Donald B. DeFranco - US grants

A glucocorticoid dependent enhancer within the long terminal repeat (LTR) of Moloney murine sarcoma virus (MoMSV) has been identified that coresides with a hormone independent enhancer. The long term goal of this project is to examine within MoMSV and related C-type retroviruses, the relationship between distinct transcriptional regulatory elements, and uncover genetic strategies by which multiple regulatory properties are superimposed. Transient transfections will be performed into different cell types with in vitro generated MoMSV mutants to identify specific sequences that are necessary for hormone dependent and independent enhancers and reveal any cell type specificity of enhancer activity. We will also analyze glucocorticoid effects on the expression of endogenous C-type proviruses. Intact and hybrid MoMSV proviruses will be generated in various cell types by stable transfections, in order to test the effect of proviral context and examine the kinetics, cell type specificity and cellular heterogeneity of the glucocorticoid response of C-type retroviruses. In addition, the transcriptional effects of glucocorticoids on endogenous xenotropic proviruses will be studied and viral sequences reponsible for the hormone response identified by cDNA cloning. Finally, we will analyze the molecular basis for the differential activity of distinct MoMSV enhancers in undifferentiated embryonal carcinoma cells. Both transient and stable transfections will be performed with MoMSV DNA to examine both cis- and trans-acting mechanisms that influence glucocorticoid regulated expression.

DESCRIPTION: (Adapted from applicant's description) Extracellular matrix (ECM) effects on gene regulatory events are hypothesized to occur, in part, through a direct effect on the nuclear matrix. The role of the ECM in several biological processes is now well established and much is known about the multiple signaling pathways that are activated by ECM interactions with the integrin family of cell surface receptors. The point of contact between the ECM and integrins occurs at specialized structures termed focal adhesions. While various second messenger systems mobilized at focal adhesion may be used to transmit ECM-directed signals to changes in gene expression, there also appears to be a "physical" link between the ECM and the nuclear matrix. This application presents evidence that HIV-5 (hydrogen peroxide-inducible clone-5), a protein associated with focal adhesions, acts to potentiate glucocorticoid receptor transactivation. Furthermore, HIC-5 does not appear to be restricted to focal adhesions, as a fraction has been localized to the nuclear matrix. Finally, LIM domains of Hic-5 were found to interact with a transactivation domain of the glucocorticoid receptor ( i.e. GR. tau2) that encodes a nuclear matrix-targeting signal. The four Specific Aims that will be pursued in this application are as follows: 1) To identify amino acids within the GR.tau2 domain that are required for Hic-5 interaction, 2) To map functional domains of Hic-5 that influence GR transactivation, 3) To identify the mechanism of Hic-5 potentiation of GR transactivation, and 4) To examine whether Hic-5 participates in ECM regulation of GR function. Cellular responses to ECM clearly play a role in physiologically relevant steroid hormone responses in specific target tissues. Furthermore, alteration in focal adhesion protein function (e.g. paxillin) has been detected in metastatic prostate cancer. Thus the mechanistic analysis of LIM domain protein involvement in steroid receptor subnuclear trafficking and transactivation proposed in this application could increase our understanding of how cell attachment to the ECM influences gene expression both during normal development and in pathophysiological conditions.

We have recently shown that the GT1-3 and GT1-7 cell lines, derived from gonadotropin-releasing hormone (GnRH) neurons of the rat hypothalamus, express immunoreactive glucocorticoid receptors (GRs). The GRs within these cell lines are functional and can activate transcription from exogenously introduced glucocorticoid responsive promoters, decrease expression of the endogenous GnRH gene, and repress transcription from transfected GnRH promoters. These results support the hypothesis that glucocorticoid effects on the reproductive axis could be mediated by the direct action of GRs contained within GnRH neurons. In this proposal we plan to investigate the molecular mechanisms of glucocorticoid repression of GnRH neurons. In this proposal we plan to investigate the molecular mechanisms of glucocorticoid repression of GnRH gene expression. One of our specific aims is to identify sequences within the mouse GnRH promoter that are responsible for glucocorticoid mediated repression. Various deletion and point mutants of the mouse GnRH promoter will be tested for glucocorticoid repression upon transient transfection into the GT1-7 cell line. These analyses should identify the minimal promoter segment required for glucocorticoid repression and reveal whether this negative glucocorticoid response element (nGRE) also possesses any other overlapping transcriptional regulatory elements. We also plan to determine whether repression of mouse GnRH promoter activity involves direct DNA binding of the GR and/or other GT1-7 cell nuclear factors. Segments of the mouse GnRH promoter, including the putative nGRE, will be tested for their ability to be bound in vitro by purified GR, or nuclear factors extracted from isolated GT1-7 nuclei. The results of these studies should reveal whether glucocorticoid repression of GnRH transcription is associated with alterations of promoter bound transcription factors, perhaps including the GR. Finally, we plan to examine the effect of glucocorticoids on GnRH secretion and gene expression in vivo. GT1-7 cells will be attached to Cytodex beads and perifused to determine whether glucocorticoids alter GnRH secretion. Effects of glucocorticoids on GnRH mRNA expression in vivo will be assessed by a sensitive solution hybridization/RNase protection assay and by in situ hybridization using two rat models, adult males and immature females treated with estradiol to induce gonadotorpin surges.

Description (From the applicant's abstract): The Hsp90-binding benzoquinoid ansamycin, geldanamycin (GA) protects Ht22 cells and immature primary rat cortical neuron cultures from glutamate induced oxidative toxicity. Furthermore, preliminary results suggest that GA given during resuscitation improves neurological outcome in rats subjected to global ischemia induced by asphyxial cardiac arrest. GA binding to HSP90 disrupts various intracellular signaling pathways and leads to induction of Hsp70, depletion of the Raf-1 protooconcogene, and reduced activation of downstream targets of Raf-1, ERK-1 and ERK-2. The downregulation of ERK activation caused by GA treatment might be an important component of its neuroprotective activity since inhibition of an ERK activating kinases (i.e. MEK-1) also protects against oxidative toxicity in Ht22 cells and primary rat cortical neuron cultures. We hypothesize that manipulation of Hsp90 function may be a useful strategy to impact various signal transduction pathways in vivo that trigger neuronal cell death. The identification of the molecular mechanisms involved in neuroprotection associated with pharmacological manipulation of Hsp90 function is the major goal of this application. In specific aim 1 we will determine the impact of Hsp90 regulated signaling pathway on glutamate-induced oxidative toxicity in the Ht22 mouse hippocampal cell line and address the following questions. Does Hsp70 induction contribute to the protective effects of Hsp90 binding drugs in vitro? Is persistent ERK activation necessary and sufficient for glutamate-induced oxidative toxicity in Ht22 cells? Does glutamate-induced oxidative toxicity affect other members of the MAPK family (i.e. JNK/SAPK and P38MAPK)? In specific aim 2 we will determine the impact of Hsp90-regulated signaling pathways on glutamate-induced oxidative toxicity in immature primary rat cortical neuron cell cultures. Are GA and U1026 protective against oxidative toxicity in immature primary rat cortical neuron cell cultures? What biochemical events are associated with neuroprotective effects of Hsp90-binding drugs in immature primary rat cortical neuron cultures? Finally in Specific Aim 3 we will determine whether Hsp90 binding drugs are effective post-treatment neuroprotective agents in rat models of global ischemia. Do Hsp90-binding drugs improve neurological outcome following asphyxial cardiac arrest or middle cerebral artery occlusion? Do Hsp90-binding drugs induce Hsp70 in vivo? Do Hsp90-binding drugs affect MAPK activation in vivo?

[unreadable] DESCRIPTION (provided by applicant): This Predoctoral Training Program is designed to provide a broad education in the Pharmacological Sciences leading to the Ph.D. degree. The Pharmacological Sciences Training Program (PSTP) is based primary in the Department of Pharmacology at the University of Pittsburgh School of Medicine, but includes faculty from four schools within the University and sixteen departments including Anesthesiology, Cell Biology and Physiology, Chemistry, Environmental and Occupational Health, Neurobiology and Pharmaceutical Sciences. Graduate students entering this program are first recruited into the Interdisciplinary Biomedical Graduate Program where they start a program that includes core didactic education and research rotations. Students then transfer into the specialized PhD program of their choice and become candidates for support by the PSTP in their second year. The training program provides graduate classes in the essential elements of modern pharmacology, including neuropharmacology, cancer pharmacology, cardiovascular pharmacology, signal transduction and drug discovery, and also the elements of quantitative pharmacokinetics, pharmacodynamics and drug metabolism. Students choose mentors from a well-funded faculty in one of four research areas: Cancer Pharmacology, Signal Transduction, Drug Discovery or Cell and Organ System Pharmacology. Following completion of the comprehensive exam and a dissertation proposal, students are engaged full time in research in the third and subsequent years of this program. The PSTP also emphasizes training in the responsible conduct of research and provides training in skills that promote professional development. The PSTP thus provides a contemporary and exciting training opportunity for motivated students within a rich research environment, and aims to generate Ph.D. graduates with a broad understanding of the discipline of pharmacology. [unreadable] [unreadable]

Project Summary: Prostatic inflammation is a common feature of symptomatic benign prostatic hyperplasia (BPH) and may alter epithelial cell proliferation and tissue homeostasis in BPH through cytokine induction of proinflammatory signaling mediators such as cyclooxygenase-2 (Cox-2). Cox-2 is overexpressed in luminal epithelial cells of BPH tissue but predominantly in regions of chronic inflammation. One clinical trial reported only a short-term benefit of a Cox-2 inhibitor (i.e. nonsteroidal anti-inflammatory agents [NSAIDs] such as rofecoxib) in reducing LUTS symptoms when combined with a 5AR inhibitor. The mechanism responsible for the limited clinical effectiveness of Cox-2 inhibition is not known. In the human BPH-1 prostate epithelial cell line (which expresses high basal levels of Cox-2), pharmacologic or molecular ablation of Cox-2 expression limits the protective effects of ERß through selective disruptions in steroidogenic enzyme expression leading to a reduced production of ERß ligands from testosterone. We therefore hypothesize that the limited effectiveness of NSAIDs in current BPH clinical trials is due to disruptions in prostatic steroidogenic pathways that generate ligands for the tissue protective ERß. Four Aims are proposed to test this hypothesis: Aim 1 will determine the impact of Cox-2 on ERß ligand production in PrECs. Aim 2 will identify genome-wide basal and ERß-regulated gene expression patterns influenced by acute or long-term adaptive responses to Cox-2 overexpression in PrECs. Aim 3 will identify the impact of Cox-2 and ERß on targets relevant to polarized epithelial cell function in 3- dimensional cultures of PrECs and ERß knockout mice. Aim 4 will determine the impact of Cox-2 on ERß signaling in human prostate explants and a rodent model of prostatic inflammation

The goal of the University of Pittsburgh School of Medicine Short-Term Medical Student Training Program in Renal, GI, Endocrine and Epithelial Biology is to foster an interest in scientific investigation as a career objective for medical students. We provide students with an opportunity to participate in biomedical research with highly qualified mentors and role models. Our program supports a summer research training experience for medical students between the first and second year of medical school. This program is designed to introduce students to a rigorous approach to scientific inquiry in basic science or clinical investigation, with a focus on three major areas of research: (1) renal and epithelial cell biology, (2) digestive and hepatic disorders, and (3) endocrinology, metabolism and diabetes. Trainees are selected by an executive committee on the basis of student-authored project proposals developed with and endorsed by faculty members of the training program. Faculty members serve both as research trainers and as mentors.

Bronchopulmonary dysplasia (BPD) remains a significant complication of prematurity affecting nearly 70% in infants born ?28 weeks. Current pharmacologic strategies to mitigate the development and progression of BPD include administration of long-acting synthetic glucocorticoids (sGCs) such as dexamethasone (Dex). Several randomized clinical trials establish that sGC therapy targeted to preterm infants with evolving lung injury decreases BPD risk substantially, but encumber significant risks related to adverse somatic growth and long-lasting alterations in brain structure and function. Therefore, there remains an urgent need for GC pharmacotherapy for BPD in neonates that will provide beneficial anti-inflammatory and lung maturation effects, but limited adverse effects on the brain. Ciclesonide (CIC) is a new generation inhaled sGC currently approved for the treatment of asthma and allergic rhinitis that does not cause systemic adverse effects often observed with other sGCs. Clinical trials also demonstrate no adverse effects of CIC beyond placebo in 2-year olds. We hypothesize that CIC will attenuate hyperoxia-mediated acute lung injury in neonates but NOT trigger the demyelination, astrogliosis, microglia activation or neuronal damage in neonatal brain caused by systemically administered sGCs such as Dex. Three Specific Aims are proposed to test this hypothesis with a multi-disciplinary team possessing the ability to simultaneously investigate in vitro mechanisms and highly relevant rodent models of neonatal lung injury. Aim 1 will determine the mechanisms by which CIC prevents hyperoxia-induced acute lung injury and alveolar remodeling in experimental BPD. Aim 2 will identify the transcriptomic responses to CIC within individual cell types of neonatal mouse lungs using scRNA-Seq. Aim 3 will compare the acute and long-term consequences of neonatal Dex versus CIC exposure on brain architecture and behavior. This study has potential to identify CIC as an effective sGC therapy for BPD with brain- sparing effects, addressing the current dearth of therapies to prevent and/or treat BPD in preterm infants.