell culture medium was purchased from Invitrogen . The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a PI3K inhibitor (LY294002), a cell-permeable AMPK activator (AICAR), and an AMPK inhibitor (compound C) were purchased from Calbiochem .Recombinant Anti-phospho-AMPKα (Thr172), anti-phospho-4E-BP1 (Thr37/46), anti-phospho-ERK, anti-ERK and anti-α-tubulin antibodies were purchased from Cell Signaling Technology . The anti-phospho-p70 S6 kinase (Thr412) antibody was purchased from Upstate . A horseradish peroxidase (HRP)-linked goat anti-rabbit second antibody was purchased from PerkinElmer Life Sciences .in vivo effect of PTTH on AMPK phosphorylation, day 7 last instar larvae were injected with 10 µl saline containing 1 µl of the original PTTH solution. Larvae injected with 10 µl saline were used as the controls.Considering the sensitive response to PTTH in prothoracic glands from day 7 last instar larvae, glands from this stage were dissected in lepidopteran saline Bombyx AMPKα with their counterparts from Drosophila, human, and mouse showed identical phosphorylation sites, indicating high conservation among species , anti-phospho-4E-BP1 (Thr37/46), anti-phospho-p70 S6 kinase (Thr412), and anti-ERK antibodies as those previously reported Phospho-specific antibodies were developed and are commercially available for human AMPKα phosphorylated at Thr172 , as phosphorylation is known to be essential for the activity of these enzymes. Comparison of deduced amino acid sequences of the phosphorylation domain of  species . ConservTo ensure that the immunoreactive signals were specific for the phospho-antibody, gland lysates were treated with lambda protein phosphatase , which removes phosphates from serine, threonine, and tyrosine residues Bombyx prothoracic glands for each time point. Total RNAs from prothoracic glands were extracted using the TRI Reagent  and contaminating genomic DNA was removed by DNase I treatment according to the manufacturer’s protocol. The quantity of extracted RNA was assessed with a UV1101 photometer , and the ratio of 260/280 was 1.8∼2.0. First-strand complementary DNA (cDNA) was synthesized using an iScript cDNA synthesis kit .Three batches of total RNAs were prepared independently from 4∼6 Bombyx ribosomal protein 49 (rp49) mRNA levels. The qRT-PCR reactions were performed using the following primers: AMPKα forward, 5′-CTGGAAGTGTTCCGAGCCAT-3′, AMPKα reverse, 5′-TACTCTGACGTGGTACGGGT-3′, AMPKβ forward, 5′-AAAGATGCTAACAAATCG-3′, AMPKβ reverse, 5′-GTTCAGTGTATTCAATATCA-3′, AMPKγ forward, 5′-GATGTACTACACGAGCCCCG-3′, AMPKγ reverse, 5′-TAGACGATGGATGCGGTTCG-3′, and RP49 forward, 5′-CAGGCGGTTCAAGGGTCAATAC-3′, RP49 reverse, 5′-TGCTGGGCTCTTTCCACGA-3′.A qRT-PCR was carried out in a 20-µl reaction volume containing 10 µl of SYBR1 Green Realtime PCR Master Mix (BIO-RAD), 2 µl of a first-strand cDNA template, and 8 µl of the primers. The iQ5 Real-Time PCR Detection System (Bio-Rad) was used according to the manufacturer’s instruction, and melting curve analysis was applied to all PCR reactions to ensure homogeneity of the amplified product. Only a single melting peak was found for all transcripts. RT-PCR primers were designed according to parameters (no primer dimers and a product length of no more than 200 bp) outlined in the manual of the SYBR1 Green Realtime PCR Master Mix. The annealing temperature for all reactions was 59.5°C. Transcript levels were normalized to the B. mori prothoracic glands was analyzed by immunoblotting with anti-phospho-AMPKα antibody, which only recognizes the phosphorylated form. B. mori gland lysates. Further evidences that the phosphorylated protein is AMPK and that the antibody specifically recognizes AMPK, comes from use of the AMPK inhibitor, compound C, and the AMPK activator, AICAR. As shown in in vitro, a great decrease in antigen recognition by the anti-phospho-AMPKα antibody was found. In contrast, treatment with AICAR greatly increased the immunoreactivity (As the first step in studying the involvement of AMPK in PTTH signal transduction in silkworm prothoracic glands, AMPK phosphorylation of activity .The effect of PTTH on AMPK phosphorylation was studied in greater detail by examining the level of AMPK phosphorylated at Thr172. Inhibition of AMPK phosphorylation by PTTH occurred after 30 min, and the inhibition persisted for 120 min . Fig. 4Bin vitro inhibition of AMPK phosphorylation of silkworm prothoracic glands by PTTH. We further examined the in vivo effect of AMPK phosphorylation of the prothoracic glands by PTTH. Larvae from day 7 last instars were injected with PTTH. Thirty minutes later, the prothoracic glands were quickly dissected out, and AMPK phosphorylation was examined and compared to that of control larvae. in vitro inhibition of AMPK phosphorylation.The above results clearly showed the B. mori, indicating the involvement of PI3K in ecdysteroidogenesis M. sextaB. moriOur previous study showed that treatment with the PI3K inhibitor, LY294002, greatly inhibited PTTH-stimulated ecdysteroidogenesis in The above results clearly showed that AMPK phosphorylation in prothoracic glands is inhibited by PTTH through the PI3K signaling pathway. To further examine whether this PTTH-mediated inhibition of AMPK phosphorylation is necessary for PTTH-stimulated ecdysteroidogenesis, we used AICAR, a pharmacological activator of AMPK Our previous study showed that PTTH stimulates the phosphorylation of 4E-BP and S6K, two known downstream signaling targets of TORC1. In order to investigate whether AMPK is involved in PTTH/TOR signaling, the effects of AICAR on PTTH-stimulated phosphorylation of 4E-BP and S6K were examined. Prothoracic glands were pre-treated with 1 mM AICAR, followed by treatment with PTTH for 60 min, then phosphorylation of 4E-BP and S6K was determined. In order to investigate whether AMPK is involved in PTTH/ERK signaling, the effect of AICAR on PTTH-stimulated ERK phosphorylation was examined. Prothoracic glands were pre-treated with 1 mM AICAR, followed by treatment with PTTH for 60 min; then ERK phosphorylation was determined. in vitro, no significant difference was detected in transcript levels of AMPKα, β, and γ between control glands and those treated with PTTH, when glands were incubated for either 1, 2 or 8 h, indicating that PTTH did not affect its transcript level.A further experiment was conducted to examine changes in mRNA expression level of AMPKα, β, and γ upon PTTH treatment. As shown in in vitro inhibition of AMPK phosphorylation of prothoracic glands by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. Pre-treatment with LY294002 partially prevented PTTH-inhibited phosphorylation of AMPK, indicating the involvement of PI3K signaling. The link between PTTH-inhibited AMPK phosphorylation and ecdysteroidogenesis was further demonstrated by AICAR, a chemical activator of AMPK. Pretreatment with AICAR increased both basal and PTTH-inhibited AMPK phosphorylation, and also greatly inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. These results clearly show that inhibition of AMPK phosphorylation plays a role in PTTH stimulation of ecdysteroidogenesis. Although the involvement of AMPK in regulating steroidogenesis was previously demonstrated in bovine granulose cells The present study clearly shows that PTTH inhibited AMPK phosphorylation in time- and dose-dependent manners. Moreover, D. melanogaster, a homologue of AMP-activated protein kinase was identified and it was shown that it is sensitive to AMP and is activated by ATP depletion D. melanogaster was lethal, resulting in severe defects in embryonic cell polarity and the cell structure Drosophila AMPK by RNAi in muscles reduced longevity and stress resistance B. mori prothoracic glands and that PTTH inhibits the phosphorylation of AMPK. In the mouse heart, it was reported that insulin treatment caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation AMPK is a serine/threonine kinase and is highly conserved throughout eukaryotes In addition, our previous study showed that PTTH stimulates the phosphorylation of 4E-BP and S6K, two TOR target proteins and that PI3K/TOR signaling is involved in PTTH-stimulated ecdysteroidogenesis B. mori prothoracic glands PTTH was shown to increase ecdysteroidogenesis of prothoracic glands through an ERK signaling pathway B. mori prothoracic glands. Based on this study and our previous studies, it is reasonable to conclude that PTTH increases ecdysteroidogenesis in B. mori prothoracic glands through an ERK-dependent pathway, and by regulating the AMPK signaling pathway through a PI3K-dependent pathway. In addition, pre-treatment with AICAR only partially prevented PTTH-stimulated 4E-BP phosphorylation, indicating AMPK-independent TOR signaling pathways.Taken together, the present study shows that AMPK, which so far is known as a controller of energy balance in cells, can also be regulated by PTTH in