Jia Luo, Ph.D. - US grants

growth factor receptors; protein tyrosine kinase; epidermal growth factor; ethanol; breast neoplasms; heregulin; neoplastic process; enzyme activity; phosphorylation; antisense nucleic acid; cell growth regulation; neoplasm /cancer pharmacology; chemical carcinogenesis; oligonucleotides; phosphoproteins; receptor expression; cell line; northern blottings;

APPLICANT'S ABSTRACT: Ethanol is a potent teratogen for the developing central nervous system (CNS). Prenatal ethanol exposure disrupts the proliferative activities of neuronal precursors and glia. Analysis of cell cycle kinetics indicate that both in vivo and in vitro ethanol treatment prolong the duration of cell cycle and in particular, the length of the G1- phase. The movement of cells through the cell cycle is regulated by a family of protein kinases known as cyclin-dependent kinases (CDKs). The activity of CDKs is regulated positively by cyclins, and negatively by CDK inhibitors (CKIS). CDK activity is controlled by extracellular signal-related kinases (ERKs). Ethanol can affect ERK activity. We hypothesize (1) that ethanol- induced inhibition of cell proliferation results from disruptions of CDK systems, and (2) that ERK mediates ethanol-induced alternations in CDK systems. The proposed project will rely on two in vitro models, B104 neuroblastoma cells and primary cortical astrocyytes, to examine the effects of ethanol on the ERK and CDK systems. The studies will investigate the effects of ethanol on the activity of ERKS. Other experiments will examine the effects of ethanol (a) on the expression and activity of G1-phase-specific CDKs and (b) the effects of ethanol on the balance between positive (cyclins) and negative (CKIS) regulators of CDKs. Each of the experiments on ethanol-induced alterations of CDK system will be performed with cells in which ERK activity is intact and depleted (using a specific ERK blocker). Thus, we will be able to determine whether the CDK activity is ERK-dependent. Together, the battery of studies represents a systematic investigation of the effects of ethanol on CDK systems and related signal pathways. It will not only explore the mechanism(s) underlying ethanol-induced cell cycle damage, but also provide an important insight into the regulation of cell cycle in neural cells.

DESCRIPTION (provided by applicant): Ethanol, a teratogen, results in central nervous system (CNS) dysfunction when developmental exposure occurs. One consequence of early ethanol exposure is irreversible depletion of neurons. However, the molecular mechanisms underlying ethanol teratogenecity remain unknown. In searching for target genes of ethanol, we identified that a gene encoding the ribosomal large subunit protein (rpL7a) is ethanol-responsive. At physiologically relevant concentrations, ethanol up-regulates the expression of rpL7a in cultured neuronal cells as well as in the developing cerebellum. The expression of rpL7a is dynamically regulated during neuronal development and ectopic over-expression of rpL7a activates p53 and induces neuronal apoptosis. Ribosomal proteins participate in various extra-ribosomal activities; rpL7a is clearly one of the ribosomal proteins involved in multiple extra-ribosomal functions. Our hypothesis is that ethanol-induced ectopic over-expression of rpL7a causes neuronal death and disruption of cell cycle kinetics. Three sets of in vitro experiments (Specific Aims 1-3) will be performed to test this hypothesis. In Specific Aim 1, we will determine whether ectopic over-expression of rpL7a alone affects neuronal survival and proliferation. Inducible rpL7a expression will be precisely controlled to a level similar to ethanol-induced alteration. Subsequently, the effect of ectopic over-expression of rpL7a on cell survival and cell cycle kinetics will be examined. In Specific Aim 2, we will determine whether ethanol-induced damages are mediated by rpL7a, rpL7a will be knocked down using RNA interference. The effect of ethanol on survival and cell cycle kinetics will be examined in the cells with suppressed expression of rpL7a. In Specific Aim 3, we will investigate the mechanisms by which rpL7a mediates ethanol-induced damages. Ectopic over-expression of rpL7a activates p53. We will determine the role of rpL7a in ethanol modulation of p53 activation. As a unit, the series of experiments will explore the novel function of rpL7a in ethanol-induced neuronal damages. The favorable outcome of proposed studies will help to identify a therapeutic target to ameliorate alcohol neurotoxicity. Furthermore, the studies will provide an important insight into the mechanisms of extra-ribosomal function of ribosomal proteins.

DESCRIPTION (provided by applicant): Two of the most striking effects of ethanol exposure during development are the disruption of neuronal migration and the deletion of neurons in certain areas of the central nervous system (CMS). The cellular/ molecular mechanisms underlying these damages remain unclear. Glycogen synthase kinase-3/beta (GSK3beta) is predominantly expressed in neurons in the developing CMS, and emerges as a critical signaling component regulating diverse events of neuronal development, such as neuronal migration and proliferation/survival. It has also been implicated in abnormalities in the adult brain, such as neurodegenerative diseases. The activity of GSK3beta is regulated by phoshorylation and subcellular localization. GSK3beta is activated by various cellular stresses including endoplasmic reticulum (ER) stress. We have demonstrated that ethanol exposure induces ER stress and significantly alters the phosphorylation state of a GSK3beta in vitro and in vivo event at modest concentrations (50-100 mg/dl); blocking GSK3beta activation mitigates ethanol-induced neuronal death. Although ethanol alone at relatively low concentrations does not cause neuronal death, it greatly potentiates apoptosis caused by various exogenous insults which also activate GSK3beta. In contrast, ethanol does not affect cell death induced by pro-apoptotic signals which are independent of GSK3beta. We hypothesize that GSK3beta is a critical mediator of ethanol neurotoxicity. In this proposal, we will characterize the effect of ethanol on phosphorylation/activity, subcellular localization and expression of GSK3beta in the developing neurons using various model systems (cultured neurons, organotypic slice culture of rat cerebellum and the developing cerebellum). Subsequently, we will determine the role of GSK3beta in ethanol-induced cell cycle arrest, apoptosis, inhibition of neurite outgrowth and aberrant migration patterns; we will determine whether blocking GSK3beta activation by selective inhibitors or over- expression of dominant negative GSK3beta (DN-GSK3beta) or GSK3beta binding protein (GBP) provides neuroprotection against ethanol-induced damage. In addition, we will investigate the role of Chop (GADD153), another ER stress responsive factor, in ethanol-induced neuronal damages. Futhermore, we will elucidate the mechanisms underlying GSK3beta activation; we will systematically investigate up-stream signaling of GSK3beta and the contribution of ER stress-responsive components, such as protein phosphatase 2A, to ethanol-induced GSK3beta activation. Finally, we will investigate whether ethanol-induced sensitization or potentiation to apoptosis caused by other insults is dependent on GSK3beta we will determine whether over- expression of DN- GSK3beta and GBP abolishes ethanol-induced potentiation to neuronal death triggered by other pro-apoptotic stimuli. As a unit, these experiments will explore a novel mechanism underlying ethanol- induced damage to the CMS using in vitro and in vivo model systems. The systematical study may establish GSK3beta as a potential therapeutic target for the treatment of alcohol neurotoxicity

DESCRIPTION (provided by applicant): Alcohol consumption promotes the development of human cancers and environmental factors play an important role in the etiology. Epidemiological studies indicate that alcohol consumption not only increases breast cancer risk, but also enhances the progression and the aggressiveness of existing breast tumors. Nonetheless, the mechanism by which alcohol contributes to breast tumor initiation or progression has yet to be established. ErbB2 is a member of epidermal growth factor receptor family. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis. We have previously demonstrated that over-expression of ErbB2 sensitized breast cancer cells to alcohol-induced tumor promotion. Recently, we identified a novel component in ErbB2 signaling pathways that may regulate cancer cell aggressiveness, the p38?. We hypothesized that alcohol enhances NOX-dependent production of ROS which activates ErbB2 or MKK6. The activation of ErbB2 and MKK6 causes selective phosphorylation of p38? which recruits SAP97/DLG. The activated SAP97/DLG promotes epithelial to mesenchymal transition (EMT), increase cancer stem cells (CSC) population and invasiveness of breast cancer cells. This leads to an enhanced aggressiveness. There will be three specific aims. Specific Aim 1 will determine the role of p38? in alcohol- induced aggressiveness in vitro. Specific Aim 2 will investigate the mechanisms underlying alcohol-induced p38? activation as well as the mechanisms how p38? mediates aggressiveness of breast cancer cells. Specific Aim 3 will investigate the role of p38? in alcohol-induced tumor aggressiveness in animal models. The study will not only explore the basic cell biology of breast cancer aggressiveness, but also elucidate the mechanisms of alcohol's tumor promotion action. The outcomes will help developing new therapeutic strategy for breast cancer treatment and alcohol-mediated tumor promotion.

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder (FASD) is one of the leading causes of mental retardation, and thus is a major public health concern. The developing central nervous system (CNS) is particularly sensitive to alcohol. One of the most deleterious effects of developmental alcohol exposure is the permanent loss of neurons in the CNS. The cellular/molecular mechanisms underlying ethanol-induced neuronal death remain unclear. During the last decade, mitochondria damage and oxidative stress have been believed to play an important role in the pathogenesis of ethanol-associated CNS injury. However, mitochondria damage does not fully explain ethanol neurotoxicity. The effects of ethanol on other cellular organelles receive little attention, and the connection between mitochondria damage and other organelle dysfunction is poorly understood. This grant proposal attempts to fill this gap and investigate the effect of ethanol on the interaction between the endoplasmic reticulum (ER) and autophagy. ER stress is induced in various physiological and pathological conditions where the accumulation of unfolded proteins or disruption of ER Ca2+ homeostasis occurs. Autophagy, a lysosomal pathway involved in the turnover of cellular macromolecules and organelles, is induced to alleviate cytotoxicity during ER stress. We have demonstrated that ethanol induces ER stress in developing neurons. We hypothesize that ethanol neurotoxicity is partially caused by the induction of ER stress and the simultaneous impairment of the protective autophagic pathway. As a corollary, we propose that activation of autophagy pathways during ethanol exposure can ameliorate ethanol cytotoxicity;contrarily, inhibition of autophagy exacerbates the effect of ethanol. To test this hypothesis, we will first determine whether ethanol inhibits ER stress- triggered autophagy. Next, we will activate or inhibit the autophagic pathway by pharmacological or genetic approaches and determine whether the modulation of autophagic pathways ameliorates or exacerbates ethanol cytotoxicity. Our hypothesis is novel and the proposed study is significant;it will offer new insight into the effect of ethanol on the endoplasmic reticulum and lysosomal degradation pathways. It will provide a potential avenue for alleviating ethanol cytotoxicity. PUBLIC HEALTH RELEVANCE: Prenatal exposure to alcohol causes profound damages to the developing brain. Fetal alcohol syndrome is the leading cause of mental retardation. One of the most deleterious effects of developmental alcohol exposure is the permanent loss of neurons in the brain. However, it remains unclear how alcohol kills immature neurons. The endoplasmic reticulum (ER) is an organelle that processes proteins and stores calcium. We have shown that ethanol causes ER injury. Autophagy, a lysosomal pathway involved in the turnover of cellular macromolecules and organelles, is induced to alleviate cytotoxicity during ER damage. Our study will test a novel hypothesis that alcohol neurotoxicity is partially caused by ER damage and simultaneous impairment of the protective autophagic pathway. Our study will offer novel insight into the effect of alcohol on the ER and lysosomal degradation pathways. It may provide a new therapeutic avenue.

DESCRIPTION (provided by applicant): Alcohol is a neuroteratogen; alcohol consumption during pregnancy may cause Fetal Alcohol Spectrum Disorders (FASD), among which, fetal alcohol syndrome (FAS) is the most severe form. The depletion of neurons in the developing CNS is the most deteriorating effect of ethanol. The loss of CNS neurons may underlie many of the behavioral deficits observed in FASD. Glycogen synthase kinase 3¿ (GSK3¿), a serine/threonine kinase, is an important mediator of neuron degeneration. We have demonstrated that ethanol activates GSK3¿, and the activation of GSK3¿ leads to neuronal death. Oxidative stress is also considered an important contributor to ethanol-induced neurotoxicity. We have purified a potent antioxidant from blackberries, cyanidin-3-glucoside (C3G). Our study indicates that C3G inhibits GSK3¿ activity and alleviates oxidative stress in cultured neuronal cells. In addition, C3G protects neuronal cells against ethanol-induced cell death. C3G-mediated neuroprotection is much more potent than other antioxidants and GSK3¿ inhibitors. C3G can cross the blood brain barrier (BBB) and distribute in the brain. C3G diminishes ethanol-induced activation of caspase-3 and Bax in the developing brain. Pharmacologically relevant concentrations of C3G are achievable through oral administration or intravenous (iv) injection in animals, and no adverse effect is observed. These findings suggest that C3G is a promising neuroprotective agent that may ameliorate/prevent ethanol-induced neuronal damage. We hypothesize that C3G's potent protection against ethanol-induced neuronal loss is mediated by the combined action of its antioxidant property and inhibition of pro-apoptotic signaling, GSK3¿/Bax pathways. To test the hypothesis, we will (1) investigate the antioxidant property of C3G and its metabolites, and their effects on GSK3¿ activity; (2) determine whether C3G protection against ethanol- induced neuronal loss is mediated by the combined action of its antioxidant property and the inhibition of GSK3¿; (3) determine whether C3G ameliorates ethanol-induced behavioral deficits. C3G is a potent natural antioxidant and has diverse potential benefits for human health. It is a promising neuroprotective agent against ethanol toxicity due to its dual functions as an antioxidant and a GSK3¿ inhibitor. As a unit, the proposed experiments will elucidate the novel role of C3G, and provide an important basis for future clinical trials to evaluate the feasibility of C3G to treat ethanol neurotoxicity. Our study will potentially offer a new therapeutic strategy.

Alcohol abuse is the major risk factor for pancreatitis. It has been shown that the drinking pattern affects the impact of alcohol-induced organ damage. However, the underlying cellular and molecular mechanisms remain unclear. The progress in this line of research has been hindered by the lack of appropriate animal models. We have recently developed a paradigm of chronic plus binge alcohol exposure in which alcohol caused pancreatic damage characteristic of acute pancreatitis. We showed neither chronic nor binge alcohol exposure alone caused significant pancreatic damage. However, chronic plus binge alcohol exposure induced drastic pancreatic damage and inflammation which was accompanied by endoplasmic reticulum (ER) stress. ER is the site for protein folding, modification and transport, and calcium storage. ER stress is caused by the alterations in ER homeostasis, such as increased protein synthesis, accumulation of misfolded proteins, or changes in the calcium levels. ER stress triggers unfolded protein response (UPR) which functions to restore ER homeostasis. However, sustained ER stress exceeds UPR?s ability to restore ER homeostasis, resulting in cell death. We further showed that chronic alcohol exposure inhibited the expression of MANF, a key ER stress responsive protein which was originally identified as a neurotrophic factor and functions primarily to maintain ER homeostasis. We hypothesize that MANF is a critical UPR component that can alleviate ER stress in response to alcohol exposure, and chronic alcohol exposure impairs MANF, resulting in increased susceptibility to ER stress. We propose two specific aims to test these hypotheses. Specific Aim 1 determines the role of MANF in chronic/binge alcohol exposure-induced damage to the pancreas. Specific Aim 2 determines whether ER stress plays a critical role in chronic/binge alcohol exposure-induced damage to the pancreas. As a unit, the proposal will use novel animal models to investigate the mechanisms underlying chronic/binge alcohol exposure-induced pancreatic damage. The study will not only gain insight into cellular/molecular mechanisms of alcoholic pancreatitis but also establish a protective role of MANF. It therefore may offer a potential new therapeutic target for the treatment of alcoholic pancreatitis.