Changes In Akt Research Articles

Lenalidomide targets clonogenic side population in multiple myeloma: pathophysiologic and clinical implications

AbstractRecurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry–based Hoechst 33342 staining to evaluate the existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.

Endothelial progenitor cell responses to hypoxia: Time‐course dependent changes in Akt signalling and gene expression

The aim of this work is to examine endothelial progenitor cells (EPCs) and their potential role as a cell based autologous therapy to revasularise the ischaemic tissue of the diabetic retina. Our group identified a distinctive human blood derived EPC with high proliferative capacity known as outgrowth endothelial cells (OECs). Characterisation of these cells highlighted their endothelial‐like, as opposed to inflammatory cell phenotype. To mimic low O2 concentrations in the diabetic retina, we have exposed OECs to 1% O2 for varying times. Induction of HIF1α was detected at 5h and maintained up to 48h at 1% O2 (in contrast to HeLa cells). Changes in Akt and p70 S6K phosphorylation were evident after 30 min exposure to hypoxia. Chronic hypoxia activated the AMPK→TORC2→p70S6K pathway leading to inhibition of p70S6K phosphorylation. Global gene array analysis using Illumina bead chip platform identified distinct cohorts of early response (8h) and late (24, 48h) response genes in OECs to hypoxia. Genes such as ADM, ANGPTL4, JMJD1A and the recently documented hypoxia inducible gene ANKRD37 were elevated in our array. A number of novel hypoxia genes including several involved in the BMP/Smad signalling pahway were also elevated. These groups of genes will then form the basis of comparison between control and diabetic OECs, to identify differences in diabetic cells that may explain their poor vaso‐reparative potential.

Androgens activate mitogen-activated protein kinase via epidermal growth factor receptor/insulin-like growth factor 1 receptor in the mouse PC-1 cell line

Androgens are the primary regulators of epididymal structure and functions. In the classical view of androgen action, binding of androgen to the intracellular androgen receptor (AR) produces the receptor-steroid complex that has high affinity for DNA response elements and regulates the transcription of target genes. In this study, we demonstrate that in epididymal cells, 5α-dihydrotestosterone (DHT) can cause an alternative and rapid response that is independent of AR-DNA interactions and is mediated by activation of signaling pathways through the AR. We examined changes in AKT and extracellular signal-regulated protein kinases (ERK1/2) activation at early time points after DHT supplementation in the mouse proximal caput epididymis-1 cell line. DHT had no significant effect on AKT activation at any time point. However, DHT activated the ERK pathway as early as at 1 min, the pathway remained activated at 10 min, but activation was not sustained at later time points. Interestingly, ERK activation was blocked by hydroxyflutamide (HF), indicating that early ERK activation was an AR-mediated response. DHT phosphorylates steroid receptor co-activator (SRC) kinase, and this activation was required for the ERK response. EGFR and IGF1R were downstream of SRC, and these two receptors together contributed to enhance ERK and cAMP response element-binding protein (CREB) phosphorylation. We postulate that this rapid action of androgen may ultimately act to modulate the transcription of genes regulated by AR in the nucleus. These results support the hypothesis that DHT can activate a pathway involving the sequential activation of MEK, ERK1/2, and CREB through the EGFR/IGF1R in an epididymal cell line.

Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation

Unilateral denervation (DNV) of rat diaphragm muscle increases protein synthesis at 3 days after DNV (DNV-3D) and degradation at DNV-5D, such that net protein breakdown is evident by DNV-5D. On the basis of existing models of protein balance, we examined DNV-induced changes in Akt, AMP-activated protein kinase (AMPK), and ERK&frac12; activation, which can lead to increased protein synthesis via mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K), glycogen synthase kinase-3β (GSK3β), or eukaryotic initiation factor 4E (eIF4E), and increased protein degradation via forkhead box protein O (FoxO). Protein phosphorylation was measured using Western analyses through DNV-5D. Akt phosphorylation decreased at 1 h and 6 h after DNV compared with sham despite decreased AMPK phosphorylation. Both Akt and AMPK phosphorylation returned to sham levels by DNV-1D. Phosphorylation of their downstream effector mTOR (Ser2481) did not change at any time point after DNV, and phosphorylated p70S6K and eIF4E-binding protein 1 (4EBP1) increased only by DNV-5D. In contrast, ERK&frac12; phosphorylation and its downstream effector eIF4E increased 1.7-fold at DNV-1D and phosphorylated GSK3β increased 1.5-fold at DNV-3D (P < 0.05 for both comparisons). Thus, following DNV there are differential effects on protein synthetic pathways with preferential activation of GSK3β and eIF4E over p70S6K. FoxO1 nuclear translocation occurred by DNV-1D, consistent with its role in increasing expression of atrogenes necessary for subsequent ubiquitin-proteasome activation evident by DNV-5D. On the basis of our results, increased protein synthesis following DNV is associated with changes in ERK&frac12;-dependent pathways, but protein degradation results from downregulation of Akt and nuclear translocation of FoxO1. No single trigger is responsible for protein balance following DNV. Protein balance in skeletal muscle depends on multiple synthetic/degradation pathways that should be studied in concert.

Study on histone acetylation modulation and Akt signaling pathway inhibition by phenyhexyle isothiocyanate in prostate cancer PC3 cell line

：Objective To investigatephenyhexyle isothiocyanate (PHI) modulating histone acetylation and inhibiting Aktsignaling pathway in prostate cancer cell line PC3 in vitro. Methods Apoptotic cells weremeasured by TUNEL assay. Histone acetylated H3, H4 and the Akt protein signaling pathway(Akt, p-Akt, mTOR, p-mTOR, p70S6K and p-p70S6K) were detected by Western blot. ResultsApoptotic cells increased after exposure to PHI with concentration dependent. PHIsignificantly induced an accumulation of histone acetylated H3, H4. The change of Akt,mTOR, p70S6K proteins was not observed. Phosphorylation of Akt (p- Akt), mTOR (p-mTOR) andp70S6K (p-p70S6K) decreased after exposure to PHI for 7 h. Conclusions PHI can inducehistone acetylation H3, H4 accumulation. PHI inhibits Akt signaling pathway resulting cellapoptosis. It might be a new anticancer agent.

Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion*

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 μg/ml) or full-length (5 μg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.

Abstract 4482: Novel and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase-1 inhibit the PDK-1/AKT signaling pathway and cell proliferation

Abstract Receptor tyrosine kinases (RTKs), PTEN (phosphatase and tensin homolog deleted on chromosome 10), and PIK3CA (encodes the p110α subunit of phosphatidylinositol 3-kinase [PI3K]) frequently contribute to tumor progression through their ability to regulate the intracellular level of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-phosphoinositide-dependent kinase-1 (PDK-1), a serine/threonine kinase, activates the catalytic domain of numerous kinases by phosphorylating their T-loop sites. PDK-1 activity is required for activation of AKT, p70S6K, and RSK which lead to cell proliferation and transformation. The interaction of the pleckstrin homology (PH) domain of AKT with the membrane bound PIP3 confers a conformational change in AKT, allowing PDK-1 to phosphorylate AKT at the residue threonine-308 (T308). This T-loop activation at T308, along with the phosphorylation of the serine-473 residue by mTOR, fully activates the AKT pathway. Although the roles of many PDK-1 substrates have yet to be characterized, the oncogenic activity of aberrant PI3K pathway signaling through PDK-1 has been extensively validated. We have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidne (CAP) compounds that are potent inhibitors of human PDK-1 (full length and kinase domain) which demonstrate more than 100-fold selectivity against P70S6K, PI3K, AKT, and mTOR. In this study, representative compounds from the CAP series were used to perform a variety of anti-tumor assays. We demonstrate PDK-1 compounds inhibit the phosphorylation of T308 on AKT as well as downstream molecules of the PI3K pathway, such as S6 ribosomal protein (S6RP) in breast, lung and colon cancer cell lines harboring a PI3KCA mutation. Additionally, blockade of AKT and molecules in the PI3K pathway leads to the inhibition of cell proliferation and cell transformation in cancer cells. Our data suggest that the inhibition of PDK-1 activity is sufficient to induce anti-tumor activity in cancer cells through the PI3K-PDK1-AKT axis, and that a potent and specific PDK-1 inhibitor could potentially be developed as a therapeutic agent against several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4482.

Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH

The AKT family is implicated in cancer progression. There are three mammalian AKT isoforms located on chromosomes 14, 19 and 1, respectively. The aim of the study was to investigate genetic alterations of AKT in breast and prostatic cancers using fluorescence in situ hybridization (FISH). In oestrogen receptor(ER)-positive breast carcinomas, AKT1 was deleted in five (4.8%) and amplified in one (1%) carcinoma. Deletions of AKT2 were seen in 19 (21.1%) cases. No AKT2 amplifications were identified. Ten (9.9%) AKT3 amplifications but no deletions were seen. In prostatic cancer, AKT1 was amplified in one carcinoma (2.6%). No genetic changes were observed for AKT2 and AKT3. High frequencies of aneusomy for all chromosomes were observed in breast and prostatic carcinomas. In breast cancer AKT3 amplifications and AKT1 and AKT2 deletions were seen, which, to our knowledge, have not been shown by FISH before. Although these two cohorts cannot be directly compared, only one AKT1 amplification was identified in prostatic carcinomas. This indicates differences in the genetic changes underlying development of breast and prostatic cancers. To evaluate further the role of genetic changes of AKT in breast cancer progression, a cohort of both ER+ and ER- patients should be evaluated.

Targeting the Clonogenic Side Population of Multiple Myeloma by Immunomodulatory Drugs (IMiDs) in the Context of Stromal Cell Microenvironment: Pathophysiologic and Clinical Implications.

Abstract 954 Introduction:Multiple myeloma (MM), a malignancy of plasma cells, remains incurable even though newly established regimens incorporating conventional and novel agents are capable of achieving complete clinical and biochemical remissions in a sizeable subset of MM patients. This indicates the presence of a distinctive drug-resistant sub-population of MM cells, which might be responsible for tumor re-growth. Methods and Results:To evaluate this phenomenon, we performed flow cytometry-based Hoechst 33342 staining assay to evaluate the existence and characteristics in MM of a population known as side population (SP), which has been identified in diverse neoplasias and has been reported to exhibit “stem-like” features. We specifically performed flow cytometry or ImageStream analyses to quantify the SP population in a panel of 18 MM cell lines. These analyses revealed heterogeneity in the proportion of SP fractions in different cell lines and indicated the lack of correlation between CD138 expression and SP fraction. We observed that, after sorting of the SP and non-SP fractions, SP cells have the potential to generate colonies in CFU assays, and regenerate a population resembling the original, pre-sorted, one. Moreover, SP cells had a high proliferation index compared to non-SP cells. We also observed that most MM cells lines express significantly higher levels of ABCG2 transcripts in their sorted SP fraction compared to their non-SP cells. The mRNA profile results were further corroborated by functional assays, where SP cells showed more pronounced efflux of the known ABCG2 substrate mitoxantrone, consistent with the association of SP phenotype with ABCG2 expression. Interestingly, at concentrations and durations of drug exposure that did not substantially affect the viability of non-SP cells, the immunomodulatory thalidomide derivative (IMiDs) lenalidomide significantly decreased the percentage and clonogenicity of SP cells. This response was accompanied by a distinct pattern of changes in diverse signaling pathways in SP cells (including phosphorylation changes in Akt, GSK-3a/b, MEK1, c-Jun, p53, p38 MAPK, p70S6K and STAT3) and increase of CD138-/low+ phenotype. Because the BM microenvironment is considered a key regulator of MM cell biological behavior, we evaluated the impact of bone marrow stromal cells (BMSCs) on the behavior of SP cells and observed that BMSCs led to increased percentage, viability, as well as proliferation potential of SP cells. Interestingly, IMiDs abrogated this stimulatory effect of stromal cells by significantly decreasing SP cell percentages. In these co-culture models, we identified several cytokines, including IL-1b, IL-10, IL-17; chemokines (MIP-1a, MIP-1b and IP-10) and growth factors such as GM-CSF and VEGF, which were up-regulated in co-culture MM cells with stromal cells compared to stroma alone or MM cells alone. Significant modulation in the production of these cytokines and chemokines was detected after IMiD treatment of MM cells only, as well as in co-culture model. Conclusion:Identifying the clonogenic population of SP cells could facilitate the development of new strategies targeting subpopulations of MM cells with drug resistant properties. Our study raises the intriguing possibility that IMiDs could represent such a strategy that can target SP cells and counteract their resistance to different conventional, and perhaps some novel, therapies. In addition, our data suggest that further studies of this tumor cell population are warranted towards to the goal of developing new strategies to treat MM. Disclosures:Laubach:Novartis: Consultancy, Honoraria. Richardson:Millenium: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

BackgroundTheca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK).MethodsOvarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells.ResultsAkt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA) does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor) suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca cells.ConclusionThese results indicate that LH stimulates CYP17 mRNA expression and androgen production in theca cells via activation of the PI3K/Akt pathway. The LH-induced Akt phosphorylation and androgen production are modulated by the MAPK signaling in bovine theca cells.

ROS-driven Akt dephosphorylation at Ser-473 is involved in 4-HPR-mediated apoptosis in NB4 cells

N-(4-hydroxyphenyl) retinamide (4-HPR), as a synthetic retinoid, has been shown to inhibit carcinogenesis in a variety of cancers. Extensive studies have indicated that ROS are involved in 4-HPR-mediated apoptosis. Herein, we provide further evidence that the Akt signaling pathway is involved in 4-HPR-mediated apoptosis. Of note is the fact that the expression of PI3K (p110) does not change obviously, and neither LY294002 nor insulin could influence the apoptosis induced by 4-HPR. These observations implicate the direct interaction between Akt and ROS. Our data also reveal that 4-HPR-mediated ROS evoke Akt conformational change by forming an intramolecular disulfide bond; N-acetylcysteine and glutathione, as thiol antioxidants, significantly abate the ROS generation in 4-HPR-exposed cells. Further experiments indicate that the conformational change in Akt not only disrupts Akt–Hsp90 binding, but also enhances Akt–PP2A interaction. All these results collectively suggest that 4-HPR-induced apoptosis is associated with a ROS-mediated conformational change in Akt, and this change, as a consequence, mediates dephosphorylation of Akt via regulating Akt–Hsp90 or Akt–PP2A complex formation.

Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano‐electrospray ionization mass spectrometry

Amide hydrogen exchange coupled to nano-electrospray ionization mass spectrometry (nano-ESI-MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D(2)O buffer, digested with pepsin, and analyzed by nano-ESI-MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four beta-strand (beta1, beta2 beta5 and beta6) regions containing membrane-binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these beta-strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning alpha-helix J region and the C-terminal locus (T450-470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival.

OVEREXPRESSION OF ALDEHYDE DEHYDROGENASE‐2 ATTENUATES CHRONIC ALCOHOL EXPOSURE‐INDUCED APOPTOSIS, CHANGE IN Akt AND Pim SIGNALLING IN LIVER

1. The liver, the main site of ethanol oxidation, is extremely vulnerable to the toxic effects of alcohol. Chronic alcohol intake has been shown to result in alcoholic liver disease, although the precise mechanism of action remains poorly understood. 2. The present study was designed to examine the impact of facilitated acetaldehyde metabolism via overexpression of aldehyde dehydrogenase-2 (ALDH2) on chronic alcohol ingestion-induced hepatic damage. Mice (wild-type Friend Virus B (FVB) and ALDH2 transgenic mice) were placed on a 4% alcohol or control diet for 12 weeks. Pro- and anti-apoptotic proteins, including p53, Omi/HtrA2, Bcl-2, Bax, X-linked inhibitor of apoptosis protein (XIAP), Akt, phosphorylated (p) Akt, the Akt downstream signalling molecule Pim and pPim, were examined using immunoblot analysis. Apoptosis and protein damage were assessed using the caspase 3 assay and protein carbonyl formation, respectively. 3. The data revealed that alcohol intake enhanced expression of p53, Omi/HtrA2, Bcl-2 and Bax without affecting XIAP expression or the Bcl-2/Bax ratio. Total Akt and pPim were downregulated in response to alcohol, whereas total Pim was upregulated in conjunction with unchanged pAkt. As a result, the pAkt : Akt and pPim : Pim ratios were elevated and reduced, respectively, in response to alcohol. All these effects that resulted from alcohol exposure were attenuated or ablated by ALDH2. 4. Collectively, the results suggest that ALDH2 may effectively ameliorate alcohol-induced hepatic apoptosis and changes in Akt as well as Pim signalling.

H017 Time dependent activation of AKT-GSK3beta and calcineurin signaling pathways during the development of TAC-induced left ventricular hypertrophy in B6D2/F1 mice

Left ventricular hypertrophy (LVH) is an adaptive response to chronic biomechanical stress that later progresses to maladaptive hypertrophy and heart failure. To better understand the mechanisms responsible for LVH development, we studied the activation of two major kinases, protein kinase B (Akt/PKB) and glycogen synthase kinase3b (GSK3b) and of a phosphatase, calcineurin (Cn) in an experimental model of thoracic aorta constriction (TAC). 4 week-old B6D2/F1 male, Sham-operated (Sham) and TAC mice were studied 3, 7, 15, 30 and 60 days post-TAC. Gene expression and activation of signalling pathways was studied by PCR and Western Blot. TAC-induced LVH (10 – 51 % at days 3-60, respectively) was associated with: 1) fetal gene reexpression characterised by an increase in mRNA levels of BNP (1.7-3 fold) and a-SK (2-4 fold), an a- to b-MHC switch seen at day 30 only ; 2) a significant increase in SERCA2a (1.7 fold), PLB, and NCX (3.6 fold) protein levels at day 7. 3) a 2 fold increase in P-Akt at day 3 and a decrease at day 15 while Akt was increased at days 7 and 15 ; 4) an increase in P-GSK3b at day 15 (1.5 fold), whereas no change in Akt and GSK3b protein levels were seen at days 30 and 60 ; 5) a significant decrease in CnAb at day 15 (2 fold). LVH develops progressively from day 3 in response to TAC, accompanied by re-expression of the fetal gene program. The alpha to beta-MHC mRNA switch occurs late, associated with the transition to HF, which occurs progressively with time. At days 3 and 15, P-Akt acts as an upstream regulator of GSK3b which may favour the development of LVH. 7 days post-TAC, a SERCA2a and NCX increase might contribute to decrease free cytosolic calcium level. At day 15, a further increase in LVH is associated with an increase in Cn activity and an increase GSK3b, which may counteract the hypertrophic response. Taken together, our data suggest a time-dependent cross-talk between Cn and GSK3b to modulate cardiac hypertrophic response to pressure overload.

New Mechanism of Rosiglitazone to Reduce Neointimal Hyperplasia

Mechanism of neointimal hyperplasia after vascular injury includes activation of signaling pathways and matrix metalloproteinases (MMPs) that are involved in cell proliferation and migration. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, was reported to inhibit neointimal hyperplasia in diabetic animals and humans. But the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone inhibited neointimal hyperplasia. The proliferation and survival of cultured rat VSMCs were reduced by rosiglitazone, which was mediated by inhibition of ERK and activation GSK-3beta, without change of Akt. The antiproliferative effect of rosiglitazone was reversed by GSK-3beta inactivation. The migration of VSMCs was also suppressed by rosiglitazone that inhibited the expression and activity MMP-9 through GSK-3beta activation. Thus migration of MMP-9(-/-) VSMCs from MMP-9 knockout mice was not affected by rosiglitazone. The underlying mechanism of MMP-9 suppression by rosiglitazone was that it inhibited NF-kappaB DNA binding activity, which was also dependent on GSK-3beta. In rat carotid artery, balloon injury significantly inactivated GSK-3beta with induction of MMP-9, which was effectively prevented by rosiglitazone. Thus, rosiglitazone significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of VSMCs at neointima, which was reversed by inactivation of GSK-3beta with adenoviral transfer of catalytically-inactive GSK-KM gene. Rosiglitazone activates GSK-3beta, which inhibits not only proliferation of VSMCs but also migration of VSMCs through blocking NF-kappaB-dependent MMP-9 activation.

Increased c-Jun-NH2-Kinase Signaling in Neurofibromatosis-1 Heterozygous Microglia Drives Microglia Activation and Promotes Optic Glioma Proliferation

Neurofibromatosis-1 (NF1) is a common tumor predisposition syndrome in which affected individuals develop benign and malignant tumors. Previous studies from our laboratory and others have shown that benign tumor formation in Nf1 genetically engineered mice (GEM) requires a permissive tumor microenvironment. In the central nervous system, Nf1 loss in glia is insufficient for glioma formation unless coupled with Nf1 heterozygosity in the brain. Our subsequent studies identified Nf1+/- microglia as a critical cellular determinant of optic glioma growth in Nf1 GEM. Using NF1 as an experimental paradigm to further characterize the role of microglia in glioma growth, we first examined the properties of Nf1+/- microglia in vitro and in vivo. Nf1+/- microglia exhibit increased proliferation and motility and express elevated levels of genes associated with microglia activation. We further show that Nf1+/- microglia harbor high levels of activated c-Jun-NH(2)-kinase (JNK) without any significant changes in Akt, mitogen-activated protein kinase (MAPK), or p38-MAPK activity. In contrast, Nf1-/- astrocytes do not exhibit increased JNK activation. SP600125 inhibition of JNK activity in Nf1+/- microglia results in amelioration of the increased proliferation and motility phenotypes and reduces the levels of expression of activated microglia-associated transcripts. Moreover, SP600125 treatment of Nf1 optic glioma-bearing GEM results in reduced optic glioma proliferation in vivo. Collectively, these findings suggest that Nf1+/- microglia represent a good model system to study the role of specialized microglia in brain tumorigenesis and identify a unique Nf1 deregulated pathway for therapeutic studies aimed at abrogating microenvironmental signals that promote brain tumor growth.

P4-251: Changes in Akt and IGF-1 receptor signaling relate to developing Abeta and tangle pathology in the triple transgenic model of Alzheimer's disease

P4-251: Changes in Akt and IGF-1 receptor signaling relate to developing Abeta and tangle pathology in the triple transgenic model of Alzheimer's disease

Chronic late-gestation hypoglycemia upregulates hepatic PEPCK associated with increased PGC1α mRNA and phosphorylated CREB in fetal sheep

Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA and phosphorylation of cAMP response element binding protein at Ser(133) were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4alpha, or CCAAT enhancer binding protein-beta. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver.

Live-cell Molecular Analysis of Akt Activation Reveals Roles for Activation Loop Phosphorylation

Activation of the serine/threonine protein kinase Akt/PKB is a multi-step process involving membrane recruitment, phosphorylation, and membrane detachment. To investigate this process in the cellular context, we employed a live-cell fluorescence imaging approach to examine conformational changes of Akt and its membrane association. A fluorescence resonance energy transfer-based reporter of Akt action (ReAktion) reveals a conformational change that is critically dependent on the existence of a phosphorylatable threonine 308 in the activation loop, because mutations to either aspartate or alanine abolished the change. Furthermore, a mutant carrying a phosphorylation mimic at this position showed diminished membrane association, suggesting that this phosphorylation plays an important role of promoting the dissociation of activated Akt from the membrane. In addition, the membrane-associating pleckstrin homology domain was found to associate with the catalytic domain when Thr308 is phosphorylated, suggesting such an interdomain interaction as a mechanism by which phosphorylation within the catalytic domain can affect membrane association. These studies uncover new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

Alcohol Accelerates Loss of Muscle and Impairs Recovery of Muscle Mass Resulting From Disuse Atrophy

Muscle disuse atrophy is observed in patients recovering from trauma and there is an increased risk and severity of injury in patients abusing alcohol (EtOH). However, the interaction of EtOH and disuse on muscle protein balance has not been examined. Therefore, the present study addressed the hypothesis that EtOH accelerates the disuse atrophy and/or impairs the accretion of muscle protein during muscle recovery. To address this aim, disuse atrophy was induced in rats by 3 days of unilateral hindlimb immobilization (casting), using the contralateral leg as control, with EtOH or saline being orally gavaged twice, each day during this period. In a separate study, EtOH-treated rats received Velcade to inhibit proteasomal degradation. Finally, in the last study, rats had 1 limb casted for 5 days, the cast removed, and EtOH or saline gavaged twice daily during a 5-day recovery period. Muscle protein metabolism was assessed using surrogate markers of protein synthesis [i.e., phosphorylation of 4E-binding protein 1 (BP1) and S6 kinase 1 (S6K1)] and protein degradation (i.e., mRNA content of the ubiquitin E3 ligases atrogin-1 and MuRF1). Ethanol alone did not decrease muscle weight in the uncasted muscle. However, the loss of mass of immobilized muscle from EtOH-gavaged rats was 80% greater than in the animals not receiving EtOH. This atrophic response was not associated with a change in Akt, 4E-BP1 or S6K1 phosphorylation among groups. In contrast, immobilization alone increased both atrogin-1 and MuRF1 mRNA, and EtOH further increased their expression in immobilized muscle. The proteasome inhibitor Velcade attenuated atrophy produced by EtOH + disuse. When administered during the recovery period, EtOH prevented the normal accretion of muscle mass. This EtOH effect was associated with increased atrogin-1 mRNA, a reduction in 4E-BP1 and S6 phosphorylation, and an increased AMP-activated kinase phosphorylation. Based on the changes in these surrogate markers, our data suggest that EtOH accelerates disuse atrophy by stimulating ubiquitin-mediated proteolysis, and blunts repletion of muscle protein during recovery from disuse by increasing proteolysis and decreasing protein synthesis.