p70S6K Pathway Research Articles

Lipid raft cholesterol and genistein inhibit the cell viability of prostate cancer cells via the partial contribution of EGFR-Akt/p70S6k pathway and down-regulation of androgen receptor

Soy isoflavones and cholesterol have been reported as dietary factors related to the incidence of prostate cancer. In this study, we investigated whether cell survival could be suppressed by a combination of the dispersion of lipid raft microdomains and treatment with genistein, a well-known potential isoflavone, in LNCaP prostate cancer cells. Cell viability was assayed by the property of reagent change upon reduction of resazurin to resorufin and apoptosis was evaluated by ethidium bromide/acridine orange (EB/AO) staining and PARP and caspase-3 expression. Signal transduction was investigated by immunoblot analysis. Cell viability decreased significantly more following successive double treatment with genistein and the cholesterol-lowering agent 2-hydroxypropyl-beta-cyclodextrin (HPCD) than in response to either agent alone. Apoptotic cell staining and cleavage of PARP and caspase-3 appeared more clearly in double-treated cells than in those treated with genistein alone. In cell signaling, both HPCD and genistein decreased the protein expressions of pAkt as well as the androgen receptors stimulated by EGF and DHT, respectively, in concentration-dependent manners. This pattern was also present in protein levels of pAkt and the androgen receptor located in the lipid raft fraction. Furthermore, the phosphorylation cascade of Akt, GSK-3β and p70S6k was markedly inhibited by the combination treatment. These data suggest that prostate cancer cells could be effectively inhibited by combination treatment of cholesterol-lowering strategies and genistein. The mechanism is likely to be partially via both the EGFR-mediated Akt or p70S6k pathways and a down-regulation of androgen receptor in the lipid raft microdomain.

Naringin inhibits matrix metalloproteinase‐9 expression and AKT phosphorylation in tumor necrosis factor‐α‐induced vascular smooth muscle cells

Citrus fruits are high in naringin, which has a beneficial effect on cardiovascular diseases. However, the matrix metalloproteinase-9 (MMP-9) regulation involved in cell migration and invasion remains to be identified. Naringin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced expression of MMP-9, under 10-25 microM concentration conditions in vascular smooth muscle cells (VSMC). The TNF-alpha-induced invasion and migration of VSMC were inhibited by naringin. Furthermore, naringin suppressed TNF-alpha-mediated release of interleukin-6 and -8 (IL-6 and IL-8). However, naringin (10-25 microM) treatment of VSMC in the presence of TNF-alpha did not affect cell growth and apoptosis. In additional experiments, naringin reduced the transcriptional activity of activator protein-1 and nuclear factor kappaB (NF-kappaB), which are two important nuclear transcription factors that are involved in MMP-9 expression. Also, naringin treatment blocked PI3K/AKT/mTOR/p70S6K pathway in TNF-alpha-induced VSMC. Treatment of aglycone naringenin (10-25 microM) had same effect on the levels of MMP-9 expression, invasion, migration, and AKT phosphorylation in TNF-alpha-induced VSMC, compared with naringin treatment. These results suggest that naringin represses PI3K/AKT/mTOR/p70S6K pathway, invasion and migration, and subsequently suppresses MMP-9 expression through the transcription factors NF-kappaB and activator protein-1 in TNF-alpha-induced VSMC. These novel findings provide a theoretical basis for the preventive use of naringin for atherosclerosis disease.

The rapamycin-derivative RAD001 (everolimus) inhibits cell viability and interacts with the Akt–mTOR–p70S6K pathway in human medullary thyroid carcinoma cells

Over-expression of the proto-oncogene Akt/PKB has been demonstrated in some neuroendocrine tumor models. Akt may activate downstream proteins such as mTOR and p70S6K, inducing tumor proliferation. The rapamycin-derivative RAD001, everolimus, interacts with this pathway by antagonizing mTOR, but its effects on neuroendocrine tumors are largely unknown. We explored the mechanism of action of RAD001 on cell proliferation, hormonal secretion and on Akt/mTOR/p70S6K pathway activation, in a human medullary thyroid carcinoma (MTC) cell-line (TT) and in cells derived from human MTCs. Treatment with RAD001 significantly inhibited cell viability in a dose- and time-dependent fashion, and diminished phosphorylation of Akt downstream targets, mTOR and p70S6K, in both TT cell-line and cultured human MTCs. Akt phosphorylation was not affected by RAD001. RAD001 induced cell-cycle arrest in the G 0/G 1 phase in TT cells, but had no effect on apoptosis. Moreover, RAD001 did not affect calcitonin and carcinoembryonic antigen secretion in TT cells and in human MTCs. RAD001 seems to have potent anti-proliferative effect in human MTC cells, which suggest that clinical trials of this agent are of considerable interest.

The PI3K/Akt and mTOR/P70S6K Signaling Pathways in Human Uveal Melanoma Cells: Interaction with B-Raf/ERK

Activated B-Raf alone cannot induce melanoma but must cooperate with other signaling pathways. The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR)/p70S6K pathways are critical for tumorigenesis. The authors investigated the role of these pathways in uveal melanoma cells. The effects of PI3K and mTOR activation and inhibition on the proliferation of human uveal melanoma cell lines expressing either activated (WT)B-Raf or (V600E)B-Raf were investigated. Interactions among PI3K, mTOR, and B-Raf/ERK were studied. Inhibition of PI3K deactivated P70S6 kinase, reduced cell proliferation by 71% to 84%, and increased apoptosis by a factor of 5.0 to 8.4 without reducing ERK1/2 activation, indicating that ERK plays no role in mediating PI3K in these processes. In contrast, rapamycin-induced inhibition of mTOR did not significantly affect cell proliferation because it simultaneously stimulated PI3K/Akt activation and cyclin D1 expression. Regardless of B-Raf mutation status, cotreatment with the PI3K inhibitor effectively sensitized all melanoma cell lines to the B-Raf or ERK1/2 inhibition-induced reduction of cell proliferation. B-Raf/ERK and PI3K signaling, but not mTOR signaling, converged to control cyclin D1 expression. Moreover, p70S6K required the activation of ERK1/2. These data demonstrate that PI3K/Akt and mTOR/P70S6K interact with B-Raf/ERK. Activated PI3K/Akt attenuates the inhibitory effects of rapamycin on cell proliferation and thus serves as a negative feedback mechanism. This finding suggests that rapamycin is unlikely to inhibit uveal melanoma growth. In contrast, targeting PI3K while inhibiting B-Raf/ERK may be a promising approach to reduce the proliferation of uveal melanoma cells.

Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling

Cognitive impairment is one of the most important negative consequences associated with cannabis consumption. We found that CB1 cannabinoid receptor (CB1R) activation transiently modulated the mammalian target of rapamycin (mTOR)/p70S6K pathway and the protein synthesis machinery in the mouse hippocampus, which correlated with the amnesic properties of delta9-tetrahydrocannabinol (THC). In addition, non-amnesic doses of either the mTOR blocker rapamycin or the protein synthesis inhibitor anisomycin abrogated the amnesic-like effects of THC, pointing to a mechanism involving new protein synthesis. Moreover, using pharmacological and genetic tools, we found that THC long-term memory deficits were mediated by CB1Rs expressed on GABAergic interneurons through a glutamatergic mechanism, as both the amnesic-like effects and p70S6K phosphorylation were reduced in GABA-CB1R knockout mice and by NMDA blockade.

Immunohistochemical analysis of the mammalian target of rapamycin signalling pathway in extramammary Paget’s disease

We have previously observed that persistent activation of the serine/threonine kinase, protein kinase B (AKT) is a frequent event in extramammary Paget's disease (EMPD). AKT promotes cell proliferation by its ability to coordinate mitogenic signalling with energy- and nutrient-sensing pathways that control protein synthesis through the atypical serine/threonine kinase, mammalian target of rapamycin (mTOR). CDK2, a member of the serine/threonine kinase family of cyclin-dependent kinases, is a key regulator of G(1)-S cell cycle progression, and has recently been shown to be one of the targets of AKT. The AKT-mTOR-p70 ribosomal protein S6 kinase (p70S6K) pathway has been described in some human malignancies, but not in EMPD. To investigate the immunohistochemical staining of the AKT-mTOR-p70S6K pathway in EMPD and to evaluate the relationships among the components. Samples of primary EMPD tissue were subjected to immunohistological staining with phosphorylated (p)-AKT, p-mTOR, p-4E-binding protein 1 (p-4EBP1), p-p70S6K/S6K1, p-ribosomal protein S6 (p-S6) and CDK2. Ten normal skin samples served as a control. Of the 32 EMPD tissue samples, 29, 27, 26, 29, 26 and 32 samples were positive for p-AKT, p-mTOR, p-4EBP1, p-p70S6K/S6K1, p-S6 and CDK2 staining, respectively. All these cell signalling molecules showed higher positivity in invasive EMPD than in EMPD in situ. There were significant correlations between p-AKT, p-mTOR, p-4EBP1, p-p70S6K/S6K1 and p-S6 and CDK2. The activation of the AKT-mTOR-p70S6K pathway may play an important role in the pathogenesis of EMPD. The high expression of the components of the pathway was highly correlated with CDK2 expression, suggesting that the AKT/mTOR pathway may induce the malignant transition through CDK2 in EMPD. The AKT-mTOR-p70S6K pathway might be a potential therapeutic target in EMPD.

PTEN Contributes to Profound PI3K/Akt Signaling Pathway Deregulation in Dystrophin-Deficient Dog Muscle

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

E013 Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50mug/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

ObjectivesSmooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. ResultsmTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50μg/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. ConclusionThese data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

Constitutive activation of p70 S6 kinase is associated with intrinsic resistance to cisplatin

Cisplatin is widely used for the treatment of solid tumors, including small cell lung cancers, but its success is often compromised due to relapse and resistance to further treatment. p70 ribosomal S6 kinase (p70S6K) has been shown to be upregulated in lung cancer cells. In the present study, we investigated whether the p70S6K pathway contributes to cisplatin resistance in human small cell lung cancer H69 cells. The levels of phosphorylated p70S6K and its downstream target S6 but not total p70S6K or S6 were elevated in the H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells. Cisplatin treatment resulted in the activation of p70S6K and downregulation of p70S6K was associated with cisplatin-induced PARP cleavage. While the ability of cisplatin to induce apoptosis was attenuated in H69/CP cells, inhibition of p70S6K by rapamycin enhanced cisplatin-induced apoptosis in these cells as evident by the increase in cisplatin-induced poly(ADP-ribose) polymerase (PARP) cleavage. The phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 alone induced PARP cleavage and further augmented cisplatin-induced PARP cleavage. In contrast, inhibition of extracellular signal-regulated kinase (ERK) by U0126 attenuated cisplatin-induced PARP cleavage. Both rapamycin and Ly294002 enhanced cisplatin-induced acti-vation of ERK1/2. Taken together, these results suggest that activation of p70S6K contributes to cisplatin resistance in small cell lung cancer H69 cells, and inhibition/downregulation of p70S6K as well as activation of ERK1/2 could circumvent cisplatin resistance.

Delphinidin inhibits cell proliferation and invasion via modulation of Met receptor phosphorylation

The HGF/Met signaling pathway is deregulated in majority of cancers and is associated with poor prognosis in breast cancer. Delphinidin, present in pigmented fruits and vegetables possesses potent anti-oxidant, anti-inflammatory and anti-angiogenic properties. Here, we assessed the anti-proliferative and anti-invasive effects of delphinidin on HGF-mediated responses in the immortalized MCF-10A breast cell line. Treatment of cells with delphinidin prior to exposure to exogenous HGF resulted in the inhibition of HGF-mediated (i) tyrosyl-phosphorylation and increased expression of Met receptor, (ii) phosphorylation of downstream regulators such as FAK and Src and (iii) induction of adaptor proteins including paxillin, Gab-1 and GRB-2. In addition, delphinidin treatment resulted in significant inhibition of HGF-activated (i) Ras-ERK MAPKs and (ii) PI3K/AKT/mTOR/p70S6K pathways. Delphinidin was found to repress HGF-activated NFκB transcription with a decrease in (i) phosphorylation of IKKα/β and IκBα, and (ii) activation and nuclear translocation of NFκB/p65. Inhibition of HGF-mediated membrane translocation of PKCα as well as decreased phosphorylation of STAT3 was further observed in delphinidin treated cells. Finally, decreased cell viability of Met receptor expressing breast cancer cells treated with delphinidin argues for a potential role of the agent in the prevention of HGF-mediated activation of various signaling pathways implicated in breast cancer.

Constitutive activation of p70 S6 kinase is associated with intrinsic resistance to cisplatin

Cisplatin is widely used for the treatment of solid tumors, including small cell lung cancers, but its success is often compromised due to relapse and resistance to further treatment. p70 ribosomal S6 kinase (p70S6K) has been shown to be upregulated in lung cancer cells. In the present study, we investigated whether the p70S6K pathway contributes to cisplatin resistance in human small cell lung cancer H69 cells. The levels of phosphorylated p70S6K and its downstream target S6 but not total p70S6K or S6 were elevated in the H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells. Cisplatin treatment resulted in the activation of p70S6K and downregulation of p70S6K was associated with cisplatin-induced PARP cleavage. While the ability of cisplatin to induce apoptosis was attenuated in H69/CP cells, inhibition of p70S6K by rapamycin enhanced cisplatin-induced apoptosis in these cells as evident by the increase in cisplatin-induced poly(ADP-ribose) polymerase (PARP) cleavage. The phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 alone induced PARP cleavage and further augmented cisplatin-induced PARP cleavage. In contrast, inhibition of extracellular signal-regulated kinase (ERK) by U0126 attenuated cisplatin-induced PARP cleavage. Both rapamycin and Ly294002 enhanced cisplatin-induced acti-vation of ERK1/2. Taken together, these results suggest that activation of p70S6K contributes to cisplatin resistance in small cell lung cancer H69 cells, and inhibition/downregulation of p70S6K as well as activation of ERK1/2 could circumvent cisplatin resistance.

High-Resolution DNA Copy Number Profiling of Malignant Peripheral Nerve Sheath Tumors Using Targeted Microarray-Based Comparative Genomic Hybridization

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition that predisposes to benign and malignant tumors. The lifetime risk of a malignant peripheral nerve sheath tumor (MPNST) in NF1 is approximately 10%. These tumors have a poor survival rate and their molecular basis remains unclear. We report the first comprehensive investigation of DNA copy number across multitude of genes in NF1 tumors using high-resolution array comparative genomic hybridization (CGH), with the aim to identify molecular signatures that delineate malignant from benign NF1 tumors. We constructed an exon-level resolution microarray encompassing 57 selected genes and profiled DNA from 35 MPNSTs, 16 plexiform, and 8 dermal neurofibromas. Bioinformatic analysis was done on array CGH data to identify concurrent aberrations in malignant tumors. The array CGH profiles of MPNSTs and neurofibromas were markedly different. A number of MPNST-specific alterations were identified, including amplifications of ITGB4, PDGFRA, MET, TP73, and HGF plus deletions in NF1, HMMR/RHAMM, MMP13, L1CAM2, p16INK4A/CDKN2A, and TP53. Copy number changes of HMMR/RHAMM, MMP13, p16INK4A/CDKN2A, and ITGB4 were observed in 46%, 43%, 39%, and 32%, respectively of the malignant tumors, implicating these genes in MPNST pathogenesis. Concomitant amplifications of HGF, MET, and PDGFRA genes were also revealed in MPNSTs, suggesting the putative role of p70S6K pathway in NF1 tumor progression. This study highlights the potential of array CGH in identifying novel diagnostic markers for MPNSTs.

The effect of MK-801 on mTOR/p70S6K and translation-related proteins in rat frontal cortex

In experimental animals, including rats, MK-801 produces characteristic behavioural changes that model schizophrenia. It has been hypothesized that these changes accompany long-term synaptic changes, which require protein neosynthesis. We observed the effect of MK-801 on the "mammalian target of rapamycin" (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway that regulates protein synthesis in the rat frontal cortex. A single injection of MK-801 (0.5, 1, or 2mg/kg) induced an acute increase in the phosphorylation of Akt (Ser-473) eIF4E-binding protein (4E-BP1) (Thr-37/46) and p70S6K (Thr-389). In contrast, after repeated treatment with MK-801 (1mg/kg for 5 or 10 days), the phosphorylation of Akt (Ser-473), mTOR (Ser-2481), 4E-BP1 (Thr-37/46), p70S6K (Thr-389), and S6 (Ser-240/244) increased. Thus, proteins in the mTOR/p70S6K pathway are modulated in chronic MK-801 animal models. These findings may suggest that repeated MK-801 treatment activates the signal transduction pathways involved in the initiation of protein synthesis in the rat frontal cortex.

Uniaxial stretch‐induced regulation of mitogen‐activated protein kinase, Akt and p70 S6 kinase in the ageing Fischer 344 × Brown Norway rat aorta

The effects of ageing on the cardiovascular system contribute to substantial alterations in cellular morphology and function. The variables regulating these changes are unknown; however, one set of signalling molecules that may be of particular importance in mediating numerous cellular responses, including control of cell growth, differentiation and adaptation, are the proteins associated with the mitogen-activated protein kinase (MAPK) signalling systems. The MAPKs, in conjunction with the p70 S6k signalling cascade, have emerged as critical components for regulating numerous mechanotransduction-related cellular responses. Here we investigate the ability of uniaxial stretch to activate the MAPK and p70 S6k pathways in adult (6-month-old), aged (30-month-old) and very aged (36-month-old) Fischer 344/NNiaHSd x Brown Norway/BiNia (FBN) rats. Western blotting of the MAPK family proteins extracellular signal-regulated kinase (Erk) 1/2, p38- and c-Jun NH(2)-terminal kinase (Jnk)-MAPKs showed differential expression and activation between these proteins with age. An acute 15 min interval of 20% uniaxial stretch using an ex vivo aortic preparation demonstrated similar regulation of Erk1/2, p38- and Jnk-MAPK. However, ageing altered uniaxial induced p70 S6k pathway signalling. These observations confirm previous data demonstrating that MAPK proteins are mechanically regulated and also suggest that p70 S6k signalling expression and activation are controlled differently with ageing. Taken together, these data may help to explain, in part, the age-related changes in vascular morphology, function and response to injury.

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Myocardial Ischaemia-reperfusion Injury is Attenuated by Intact Glucagon Like Peptide-1 (GLP-1) in the In Vitro Rat Heart and may Involve the p70s6K Pathway

Glucagon Like Peptide-1 (GLP-1), one of the most potent incretin hormones, has potential beneficial actions on the ischaemic and failing heart. This study sought to further identify the mechanisms of action of GLP-1 on the ischaemic heart using an in vitro isolated perfused rat heart model of ischaemic-reperfusion injury (measuring infarct size to area of risk (%)) subjected to 35 min regional ischaemia and 2 h reperfusion. To examine the effect of intact GLP-1 we used an inhibitor of GLP-1 breakdown, Valine pyrrolidide (VP). The downstream target of phosphatidylinositol 3-kinase includes the mTOR/p70s6 kinase pathway which was pharmacologically inhibited by rapamycin. GLP-1 alone did not decrease myocardial infarction (54.4 +/- 3.1%). VP alone did not decrease myocardial infarction (52.5 +/- 4%). GLP-1 in the presence of VP produced significant reduction in myocardial infarction compared to control hearts (28.4 +/- 2.7% vs. 56.4 +/- 3.9% vs. P < 0.05). Inhibiting p70s6 Kinase with rapamycin completely abolished GLP-1 induced protection (57.1 +/- 4.9% vs. 28.4 +/- 2.7% P < 0.05). There was no detectable increase in the phosphorylated p70s6k after either 5 or 10 min of treatment with GLP-1/VP or with VP alone in comparison to control blots. In conclusion we show for the first time that the protective effects of GLP-1 are mediated by intact GLP-1 and can be inhibited by blocking the p70s6 kinase.

Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamycin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry of 94:1 lipid/protein. Polyphosphoinositides PI4-P and PI4,5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS-7 cells with a wild-type myc-(pcDNA)-PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6K's natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS-7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR-independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.

Refeeding and insulin activate the AKT/p70S6 kinase pathway without affecting IRS1 tyrosine phosphorylation in chicken muscle

p70 S6 kinase (p70S6K) is a key enzyme involved in the control of protein synthesis. We have previously shown that this kinase is insulin sensitive in chicken muscle despite a relative insulin resistance in the early steps of insulin receptor signaling in this tissue, particularly with no change in tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1). The aim of the present study is to further study the p70S6K pathway in chicken muscle. By analyzing in silico several kinases involved in the protein kinase B (PKB also called AKT)/target of rapamycin (TOR)/p70S6K pathway in the chicken, we showed that the amino acid sequence of the proteins exhibited a very high identity with their homologs in mammalian species and Drosophila. We investigated the regulation of these kinases in vivo or in vitro. Refeeding and insulin treatment significantly ( P < 0.05) increased the phosphorylation and/or activity of kinases upstream of p70S6K such as AKT and TOR. Similarly, refeeding and insulin increased the phosphorylation of p70S6K on key residues (i.e. T389, T229 and T421/S424) and the phosphorylation of a p70S6K downstream target, the ribosomal protein S6 (by 3–10-fold, P < 0.05). Interestingly, we also showed an increase in the phosphorylation level of IRS1 on S632/S635, sites involved in insulin resistance. In conclusion, the AKT/TOR/p70S6K pathway is activated by refeeding and insulin injection, which might negatively regulate IRS1 tyrosine phosphorylation. These results indicate some particularities of the insulin signaling in chicken muscle and suggest the involvement of p70S6K in these features.

Thyroid hormone and Na+-K+-ATPase: more than simple transcription

FLUID SECRETION INTO THE DEVELOPING lungs’ air spaces is essential for normal lung development, but this fluid must be cleared at birth so that effective gas exchange can occur. Impaired clearance of this fetal lung liquid, which arises from immaturity of the distal lung epithelium’s active Na transport system (for review, see Ref. 17), causes transient tachypnea of the newborn (2, 12, 13), and, if combined with immaturity of the surfactant system, neonatal respiratory distress syndrome (nRDS) (4, 17). Clearance of air space fluid is driven by active transepithelial Na transport resulting from synchronized activity of apical Na permeant ion channels (e.g., epithelial Na channel, ENaC) and basolateral Na-K-ATPase. The chemical and electrical gradients for Na absorption are, respectively, maintained by Na-K-ATPase and K channels. Perinatal changes in PO2, glucocorticoids, -agonists, and thyroid hormones interact to increase transepithelial Na absorption, to bring about the switch from net fluid secretion to net absorption as lung liquid is cleared at birth. However, despite considerable data from molecular, transgenic, cell culture, and in vivo studies, the fundamental mechanisms of this crucial developmental shift are incompletely understood (5, 19). Barker and coworkers (6) demonstrated that thyroid hormone [triiodothyronine (T3)] played an important role in maturation of the fetal lungs’ ability to respond to an infusion of epinephrine by switching to fluid absorption, and others showed that thyroid hormone in concert with glucocorticoids increased the expression of ENaC (18, 22). Although T3 classically acts as a direct transcriptional activator of target genes and has been shown to potentiate dexamethasone-stimulated transcription of the ENaC promoter (20), T3-induced upregulation of Na-K-ATPase activity in adult rat alveolar epithelial cells was recently shown to be insensitive to actinomycin D (15). This has functional significance. For example, it has been demonstrated that overexpression of Na-KATPase via adenoviral-mediated gene transfer is sufficient to increase alveolar fluid clearance (21). It is known that thyroid hormones have profound effects on the maturation of the fetal lung and that premature infants with nRDS have lower thyroid hormone levels than well preterm infants (9, 10). However, when clinical trials have been conducted wherein augmentation of the thyroid hormonal axis has taken place, there has been no demonstrable benefit. The THORN trial of T3 and hydrocortisone supplementation in preterm infants of less than 30 wk gestation found that low thyroid hormone levels were associated with higher mortality and ventilator dependence (7, 8). However, this trial (8) was unable to demonstrate beneficial effects of T3 and hydrocortisone infusion. Similarly, the provision of thyroid-releasing hormone to infants who had received antenatal glucocorticoid