Hongbing Wang, Ph.D - US grants

DESCRIPTION (provided by applicant): Induction of cytochrome P450 (CYP) enzymes by xenobiotics is a major concern in clinical practice because of the potential for drug-associated attenuated efficacy, or increased metabolite-associated toxicity, in the presence of concomitantly administered drugs. CYP2B6 is a highly-inducible enzyme in human liver. Recent studies have shown that the relative contribution of CYP2B6 to hepatic CYP content and substrate metabolism are much higher than estimated previously, and suggest that induction of this enzyme may be of therapeutic importance. The long-term objective of this ongoing project is to elucidate the molecular mechanisms governing drug-induced regulation of human hepatic CYP2B6. Results from the current funding period established human (h) PXR as a major transcriptional factor capable of regulating CYP2B6 induction by many compounds. Although it is widely accepted that CAR mediates induction of rodent CYP2B genes, the role of hCAR in human CYP2B6 regulation is far from clear, and direct extrapolation of rodent data to humans is risky due to obvious species differences. The central hypothesis of the proposed studies is that hCAR plays a distinct role compared with hPXR in human CYP2B6 induction, and differs from hPXR in its preferred induction of CYP2B6 over CYP3A4. The specific aims of the proposed studies are to: 1) define the role of hCAR in the regulation of hepatic CYP2B6 expression, 2) elucidate the molecular mechanisms of differential regulation of CYP2B6 and CYP3A4 by hCAR, 3) characterize the differences between direct and indirect hCAR activators in the mechanisms of hCAR nuclear activation and CYP2B6 induction, and 4) assess the utility of selective hCAR activators to facilitate cyclophosphamide therapeutic activation. A multifaceted experimental approach incorporating protein-protein and protein-DNA binding assays, transfection and hCAR translocation assays in immortalized cell lines and/or human primary hepatocytes, siRNA knockdown of hCAR and hPXR in human hepatocytes, and transient in vivo expression of hCAR in CAR-/- mice will be employed to delineate the overlapping and distinct roles of hCAR versus hPXR in CYP2B6 induction, to compare indirect and direct mechanisms of hCAR nuclear activation and their impact on CYP2B6 induction, and to evaluate the roles of selective hCAR activators in facilitating cyclophosphamide (CPA) bioactivation. Data generated from the proposed in vitro and in vivo studies will provide important insight into the molecular mechanisms of CYP2B6 induction in humans, allowing for improved prediction of drug-drug interactions. In addition, these studies will explore selective modulation of drug metabolism via hCAR-mediated CYP2B6 induction as a means to increase therapeutic efficacy and decrease toxicity of a model CYP2B6 substrate, CPA.

DESCRIPTION (provided by applicant): By traditional definition, brain-derived neurotrophic factor (BDNF) was initially identified to mediate cell proliferation, differentiation and apoptosis in the nervous system. Recently, a large body of evidence strongly suggested an essential role of BDNF in regulating neuroplasticity, including long-term potentiation (LTP) and learning and memory. Importantly, BDNF expression is tightly regulated by neuronal activity. Molecular analysis of its promoter regions (promoter 1 and 3, or P1 and P3) has revealed several calcium-responsive elements (CaREs), indicating that BDNF transcription may be induced by Ca, the major second messenger in neurons. In addition, a number of transcription factors, such as CREB, CaRF and USF, were identified to bind different CaRE. The existence of these multiple control elements in BDNF promoters is believed to achieve specific regulation in different populations of neurons and upon different neuronal activities. We found that CREB- and CaRF-mediated transcription was differentially regulated by PKA and ERK. Knowing that both PKA and ERK activity are induced by Ca and neuronal activity, we hypothesize that P1 and P3 are specifically activated by neural activity in a tissue specific manner, and differentially regulated by different Ca-stimulated protein kinases. Although the role of PKA, ERK and calmodulin-dependent kinases (CaMK) were implicated in CREB-mediated transcription, their function in regulating native BDNF promoters is not clear. It is also not clear which promoter is regulated by neural activities in intact animals. Furthermore, the in vivo regulator for the activity-dependent BDNF up-regulation is unknown. We will measure reporter gene expression under the control of the individual CaRE or native P1 and P3. The effects of Ca-stimulated kinases will be tested by using inhibitors, dominant negative form of the individual kinase, and in neurons from mutant mice. BDNF expression will be examined in cultured neurons, brain slices, and intact animals after training for certain learning-related behavioral paradigms. Importantly, the physiological relevance of BDNF will also be addressed with mutant and aged mice. Because abnormal BDNF expression has been implicated in aging, depression, stress, and neurological diseases, the proposed study will not only enhance the general understanding on gene expression in neuroplasticity, but also may lead to the development of a potential neurotherapeutic.

DESCRIPTION (provided by applicant): Overactivation of N-methyl D-aspartate receptor (NMDAR) by massive glutamate release during acute brain insults represents a major mechanism for neuronal loss in ischemic stroke and brain trauma. While proper activation of NMDAR is required for cell survival, overactivation of NMDAR stimulates cell death signaling and causes the loss of calcium homeostasis. There are significant interests and needs to target NMDAR as therapeutic interventions for ischemic stroke and other forms of neurodegeneration. Because general suppression of all NMDAR function may be detrimental to normal brain functions, targeting specific pools or subtypes of NMDAR may improve the therapeutic values. For example, inhibiting extrasynaptic NMDAR may attenuate receptor overactivation without disrupting normal synaptic functions. We hypothesize that the pharmacological property is dramatically different between synaptic and extrasynaptic NMDAR. To test the hypothesis and better understand the function of extrasynaptic NMDAR function, we will pursue 3 specific aims: Aim 1) Determine the function of extrasynaptic NMDAR in cell death;Aim 2) Determine the function of extrasynaptic NMDAR in Ca dysregulation;Aim 3) Determine the difference in pharmacological property between extrasynaptic and synaptic NMDAR. This application proposes to use calcium imaging and molecular characterization to identify the function and property of extrasynaptic NMDAR. The outcome of this proposal is expected to set the foundation for future therapeutic strategies to attenuate neurodegeneration, such as stroke, via specific blockade of extrasynaptic NMDAR. PUBLIC HEALTH RELEVANCE: Stroke is one of the major causes of mortality and adult disability in the United States. The long-term goal of our research is to develop molecular therapies for stroke. In addition, our study on molecular and cellular mechanisms may also lead to therapeutic development for other forms of neurodegeneration.

DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is a common form of mental disorder caused by genetic mutation in Fmr1 gene that leads to lack of expression of FMRP (fragile X mental retardation protein). Tremendous advances in understanding FXS are made from pre-clinical studies using animal models. The Fmr1 knockout mouse model replicates many aspects of phenotypes associated with FXS, including cognition deficits, hyperactivity, hyperarousal, and impaired social ability. One major pathophysiology associated with FXS, the enhanced group I metabotropic glutamate receptor (mGluR)-mediated synaptic long-term depression (mGluR-LTD), implicates that overactivation of mGluR signaling may play a role in FXS etiology. Thus, there is significant need to identify key molecular components in the mGluR signaling cascade and develop therapeutic strategies. Here, we found that lowering basal cAMP level caused an opposite synaptic phenotype to that of FXS. However, it is not known whether cAMP is a functional component in the mGluR signaling cascade. It is also important to identify specific approaches to manipulate basal cAMP level in FXS and achieve therapy. Consistent with the mission of NIH, this proposal aims to identify a novel component in the mGluR signaling cascade and validate a new therapeutic strategy for the treatment of FXS in mouse animal model. The goals of this proposal are 1) to determine how mGluR and cAMP are coupled, 2) to determine how to manipulate the basal, as well as mGluR-stimulated cAMP level in the mouse model of FXS, and 3) to determine the therapeutic value of cAMP manipulation in FXS. We will use genetic approaches to manipulate the cAMP level. We will further examine the therapeutic value by measuring mGluR-LTD, the core FXS-associated behavioral phenotypes, and the key molecular and cellular mechanisms underlying the pathology of FXS. We expect that this work will identify novel mechanism and suggest new strategy to treat FXS. Our method of manipulating cAMP and its therapeutic value in FXS will be validated in pre-clinical studies using an FXS animal model. Because mutation of Fmr1 gene is also a leading cause for autism, it has been suggested that FXS and autism may share some common mechanisms. Thus, we expect that the outcome of this proposal may also help understanding the signal transduction involved in autism.

DESCRIPTION (provided by applicant): Cyclophosphamide (CPA), an alkylating prodrug, has been used extensively in the treatment of hematologic malignancies, in particular, as an important component in the front-line regimens for non-Hodgkin lymphoma and chronic lymphocytic leukemia. Unfortunately, despite aggressive chemotherapy, a significant number of patients remain uncured due to development of drug resistance and/or intolerable toxicities. The need for further optimization of the current regimens is evident. The overall goal of this proposal is to improve the therapeutic efficacy of CPA-based chemotherapy by enhancing metabolic conversion of CPA to the pharmacologically active 4-hydroxylcyclophosphamide (4-OH-CPA) via CYP2B6, but not to the N- dechloroethyl-cyclophosphamide and the toxic chloroacetaldehyde by CYP3A4. Towards this end, we have shown that activation of the human constitutive androstane receptor (hCAR) preferentially induced the expression of hepatic CYP2B6 over CYP3A4 and increased the formation of 4-OH-CPA. We have also developed a unique human primary hepatocyte (HPH)-leukemia coculture model and demonstrated that co- administration of CPA with a selective hCAR activator leads to significantly enhanced apoptosis in leukemia cells without increasing hepatotoxicity. In this application, we hypothesize that activation of hCAR can selectively enhance systemic exposure to 4-OH-CPA and increase the efficacy:toxicity ratio of CPA-based treatment for lymphoma and leukemia. This central hypothesis will be tested by the following specific aims: Aim #1. Evaluate the role of hCAR in the bioactivation of CPA in HPH and lymphoma/leukemia cells; Aim #2. Assess the metabolism and anticancer activity of CPA in the HPH-lymphoma/leukemia coculture model; and Aim #3. Examine the influence of hCAR activation on CPA-based treatment of lymphoma/leukemia in an hCAR-transgenic mouse model. The outcomes are expected to establish hCAR as a novel therapeutic target facilitating the CPA-based chemotherapy for hematopoietic malignancies. Improved understanding of the underlying mechanism(s) associated with hCAR-mediated enhancement of the anticancer activity of CPA may ultimately benefit the development of new therapeutic strategies.

Project Summary Citrate is a key energy sensor that plays a central role in carbohydrate metabolism, energy production, and histone acetylation. The intracellular level of citrate is tightly controlled through a balance of biosynthesis and transport. In the liver, the solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, is essential for the import of citrate from the circulation to hepatocytes, a process that can be perturbed by both xenobiotic and endobiotic stimuli. Recent studies have shown that expression of SLC13A5 was increased in obese, non-alcoholic fatty liver disease (NAFLD) patients, high-fat diet (HFD)-treated rhesus monkeys, and in xenobiotic-treated human and rat hepatocytes, suggesting upregulation of SLC13A5 can be a risk factor for metabolic disorders. In contrast, deletion of SLC13A5 protects mice from HFD-induced hepatic steatosis and mutations of the SLC13A5 ortholog in D. melanogaster promote longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, the mechanism(s) by which the SLC13A5 gene is transcriptionally regulated and whether clinically used drugs disturb the expression of this transporter are not well characterized. Moreover, whether SLC13A5 affects hepatic functions beyond lipid homeostasis is largely unknown. The overall objective of this proposal is to understand the molecular mechanisms governing hepatic SLC13A5 gene expression and to delineate the role of SLC13A5 in human liver cell proliferation. To this end, we have shown that 1) prototypical activators of the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) robustly induce expression of human SLC13A5; 2) knockdown of SLC13A5 attenuates the proliferation of hepatocellular carcinoma cells; and 3) expression of SLC13A5 is inversely correlated with the activation of AMPK signaling. Building on these preliminary results, we hypothesize that CAR and PXR are key regulators of the inductive expression of SLC13A5 in the liver, and SLC13A5 functions as a nutrient regulator altering the proliferation of hepatoma cells by modulating AMPK/mTOR signaling pathways. This central hypothesis will be tested in the following specific aims: Aim 1. Define the role of CAR and PXR in xenobiotic- induced expression of SLC13A5; Aim 2. Elucidate the mechanism(s) underlying CAR- and PXR-mediated induction of SLC13A5; Aim 3. Determine the effects of SLC13A5 on hepatoma cell proliferation. The outcomes are expected to provide fundamental novel knowledge on the transcriptional regulation of SLC13A5 in the liver, and to delineate a crucial role of SLC13A5 in bridging energy metabolism with liver cancer progression.

Abstract/Summary    As  a  functionally  important  aspect  of  cognitive  flexibility,  reversal  learning  leads  to  inhibition/suppression  of  the  previously  established  memory.  Effective  reversal  learning  is  fundamental  for  information  updating  and  essential  for  adaptation  to  changing  environmental cues. Regarding its impact on mental health, deficits in cognitive flexibility  and  reversal  learning  are  prevalent  in  psychological  and  mood  disorders,  and  are  considered  as  an  emerging  therapeutic  target.  However,  there  is  limited  understanding  of  mechanisms  underlying  cognitive  flexibility.  Our  recent  experimental  data  revealed  that,  contrary  to  the  previously  recognized  role  of  cAMP  signaling  in  regulating  broad  spectrum  of  learning  and  memory,  type  8  adenylyl  cyclase  (ADCY8)  specifically  regulates  the  activity-­dependent  suppression  of  old  memory  following  reversal  learning.  With  our  recently  developed  Adcy8  conditional  knockout  mice,  we  will  determine  the  effects  of  region-­  and  cell  type-­specific  ADCY8  deficiency  on  synaptic  and  cognitive  flexibility:  reversal/suppression  of  the  previously  established  synaptic  potentiation  (i.e.  depotentiation) and reversal/suppression of the previously established memory. Further,  computational  analysis  with  transcriptome  landscape  predicts  that  the  PI3K  (phosphatidylinositide  3-­kinase)/Akt  (protein  kinase  B)-­GSK3?  (glycogen  synthase  kinase  3?)  signaling  cascade  is  the  molecular  substrate  of  ADCY8.  We  will  determine  whether  restoration  of  the  ADCY8-­PI3K/Akt-­GSK3?  signaling  cascade  causally  corrects  the  defective  synaptic  depotentiation  and  reversal/suppression  of  old  memory.  Finally,  we  will  determine  the  causal  effect  of  synaptic  depotentiation  on  old  memory  suppression  and  its  dependency  on  the  ADCY8-­PI3K/Akt-­GSK3b?  signaling  cascade.  Considering  that  there  are  10  different  ADCYs  in  mammalian  system,  the  outcome  of  this  project  will  delineate  a  unique  of  role  of  ADCY8  in  regulating  a  specific  domain  of  cAMP  signaling  that  is  functionally  linked  to  cognitive  and  synaptic  flexibility.  We  also  expect  that  the  mechanisms  learned  from  this  study  may  suggest  targeted  therapeutic  strategies to attenuate reversal learning deficits in certain patient population with altered  cAMP-­PI3K/Akt-­GSK3? signaling.     

This project will delineate a non-conventional role of adenylyl cylcase (ADCY) in regulating Gq-mediated signaling and synaptic long-term depression LTD (LTD) in normal brain and pathophysiology associated with Fragile X syndrome (FXS). Activation of specific groups of G protein-coupled receptors stimulates Gq, and in turn triggers signal transduction cascade, leading to translation-dependent synaptic plasticity such as LTD. Relevant to neurological disorders, hyper-function of Gq-coupled metabotropic glutamate receptor 5 (mGluR5) and muscarinic acetylcholine receptor (Gq-mAchR) as well as elevated translation underlie multiple aspects of neuronal dysfunction in FXS. We recently found that type 1 adenylyl cylcase (ADCY1) level is aberrantly increased in FXS mouse model (i.e. Fmr1 knockout mice). Genetic deletion or pharmacological inhibition of ADCY1 corrects core cellular and behavioral symptoms. Intriguingly, inconsistent with the current understanding on Gq, of which the functions of ADCY and cAMP-mediated signaling are not considered, we found that ADCY1 is essential for LTD following activation of mGluR5. Based on these results, our central hypothesis is that the Ca2+-stimulated ADCY1 is a functional component of Gq signaling, and thereby the abnormally elevated ADCY1 expression in FXS accounts for the exaggerated Gq-mediated synaptic dysfunction and aberrantly elevated translation. This project will first address how ADCY1 regulates Gq signaling, translation, and Gq-LTD in normal neurons. Second, it will address how elevated ADCY1 governs alterations in distinct translation process, and whether elevated ADCY1 is causal for Gq-mediated synaptic dysfunction in FXS neurons. Considering that the conventional view emphasizes the role of PLC (phospholipase C)-Ca2+/PKC (protein kinase C) cascade rather than ADCY/cAMP in Gq signaling, validation of ADCY1 function in Gq-mediated signaling and Gq-LTD will suggest a substantial paradigm shift/modification and re-define how Gq functions in neurons. The results of this project will also provide new insights into pathophysiology and disease mechanism in FXS. It will reveal that ADCY1, as a key target of FMRP (Fragile X mental retardation protein), connects altered Gq signaling cascades with abnormal translation and synaptic dysfunction in FXS. It will uncover a new concept that the abnormal ADCY1-mediated signaling contributes to altered global translation via distinct aspects of translation processes such as translation capacity and efficiency, and thereby advance our understanding on FXS pathology. Considering that ADCY1 is only expressed in the central nervous system and functionally connected to multiple signaling molecules that are altered in FXS, the results will also suggest an attractive and mechanism-based therapy.

Project Summary: Excessive consumption of alcohol is a major contributor to the global burden of morbidity and mortality, and is the cause of alcoholic liver disease (ALD) that accounts for up to 25% of alcohol-associated deaths worldwide. The spectrum of ALD ranges from relatively mild hepatic steatosis, alcoholic steatohepatitis and fibrosis, to irreversible cirrhosis and hepatocellular carcinoma. Epidemiological studies reveal that binge drinking, a high- risk alcohol consumption style, has become increasingly popular among young adults; and frequent binge drinkers are at higher risk for developing severe ALD. However, the mechanisms underlying alcohol binge- induced liver injury and whether there is an adaptive enzymatic system that metabolizes high concentrations of ethanol after binge drinking are poorly understood. Our preliminary data demonstrate that chronic ethanol feeding plus binge administration drastically induces hepatic expression of the cytochrome P450 2b10 (Cyp2b10) in mice. After alcohol binge ingestion blood ethanol levels of Cyp2b10-null mice were significantly higher than that of wild-type mice. Moreover, chronic-plus-binge ethanol feeding resulted in greater liver damage in Cyp2b10-null mice than that in wild-type mice. These exciting findings have led to the overarching hypothesis that human CYP2B6, the analog of murine Cyp2b10, plays an important role in binge drinking- induced ALD, and that adaptive induction of CYP2B6 enzyme coordinates a novel protective mechanism underlying ALD. We will test this hypothesis in two proposed specific aims: Aim 1 is to determine CYP2B6- mediated metabolism of ethanol in human liver cells; and Aim 2 will investigate the role of CYP2B6 in ethanol binge-induced liver injury in humanized mouse model. These studies will provide necessary groundwork for identifying and validating a novel metabolic pathway-mediated by CYP2B6/Cyp2b10 for adaptive metabolism and detoxification of excessive alcohol after binge ingestion.

Project Summary The constitutive androstane receptor (CAR; NR1i3) is a well-established xenobiotic sensor that regulates the expression of numerous genes encoding proteins important for drug metabolism and clearance. Accumulating evidence suggests that CAR also plays noncanonical roles in coordinating diverse physiological and pathophysiological responses associated with energy homeostasis and cell proliferation. Studies in rodents have established activation of CAR as a key event promoting liver tumor formation. In contrast, CAR activation- induced replicative DNA synthesis and hepatocyte proliferation in rodents were not observed in either cultured human liver cells in vitro or in chimeric mice with humanized liver in vivo. Moreover, epidemiological studies have shown that even after long-term clinical use, phenobarbital, a prototypical CAR activator, does not increase the incidence of liver tumors in humans. Yet, the role of human CAR (hCAR) in hepatoma cell proliferation and liver cancer development remains poorly understood. The overall objective of this application is to delineate the role of hCAR in liver tumor progression and to develop a comprehensive understanding of the molecular mechanisms underlying the effects of hCAR on hepatoma cell proliferation. To this end, we have shown that 1) expression of hCAR was significantly lower in hepatocellular carcinoma (HCC) compared to normal liver and, importantly, hCAR expression is inversely correlated with HCC outcomes; 2) ectopic expression of the reference hCAR but not a splicing variant isoform (hCAR3) in hepatoma cells markedly repressed cell proliferation, soft agar colony formation, and the growth of hepatoma xenografts in nude mice; 3) RNA-seq analyses revealed that hCAR alters the expression of a cluster of tumor suppressors and oncogenes including the downregulation of erythropoietin (EPO), a pleiotropic growth factor that exhibits cell proliferation and anti-apoptosis functions; and 4) activation of human and mouse CAR differentially alters the expression of cell proliferation genes in vivo. Based on these exciting preliminary findings, we hypothesize that in stark contrast to its rodent counterparts, hCAR exhibits anticancer functions that repress the progression of HCC by downregulating EPO. This central hypothesis will be tested in two Specific Aims: Aim 1. Define the role of hCAR isoforms in hepatoma cell proliferation and HCC progression; and Aim 2. Delineate the mechanisms by which hCAR represses HCC progression. Our findings are expected to determine the role of hCAR in HCC development and provide novel mechanistic insights into hCAR-mediated suppression of HCC progression that will open the door to novel biomarkers and therapeutics.