Phosphoinositol 3-kinase Pathway Research Articles

Atrial natriuretic peptide inhibits angiotensin II‐stimulated proliferation in fetal cardiomyocytes

The role of atrial natriuretic peptide (ANP) in regulating fetal cardiac growth is poorly understood. Angiotensin II (Ang II) stimulates proliferation in fetal sheep cardiomyocytes when growth is dependent on the activity of the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. We hypothesized that ANP would suppress near-term fetal cardiomyocyte proliferation in vitro and inhibit both the MAPK and PI3K pathways. Forty-eight hour 5-bromodeoxyuridine (BrdU) uptake (used as an index of proliferation) was measured in cardiomyocytes isolated from fetal sheep (135 day gestational age) in response to 100 nm Ang II with or without ANP (0.003-100 nm) or 1 microm 8-bromo-cGMP. The effects of these compounds on the MAPK and PI3K pathways were assessed by measuring extracellular signal-regulated kinase (ERK) and AKT phosphorylation following 10 min of treatment with Ang II, ANP or 8-bromo-cGMP. In right ventricular myocytes (RV), the lowest dose of ANP (0.003 nm) inhibited Ang II-stimulated BrdU uptake by 68%. Similarly, 8-bromo-cGMP suppressed Ang II-stimulated proliferation by 62%. The same effects were observed in left ventricular (LV) cardiomyocytes but the RV was more sensitive to the inhibitory effects of ANP than the LV (P < 0.0001). Intracellular cGMP was increased by 4-fold in the presence of 100 nm ANP. Ang II-stimulated ERK and Akt phosphorylation was inhibited by 100 nm ANP. The activity of ANP may in part be cGMP dependent, as 8-bromo-cGMP had similar effects on the cardiomyocytes.

Abstract 1650: Targeting of compensatory signaling pathways identified in a synthetic lethal screen leads to synergistic growth inhibition in bladder cancer cells

Abstract Despite promising preclinical results, monotherapies that target deregulated signal transduction pathways often are insufficient to slow or shrink tumors in human patients. We hypothesize that multiple pathways within the cellular signaling network are altered in cancer cells, and that these pathways are acting in a redundant or compensatory manner to provide resistance against drugs targeting a single protein. A treatment plan consisting of multiple small molecules that inhibit these compensatory and redundant pathways should overcome this resistance. To discover pathways that act in a redundant or compensatory manner, we performed a screen that allows us to examine the cellular effects of blocking pathways both individually and in combination. We found that the combination of the epidermal growth factor receptor (EGFR) inhibitor lapatinib in combination with the phosphoinositol-3-kinase (PI3K) inhibitor LY294002 produced synergistic growth inhibition in UMUC-6 bladder cancer cells. In order to confirm that this effect was due to inhibition of the putative targets of these compounds, we substituted structurally distinct EGFR and PI3K inhibitors for lapatinib and LY294002 and obtained similar results. Cell cycle analysis of cells treated with this combination revealed an arrest in the G1 phase, which could explain the synergistic inhibition observed in the screen. In order to find a molecular mechanism for this synergistic effect, we performed Western blotting analysis of the cells treated with each drug individually and in combination. We found that treatment with LY294002 caused an increase in levels of phosphorylated SHC, and ERK, indicating that receptor tyrosine kinase signaling, and possibly EGFR signaling is activated to compensate for PI3K pathway inhibition. Inhibition of ERK activity with PD325901 produced the same effect as EGFR inhibition, confirming the role of ERK as a compensatory signaling molecule for PI3K inhibition. Parallel RNAi experiments against SHC are currently ongoing. These data suggest that synthetic lethal screening with a library of targeted inhibitors of signaling proteins can be used to search for combinatorial effects and functionally identify compensatory and redundant relationships between different signaling components. This work paves the way for the development of rational combinatorial therapies for the effective treatment of bladder cancer. 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 1650.

Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing links. The Claude Bernard Lecture 2009.

Insulin resistance is a hallmark of type 2 diabetes mellitus and is associated with a metabolic and cardiovascular cluster of disorders (dyslipidaemia, hypertension, obesity [especially visceral], glucose intolerance, endothelial dysfunction), each of which is an independent risk factor for cardiovascular disease (CVD). Multiple prospective studies have documented an association between insulin resistance and accelerated CVD in patients with type 2 diabetes, as well as in non-diabetic individuals. The molecular causes of insulin resistance, i.e. impaired insulin signalling through the phosphoinositol-3 kinase pathway with intact signalling through the mitogen-activated protein kinase pathway, are responsible for the impairment in insulin-stimulated glucose metabolism and contribute to the accelerated rate of CVD in type 2 diabetes patients. The current epidemic of diabetes is being driven by the obesity epidemic, which represents a state of tissue fat overload. Accumulation of toxic lipid metabolites (fatty acyl CoA, diacylglycerol, ceramide) in muscle, liver, adipocytes, beta cells and arterial tissues contributes to insulin resistance, beta cell dysfunction and accelerated atherosclerosis, respectively, in type 2 diabetes. Treatment with thiazolidinediones mobilises fat out of tissues, leading to enhanced insulin sensitivity, improved beta cell function and decreased atherogenesis. Insulin resistance and lipotoxicity represent the missing links (beyond the classical cardiovascular risk factors) that help explain the accelerated rate of CVD in type 2 diabetic patients.

Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth Factor-I/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Genetic Disruption of the PI3K Regulatory Subunit, p85α Partially Normalizes Gain-of-Function PTPN11-Induced Hypersensitivity to GM-CSF in Hematopoietic Progenitors.

Abstract 3968Poster Board III-904Juvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPD) of children, characterized by hyperproliferation of myelomonocytic cells and hypersensitivity to Granulocyte-Monocyte Colony-Stimulating Factor (GM-CSF). Most patients show hyperactivation of Ras via one or more mutations, including in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. It has been demonstrated that gain-of-function mutant Shp2 (Shp2 E76K and Shp2 D61Y) causes hyperactivation of the Mitogen-Activated Protein Kinase (MAPK) pathway. Additionally, we have previously shown that macrophage progenitor cells transduced with Shp2 D61Y or Shp2 E76K showed elevated levels of phospho-Akt, both at baseline and following 1 hour of GM-CSF stimulation, indicating a role for the Phospho-Inositol-3-Kinase (PI3K)/Akt pathway in the phenotype of elevated proliferation and survival in mutant Shp2-expressing cells (Yang, et al, 2008). However, it remains to be elucidated how PI3K contributes to the phenotype of increased proliferation and survival in cells bearing gain-of-function mutations in Shp2. Class IA PI3K is a lipid kinase heterodimer consisting of one of three catalytic subunits (p110α, p110β, or p110δ) and one of two regulatory subunits (p85α or p85β). It has been demonstrated that knocking out the main regulatory subunit, p85α, abrogated the hyperproliferative phenotype in mast cell progenitors bearing an oncogenic mutation in Kit in a model of another MPD, Systemic Mastocytosis (Munugalavadla, et al, 2007). In order to examine whether eliminating expression of p85α would cause a similar correction in cells expressing gain-of-function mutant Shp2, we performed timed matings of mice heterozygous for the knock-out of Pik3r1, which encodes the p85α subunit as well as its isoforms, p55α and p50α, since homozygous Pik3r1-/- is lethal in utero. WT and Pik3r1-/- fetal liver cells were isolated from 14.5 day embryos and transduced with either WT Shp2 or mutant Shp2 E76K. Transduced cells were subjected to serum deprivation followed by a 24-hour treatment with increasing doses of GM-CSF, and proliferation was then measured with H3-thymidine incorporation assays. We found that the absence of all the Pik3r1 isoforms resulted in a significant but incomplete correction of GM-CSF hypersensitivity in Shp2 E76K-expressing cells. To further investigate this observation, WT Pik3r1 and Pik3r1-/- macrophage progenitors, transduced with WT Shp2 or mutant Shp2 E76K as described above, were serum- and growth factor-deprived and then stimulated for 1 hour with GM-CSF. Western blot analysis showed that there was an expected increase in phospho-Akt in WT Pik3r1 cells following GM-CSF stimulation and that this increase was larger in Shp2 E76K-expressing cells than in WT Shp2-expressing cells, as previously observed. Upon genetic disruption of Pik3r1, Akt activation was reduced in both WT Shp2- and Shp2 E76K-expressing cells; however, the phospho-Akt in the Shp2 E76K-expressing cells was not reduced to WT levels. The phospho-Akt levels mirrored the proliferation pattern displayed by these cells in the H3-thymidine incorporation assays, where a modest reduction in proliferation in Pik3r1-/-, Shp2 E76K cells corresponded to the modest reduction in phospho-Akt levels in the same cells. Additionally, we found that Pik3r1-/-, Shp2 E76K cells also showed a blunted increase in phospho-Erk levels following GM-CSF stimulation compared to the WT Pik3r1, Shp2 E76K cells, indicating that knocking out Pik3r1 affects the MAPK pathway as well, which likely also contributes to the reduction in proliferation seen in Pik3r1-/-, Shp2 E76K cells following GM-CSF stimulation. Based on these data, we conclude that: (1) gain-of-function Shp2 activity results in dysregulated PI3K signaling, contributing to the leukemic phenotype of increased proliferation and survival; (2) PI3K signaling is reduced but not completely normalized in the absence of the main regulatory subunit, p85α and its isoforms, in gain-of-function mutant Shp2-expressing cells; and (3) there is cross-talk between the PI3K and MAPK pathways in the presence of Shp2 activating mutations, which is revealed by knocking out Pik3r1. Disclosures:No relevant conflicts of interest to declare.

Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) Directs Prostaglandin E2-mediated Fibroblast Responses via Regulation of E Prostanoid 2 Receptor Expression

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite that counters transforming growth factor-beta-induced fibroblast activation via E prostanoid 2 (EP2) receptor binding. Phosphatase and tensin homologue on chromosome 10 (PTEN) is a lipid phosphatase that, by antagonizing the phosphoinositol 3-kinase (PI3K) pathway, also inhibits fibroblast activation. Here, we show that PTEN directly regulates PGE(2) inhibition of fibroblast activation by augmenting EP2 receptor expression. The increase in collagen production and alpha-smooth muscle actin expression observed in fibroblasts in which PTEN is deficient was resistant to the usual suppressive effects of PGE(2). This was due to marked down-regulation of EP2, a G(s) protein-coupled receptor (GPCR) that mediates the inhibitory actions of this prostanoid via cAMP. pten(-/-) or PTEN-inhibited fibroblasts in which the PI3K pathway was blocked demonstrated a restoration of EP2 receptor expression, due to augmented gene transcription and mRNA instability. Importantly, restoration of the balance between PI3K and PTEN reestablished the inhibitory effect of PGE(2) on fibroblast activation. No such influence of PTEN was observed on alternative E prostanoid GPCRs. Moreover, our studies identified a positive feedback loop in which cAMP signaling enhanced EP2 receptor expression, independent of PTEN. Therefore, our findings indicate that PTEN regulates the antifibrotic effects of PGE(2) by a specific and permissive effect on EP2 receptor expression. Further, our data imply that cAMP signaling circumvents EP2 down-regulation in pten-deficient cells to restore EP2 receptor expression. This is the first description, to our knowledge, of PI3K/PTEN balance directing GPCR expression, and provides a novel mechanism for cellular effects of PTEN.

Thyroid-stimulating hormone/cAMP-mediated proliferation in thyrocytes

Current research on thyrotropin-activated proliferation in the thyrocyte needs to be aimed at a better understanding of crosstalk and negative-feedback mechanisms with other proliferative pathways, especially the insulin/IGF-1-induced phosphoinositol-3 kinase pathway and the serum-induced MAPK or Wnt pathways. Convergence of proliferative pathways in mTOR is a hotspot of current research, and combined treatment using double class inhibitors for thyroid cancer may bring some success. New thyroid-stimulating hormone receptor (TSHR)-interacting proteins, a better picture of cAMP targets, a deeper knowledge of the action of the protein kinase A regulatory subunits, especially their interactions with the replication machinery, and a further understanding of mechanisms that lead to cell cycle progression through G1/S and G2/M checkpoints are areas that need further elucidation. Finally, massive information coming from microarray data analysis will prompt our understanding of thyroid-stimulating hormone-promoted thyrocyte proliferation in health and disease.

Upregulation of Hypoxia-Inducible Factors in Normal and Psoriatic Skin

Angiogenesis induced by vascular endothelial growth factor (VEGF) plays an important role in psoriasis. Hypoxic adaptation is conferred through hypoxia-inducible transcription factors (HIFs). VEGF and its receptor Flt-1 are HIF target genes. Growth factors and inflammatory cytokines activate the phosphoinositol-3 kinase pathway, and via activated protein kinase B (phospho-Akt) augment HIF activity. Here, we demonstrate that the major oxygen-dependent HIF isoforms are strongly upregulated in psoriatic skin: HIF-1alpha mainly in the epidermis, in an expression pattern similar to VEGF mRNA; HIF-2alpha in both the epidermis and in capillary endothelial cells of the dermis. In contrast, normal human skin shows low expression of HIF-alpha proteins, with the exception of hair follicles, and glands, which strongly express HIF-1alpha. In normal human skin, phospho-Akt appeared in the basal epidermal layer, in hair follicles, and in dermal glands. In contrast, in psoriasis, phospho-Akt expression was low in the epidermis, but markedly enhanced in the dermal capillaries and in surrounding interstitial/inflammatory cells. Our data suggest that hypoxia initiates a potentially self-perpetuating cycle involving HIF, VEGF, and Akt activation, which could drive physiologic growth of hair follicles and skin glands. Furthermore, such a cycle may exist in psoriasis in dermal capillaries and contribute to disease progression.

Protection against β-Amyloid-induced Apoptosis by Peptides Interacting with β-Amyloid

beta-Amyloid peptide produces apoptosis in neurons at micromolar concentrations, but the mechanism by which beta-amyloid exerts its toxic effect is unknown. The normal biological function of beta-amyloid is also unknown. We used phage display, co-precipitation, and mass spectrometry to examine the protein-protein interactions of beta-amyloid in normal rabbit brain in order to identify the biochemical receptors for beta-amyloid. beta-Amyloid was found to bind primarily to proteins involved in low density lipoprotein and cholesterol transport and metabolism, including sortilin, endoplasmic reticulum-Golgi intermediate compartment 2 (ERGIC2), ERGIC-53, steroid 5alpha-reductase, and apolipoprotein B. beta-Amyloid also bound to the C-reactive protein precursor, a protein involved in inflammation, and to 14-3-3, a protein that regulates glycogen synthase kinase-3beta, the kinase involved in tau phosphorylation. Of eight synthetic peptides identified as targets of beta-amyloid, three were found to be effective blockers of the toxic effect of beta-amyloid on cultured neuronal cells. These peptides bound to the hydrophobic region (residues 17-21) or to the nearby protein kinase C pseudo-phosphorylation site (residues 26-30) of beta-amyloid, suggesting that these may be the most critical regions for beta-amyloid effector action and for aggregation. Peptides or other small molecules that bind to this region may protect against beta-amyloid toxic effect by competitively blocking its ability to bind beta-amyloid effector proteins such as sortilin and 14-3-3.

Mitochondrial Reactive Oxygen Species Signal Hepatocyte Steatosis by Regulating the Phosphatidylinositol 3-Kinase Cell Survival Pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.

Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. We have previously shown that PTHrP increases colon cancer cell proliferation, extracellular matrix adhesion, and cell-surface integrin α6β4 expression. Since cancer cell migration, invasion, and survival are crucial components of metastasis, and colon cancer has a high metastatic potential, in this study we used the human colon cancer cell line LoVo as a model system to study the effects of PTHrP on these parameters. PTHrP expression was modulated by stable transfection with a construct expressing PTHrP (− 36 to + 139). We report that PTHrP increases cell migration, invasion, and survival. PTHrP altered cell morphology, with PTHrP-overexpressing cells exhibiting increased spreading and several long protrusions. PTHrP also increased the steady-state mRNA levels of the integrin α6 and β4 subunits, indicating a direct and/or indirect effect of PTHrP on the transcriptional and/or post-transcriptional regulation of integrin α6 and β4 expression. Integrin α6β4 activates the phosphoinositol 3-kinase (PI3-K)/Akt pathway, leading to glycogen synthase kinase-3 (GSK-3) deactivation. PTHrP overexpression also led to an increase in active Akt and inactive GSK-3 levels, indicating that the PTHrP-mediated upregulation of integrin α6β4 expression may activate the PI3-K pathway, resulting in increased cell survival, migration and invasion.

Role of activator protein-1 on the effect of arginine-glycine-aspartic acid containing peptides on transforming growth factor-β1 promoter activity

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-β1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-β1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-β1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-β1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

S4-02-04: From neuropsychology to neuroimaging in neurodegenerative diseases

not available. S4-02-06 REGULATORY PERSPECTIVE OF MCI Karl Broich, Federal Institute of Drugs and Medical Devices, Bonn, Germany. Contact e-mail: k.broich@bfarm.de Abstract not available. WEDNESDAY, JULY 19, 2006 SYMPOSIA S4-03 DISEASE MECHANISMS (SIGNAL TRANSDUCTION)not available. WEDNESDAY, JULY 19, 2006 SYMPOSIA S4-03 DISEASE MECHANISMS (SIGNAL TRANSDUCTION) S4-03-01 EVIDENCE THAT ABERRANT POST-TGN TRAFFICKING OF ALZHEIMER’S APP IS SUFFICIENT TO ELEVATE BRAIN A-BETA 42 AND MAY UNDERLIE MUTANT PRESENILIN ACTION Sam Gandy, Yun-wu Zhang, Annat Ikin, Stephen Schmidt, Roxanne Sheffield, Ralph Nixon, Francesca-Fang Liao, Paul Matthews, Huaxi Xu, Michelle Ehrlich, Farber Institute for Neurosciences at Thomas Jefferson University, Philadelphia, PA, USA; Burnham Institute, La Jolla, CA, USA; Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: samgandy@earthlink.net Mutations in presenilin 1 (PS1) cause familial Alzheimer’s disease (FAD) by increasing cerebral amyloidogenic A-beta peptide ratios, expressed as A-beta 42/40 42. We investigated A-beta speciation in mice expressing an A-beta precursor protein (APP):furin chimera highly concentrated in the neuronal trans-Golgi network (TGN). Brain A-beta 42/40 42 ratios from APP:furin mice were similar to reported ratios associated with FADmutant PS1, lending plausibility to the model that altered trafficking of APP out of the TGN could underlie PS1-linked FAD. The association of mutant PS1 with delayed TGN exit was confirmed using a cell-free assay based on APP transport vesicle biogenesis from TGN-rich fractions prepared from cerebrocortical neurons of PS1 M146V knock-in mice. In intact neurons, reduced cell-surface arrival of newly synthesized APP was associated with the presence of PS1 M146V. These data support the notion that selective localization of APP to the TGN is sufficient to elevate the A-beta 42/40 42 ratio, and that delayed exit of APP from the cerebrocortical neuronal TGN is a phenotype associated with the presence of mutant PS1. S4-03-02 ROLE OF P70 S6 KINASE IN ALZHEIMER’S DISEASE Jin Jing Pei, Wen-Lin An, Cecilia Bjorkdahl, Xu Li, Xinwen Zhou, Bengt Winblad, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: Jin-Jing.Pei@neurotec.ki.se The cytoplasmic isoform of ribosomal S6 kinase 1 (RSK1), the 70-kDa S6 kinase (p70S6K), is a Ser/Thr (S/T) -directed kinase that regulates the phosphorylation of the 40S ribosomal protein S6. Phosphorylation and activation of p70S6K have been suggested to be mediated by both phosphoinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Our previous studies indicated that both PI3K and MAPK pathways are aberrantly regulated in Alzheimer’s disease (AD) brains. In the study, we wanted to investigate the role of p70S6K in AD pathogenesis. In a series of studies, we found: 1) an increased immunoreactivity of the phosphorylated (p) / activated form of p70S6K, and its upstream kinase: mammalian target of rapamycin, in AD as compared with control brains; 2) a colocalization of p-p70S6K with PHF-tau in neurons bearing neurofibrillary tangles (NFTs) and pre-tangles; 3) that p70S6K activation and tau hyperphosphorylation can be induced by a selective protein phosphatase (PP) -2A inhibition in rat brain slices, as well as 100 M zinc in SH-SY5Y cells and primary cultured neurons; 4) that phosphorylation and activation of p70S6K are preferentially regulated by the PI3K pathway, together with the MAPK pathway; 5) that p70S6K can mediate tau phosphorylation and synthesis; and 6) that p70S6K activation does not increase phosphorylation of neurofilament. These suggested p70S6K might play a significant role in tau abnormalities such as hyperphosphorylation, accumulation, assembly into paired helical filaments and neurofibrillary tangle formation, accompanied by microtubule disruption. Activation of p70S6K in AD might be caused by either upregulated PI3K and MAPK pathways, or downregulated PP-2A. S71 Symposia S4-03: Disease Mechanisms (Signal Transduction)

Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity

Deprivation of estrogen causes breast tumors in women to adapt and develop enhanced sensitivity to this steroid. Accordingly, women relapsing after treatment with oophorectomy, which substantially lowers estradiol for a prolonged period, respond secondarily to aromatase inhibitors with tumor regression. We have utilized in vitro and in vivo model systems to examine the biologic processes whereby long-term estradiol deprivation (LTED) causes cells to adapt and develop hypersensitivity to estradiol. Several mechanisms are associated with this response, including up-regulation of estrogen receptor-alpha (ERalpha) and the MAP kinase, phosphoinositol 3 kinase (PI3-K) and mammalian target of rapamycin (mTOR) growth factor pathways. ERalpha is four- to tenfold up-regulated and co-opts a classical growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. The molecules involved in the non-genomic signaling process have been identified. Estradiol binds to cell membrane-associated ERalpha, which physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which result in the rapid activation of MAP kinase. These non-genomic effects of estradiol produce biologic effects as evidenced by Elk-1 activation and by morphologic changes in cell membranes. Additional effects include activation of the PI3-K and mTOR pathways through estradiol-induced binding of ERalpha to the IGF-I and epidermal growth factor receptors. A major question is how ERalpha locates in the plasma membrane since it does not contain an inherent membrane localization signal. We have provided evidence that the IGF-I receptor serves as an anchor for ERalpha in the plasma membrane. Estradiol causes phosphorylation of the adaptor protein, Shc and the IGF-I receptor itself. Shc, after binding to ERalpha, serves as the 'bus' which carries ERalpha to Shc-binding sites on the activated IGF-I receptors. Use of small inhibitor (si) RNA methodology to knockdown Shc allows the conclusion that Shc is needed for ERalpha to localize in the plasma membrane. In order to abrogate growth factor-induced hypersensitivity, we have utilized a drug, farnesylthiosalicylic acid, which blocks the binding of GTP-Ras to its membrane acceptor protein, galectin 1, and reduces the activation of MAP kinase. We have also shown that this drug is a potent inhibitor of mTOR as an additional mechanism of inhibition of cell proliferation. The concept of 'adaptive hypersensitivity' and the mechanisms responsible for this phenomenon have important clinical implications. The efficacy of aromatase inhibitors in patients relapsing on tamoxifen could be explained by this mechanism and inhibitors of growth factor pathways should reverse the hypersensitivity phenomenon and result in prolongation of the efficacy of hormonal therapy for breast cancer.

Transient Phosphatidylinositol 3-Kinase Inhibition Protects Immature Primary Cortical Neurons from Oxidative Toxicity via Suppression of Extracellular Signal-regulated Kinase Activation

Oxidative stress has been shown to underlie a diverse range of neuropathological conditions. Glutamate-induced oxidative toxicity is a well described model of oxidative stress-induced neurodegeneration that relies upon the ability of extracellular glutamate to inhibit a glutamate/cystine antiporter, which results in a depletion of intracellular cysteine and the blockade of continued glutathione synthesis. Glutathione depletion leads to a gradual toxic accumulation of reactive oxygen species. We have previously determined that glutamate-induced oxidative toxicity is accompanied by a robust increase in activation of the mitogen-activated protein kinase (MAPK) member extracellular-signal regulated kinase (ERK) and that this activation is essential for neuronal cell death. This study demonstrates that delayed ERK activation is dependent upon the activity of phosphoinositol-3 kinase (PI3K) and that transient but not sustained PI3K inhibition leads to significant protection of neurons from oxidative stress-induced neurodegeneration. Furthermore, we show that transient PI3K inhibition prevents the delayed activation of MEK-1, a direct activator of ERK, during oxidative stress. Thus, this study is the first to demonstrate a novel level of cross-talk between the PI3K and ERK pathways in cultured immature cortical neuronal cultures that contributes to the unfolding of a cell death program. The PI3K pathway, therefore, may serve opposing roles during the progression of oxidative stress in neurons, acting at distinct kinetic phases to either promote or limit a slowly developing program of cell death.

Signalling pathways involved in the short-term potentiation of dopamine release by BDNF

Brain-derived neurotrophic factor (BDNF) has been shown to modulate synaptic plasticity in the corpus striatum in vitro by activation of the tyrosine kinase linked receptor, TrkB. However, the signalling pathways that mediate this modulation of plasticity are poorly understood. Three proteins mediating signalling pathways are activated by the binding of BDNF to TrkB: phosphoinositol-3 kinase (PI3K); Ras-MEK and phospholipase C-γ (PLCγ). The present study investigates which of these pathways are necessary for BDNF-mediated potentiation of synaptic output of dopamine from slices and synaptosomes of rat corpus striatum. The results indicate that activation of the PI3K and Ras-MEK pathways, but not PLCγ, are involved. Inhibitors of transcription and translation had no effect on the potentiation of depolarisation-stimulated (15 mM KCl) dopamine release mediated by BDNF.

Insulinlike growth factor–I signaling in multiple myeloma: downstream elements, functional correlates, and pathway cross-talk

In multiple myeloma cells, insulinlike growth factor-I (IGF-I) activates 2 distinct signaling pathways, mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI-3K), leading to both proliferative and antiapoptotic effects. However, it is unclear through which of these cascades IGF-I regulates these different responses. The present studies identify a series of downstream targets in the PI-3K pathway, including glycogen synthase kinase-3beta, p70S6 kinase, and the 3 members of the Forkhead family of transcription factors. The contribution of the MAPK and PI-3K pathways and, where possible, individual elements to proliferation and apoptosis was evaluated by means of a series of specific kinase inhibitors. Both processes were regulated almost exclusively by the PI-3K pathway, with only minor contributions associated with the MAPK cascade. Within the PI-3K cascade, inhibition of p70S6 kinase led to significant decreases in proliferation and protection from apoptosis. Activation of p70S6 kinase could also be prevented by MAPK inhibitors, indicating regulation by both pathways. The Forkhead transcription factor FKHRL1 was observed to provide a dual effect in that phosphorylation upon IGF-I treatment resulted in a loss of ability to inhibit proliferation and induce apoptosis. The PI-3K pathway was additionally shown to exhibit cross-talk and to regulate the MAPK cascade, as inhibition of PI-3K prevented activation of Mek1/2 and other downstream MAPK elements. These results define important elements in IGF-I regulation of myeloma cell growth and provide biological correlates critical to an understanding of growth-factor modulation of proliferation and apoptosis.

Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction.

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPKpathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.

Insulin-like Growth Factor-1-mediated Neuroprotection against Oxidative Stress Is Associated with Activation of Nuclear Factor κB

The role of insulin-like growth factor 1 (IGF-1) for the treatment of neurodegenerative disorders, such as Alzheimer's disease, has recently gained attention. The present study demonstrates that IGF-1 promotes the survival of rat primary cerebellar neurons and of immortalized hypothalamic rat GT1-7 cells after challenge with oxidative stress induced by hydrogen peroxide (H2O2). Neuroprotective concentrations of IGF-1 specifically induce the transcriptional activity and the DNA binding activity of nuclear factor kappaB (NF-kappaB), a transcription factor that has been suggested to play a neuroprotective role. This induction is associated with increased nuclear translocation of the p65 subunit of NF-kappaB and with degradation of the NF-kappaB inhibitory protein IkappaBalpha. IGF-1-mediated protection of GT1-7 cells against oxidative challenges was mimicked by overexpression of the NF-kappaB subunit c-Rel. Partial inhibition of NF-kappaB baseline activity by overexpression of a dominant-negative IkappaBalpha mutant enhanced the toxicity of H2O2 in GT1-7 cells. The pathway by which IGF-1 promotes neuronal survival and activation of NF-kappaB involves the phosphoinositol (PI) 3-kinase, because both effects of IGF-1 are blocked by LY294002 and wortmannin, two specific PI 3-kinase inhibitors. Taken together, our results provide evidence for a novel molecular link between IGF-1-mediated neuroprotection and induction of NF-kappaB that is dependent on the PI 3-kinase pathway.