Juanita L. Merchant - US grants

Gastrin is a potent regulator of normal gastrointestinal cell growth and acid secretion; therefore elucidation of mechanisms that control its synthesis may be applied to understanding such disease states as neoplastic transformation and peptic ulceration. Although gastrin release from enriched antral G cell populations is known to be regulated by cholinergic agonists and the neuropeptide bombesin, the direct cellular mechanisms controlling gastrin synthesis at the level of transcription are poorly understood. This proposal addresses the hypothesis that direct activation of the Epidermal Growth Factor (EGF) receptor on antral G cells stimulates gastrin gene expression. The specific aims focus on EGF as a physiologic regulator of gastrin gene expression and use this observation to dissect the nuclear events that occur in response to growth factor stimulation. Enriched primary G cells will be isolated from dog and stimulated with EGF, Transforming Growth Factor alpha (TGFalpha), bombesin, and phorbol esters prior to measuring changes in gastrin mRNA on Northern blots. Nuclear runoff assays and intron probe analysis will be used to determine if the increase in gastrin mRNA levels is the result of an increase in new transcript synthesis or mRNA stability. To demonstrate that the antral G cell is capable of responding directly to EGF, the EGF receptor and gastrin antibodies were co-localized to the G cell in dog antral tissue. Prior work in a pituitary cell line (GH4) using gastrin-reporter gene constructs identified a novel DNA element, -68GGGGCGGGGTGGGGGG-53, that is required for both EGF and phorbol ester regulation of gastrin gene expression. Two nuclear proteins bind specifically to this element: Sp1 and a previously uncharacterized transcription factor called gERP (gastrin EGF Responsive Protein). The cDNA for Sp1 has been previously cloned. Therefore, the gERP cDNA will be cloned by screening an expression lambdagt11 phage library (Southwester method). To determine whether Sp1 and gERP bind separately or cooperatively to confer the EGF response point mutations of gERE will be tested in gel shift and transient transfection assays. Transcriptional control by both EGF and phorbol ester-activated protein kinase C (PKC) converge upon the gastrin EGF Response Element (gERE); therefore, it is likely that changes in the phosphorylation state of these regulatory proteins are responsible for activation of the gastrin gene. To determine if changes in the phosphorylation state of Sp1 or gERP alter binding to gERE, specific phosphatase inhibitors (e.g. okadaic acid) will be added to the gel shift assays. If blocking phosphatase activity does indeed alter transcription factor binding in vitro, then the effect of these phosphatase inhibitors on transient transformants containing gastrin-reporter constructs will be examined. Therefore these studies hope to explore the molecular mechanisms by which the gastrin is regulated, in particular by growth factors.

[unreadable] DESCRIPTION (provided by applicant): Hypergastrinemia has enormous clinical implications with respect to acid secretion, ulcer development and epithelial transformation. It is known that to achieve induction of gastrin (GAS) gene expression and subsequently secretion, the inhibitory effects exerted on GAS-expressing enteroendocrine cells must be suppressed. Somatostatin (SOM) is the most effective physiologic suppressor of GAS expression. More recently, studies have revealed that menin, a tumor suppresor and gene product of the MEN1 locus, is also a strong repressor of gastrin gene expression. The OVERALL GOAL of the proposed studies is to understand the role of menin in mediating the inhibitory effect of SOM on GAS gene expression. Three SPECIFIC AIMS are proposed: 1) To determine how somatostatin inhibits gastrin through regulation of menin; 2) To determine how menin inhibits AP1 induction of gastrin gene expression; 3) To determine how the genetic deletion of menin induces gastrin in vivo. The central hypothesis to be tested is whether there is crosstalk between the SOM inhibitory pathway and the repression induced by menin. SOM blocks cAMP generation inhibiting protein kinase A (PKA) so, we will first examine whether PKA activation must be suppressed to induce menin. A mechanism by which menin mediates its transcriptional control is through direct binding to the AP1 family member JunD. Therefore we mapped the AP1 regulatory element to the proximal gastrin promoter by deletion analysis and site-directed mutagenesis. Surprisingly, the AP1 induction required two proximal Sp1 elements. These are the same elements shown previously to mediate EGF induction of the gastrin promoter. Since AP1 family members directly bind Sp1 at its basic domain and are induced by EGF and ERKs, we will dissect how menin modulates this interaction to suppress EGF induction of gastrin. Specifically, HDAC complexes modulated by menin and known to bind Sp1 will be analyzed. Since MEN1 gastrinomas are autosomal dominant GAS-expressing tumors primarily of the duodenum, a gastrinoma mouse model will be generated by conditional gene targeting so as to study the transcriptional control of GAS in this tumor compared to physiologic GAS regulation in SOM null mice. Relevance to PUBLIC HEALTH include furthering our understanding of peptic ulcer disease, neuroendocrine transformation and the molecular basis underlying the effects of SOM analogs used to treat neuroendocrine disorders [unreadable] [unreadable] [unreadable]

Gastrin is a regulatory peptide expressed in adult antrum. Hypergastrinemic states correlate with gastric acid hypersecretion that result in duodenal ulcerations. Within the last 30 years, progress has been made in understanding the biology and biochemistry of gastrin but little has been elucidated regarding the specific molecular mechanism regulating targets of this hormone nor how the expression of this gene is regulated. The proposal outlined is to support the Third Conference on Gastrin. The first two conferences held in 1968 and 1992 were directed towards understanding the biochemistry of gastrin synthesis and its role in disease states. The conference currently being organized will focus on the translation of gastrin physiology and pathogenesis into animal models and molecular paradigms. A central feature of the conference structure will be the data presentations from Young Investigators and Practical Workshops designed to teach the methods applicable to the projects presented. Several sessions will be devoted to the relationship between colon cancer and Helicobacter pylori. Moreover, sessions given by top epidemiologists in the area will address on the clinical impact of Helicobacter pylori and hypergastrinemia.

DESCRIPTION (provided by applicant): The intestinal enterochromaffin cell (EC) compartment expands in response to acute injury, microbial infection and colitis to produce serotonin (5-hydroxytryptamine, 5HT). Elevated levels of plasma serotonin stimulate fluid secretion and gut motility to expel the infectious or toxic irritants. Although an essential component of the innate immune response, these gut functions contribute to patient discomfort and if sustained become a source of significant morbidity and possible mortality. Despite its central role, it is not understood what regulates EC cell function. During the prior funding cycle, we found that the zinc finger transcription factor ZBP-89 interacts directly with the tumor suppressor protein ataxia telangiectasia mutated (ATM) in response to histone deacetylase inhibition (HDACi), e.g., butyrate or trichostatin A (TSA). Butyrate induces ZBP-89 expression and binding to GC-rich DNA elements in several promoters including the cyclin-dependent kinase inhibitor (CDKI) p21. Butyrate also triggers auto-phosphorylation of ATM at serine 1981 (pATMS1981) that subsequently complexes with ZBP-89 to activate p21. In the current proposal, pATMS1981 positive expression in the gut was found to occur exclusively in the cytoplasm of intestinal EC cells suggesting that pATMS1981 participates in an essential function of these cells. Mice conditionally null for ZBP-89 in the colon exhibited reduced numbers of EC cells and reduced tryptophan hydroxylase 1 gene (TPH1) transcripts on a microarray. Indeed, we found GC-rich DNA elements in the proximal promoter of the TPH1 gene, the rate-limiting synthetic enzyme for 5HT biosynthesis. In addition, pATMS1981-expressing cells in APCmin polyps were absent suggesting that excess Wnt signaling prevents EC cell differentiation. In the current application, three aims are proposed to test the overarching hypothesis that ZBP-89 stimulates serotonin production in gut EC cells by regulating TPH1 gene expression while pATM in the cytoplasm regulates 5HT release. Moreover, we hypothesize that inflammation perturbs their function setting the stage for neoplastic transformation. In Aim 1, we will determine how ZBP-89- regulates TPH1 gene expression. In Aim 2, we will dissect the role of pATMS1981 in butyrate-mediated secretion of 5HT. In Aim 3, we will study the role of these two proteins in 5HT biosynthesis during chronic inflammation and colonic transformation. Collectively, these studies will establish a link between ZBP-89 and pATMS1981, in the biosynthesis and function of 5HT in response to luminal butyrate. PUBLIC HEALTH RELEVANCE: Enterochromaffin (EC) cells secrete serotonin into the circulation to exert a number of clinical effects including increased gut motility and diarrhea. Both the transcription factor ZBP-89 and the tumor suppressor protein ATM are regulated by luminal butyrate from commensal bacteria and are important in maintaining 5HT biosynthesis and secretion from EC cells.

DESCRIPTION (provided by applicant): The Program Project Grant (PPG), " Cellular Decisions of Differentiation in the GI Tract" integrates the efforts of four investigators (two basic science and two clinical) from three Departments of the University of Michigan Medical School. The central goals of the work proposed in the PPG are: (a) To understand how epithelial cells in the upper gastrointestinal tract acquire their identity, both with respect to tissue identity (gastric vs. small intestine) and lineage identity (enteroendocrine cell vs. enterocyte or goblet cell) during ontogeny; (b) To investigate how the patterns of cellular differentiation in the acidsecreting epithelium of the stomach are normally controlled through specific intracellular pathways and how these pathways are altered by pathological insults (such insults can cause an alteration in identity of the gastric epithelium, such that it acquires a small intestinal phenoytpe). Subproject #1 examines the cis and trans factors that control identity in the intestinal epithelial cell, from a tissue-specific standpoint (stomach versus intestine), a regional standpoint (duodenum vs. distal intestine) and a differentiation standpoint (crypt vs. tip). In addition, in conjunction with Subprojects #2 and #4, the question of how gene regulation changes when stomach cells acquire intestinal identity (intestinal metaplasia) after pathological insult will be examined. Subproject #2 will trace the development of the enteroendocrine cell lineage within the intestine using cholecystokinin (CCK) as an early marker for this cell compartment. This work will utilize regulatory transgenes generated in Subproject #1. Subproject #3 will focus on how endogenous growth factors present in the stomach control the pattern of differentiation of the parietal cell. Subproject #4 examines the pathways through which the gastric epithelium responds to inflammation and/or bacterial overgrowth in the stomach through the generation of intestinal metaplasia, an alteration in gastric cell identity; the role of the pathways identified in Subproject #3 will be examined. Two Cores will assist the PPG investigators in the performance of Cell Biology techniques (Core A) and with Administration of the program (Core B). Both Cores will enhance the already strong interaction between these four investigators across Departmental lines. Overall, this PPG application will further our understanding of how cells make decisions of identity and differentiation in the stomach and intestine of the GI tract and will provide clues as to how this identity may be altered in pathological states.

[unreadable] DESCRIPTION (provided by applicant): Chronic inflammation in the stomach (gastritis) is usually associated with Helicobacter pylori, but may occur from bacterial overgrowth because of hypochlorhydria. Chronic gastritis also results in initial increase then loss of parietal cells over time (chronic atrophic gastritis). A recurring theme is that disruption of parietal cell function eventually results in fewer parietal cells followed by an expansion of the mucous and undifferentiated cell types in the stomach. Interestingly, destruction of the parietal cell through ectopic expression of toxins has also been reported to generate the same phenotype. In some instances, these phenotypic alterations progress to the point where mucous cell types emerge, a subset of which express intestine-specific genes (intestinal metaplasia). Intestinal metaplasia is a condition that predisposes the gastric mucosa to cancer. Central to initiating these important alterations are changes in the parietal cell population. In this proposal, we hypothesize that an important trigger altering the normal phenotypic pattern of gastric epithelial cells is inflammation generated from bacterial colonization. The primary goal of this proposal is to understand how components of a bacterial infection trigger parietal cell atrophy and subsequently pre-neoplastic changes. The preliminary results show that both CagA and INF( alter gastric architecture. First, the experiments proposed use a transgenic mouse model expressing CagA (Aim 1) or treatment of mice with pro-inflammatory cytokines (Aim 2) to alter parietal and mucous cell populations. Second, in vitro studies in primary parietal and mucous cells cultures, will be used to dissect the signaling pathways activated (Aim 3) and will study the target proteins regulated during the transformation of the mucosa from chronic atrophy to dysplasia (Aim 4). We will examine whether Sonic hedgehog expressed primarily in parietal cells may be lost during parietal cell atrophy and contribute to the increase in mucosal proliferation and subsequently transformation. These studies will further our understanding of how corpus atrophy predisposes the gastric mucosa to neoplastic transformation. [unreadable] [unreadable]

The molecular basis for the gene specific effects of butyrate remains poorly defined. Butyrate's major known function involves inhibition of histone deacetylases (HDACs) resulting in increased acetylation. In addition to histone acetylation, it is now known that DNA binding proteins become acetylated. The proposed function of acetylated transcription factors varies and includes increased or decreased DNA binding as well as protein stability. In many instances, the genetic targets of butyrate are GC-rich sequences that bind Sp1 and Sp3. Recruitment of the histone acetyltransferase (HAT) p300 cooperates with Sp1 and Sp3 to mediate the effects of butyrate to the p21waf1 promoter. However, Sp1 does not complex with p300, but instead binds HDAC1. We have shown previously that ZBP-89 is another DNA binding protein that binds GC-rich sites and mediates butyrate induction of p21waf1. Understanding the mechanisms by which butyrate suppresses growth through ZBP-89 is the focus of this competing renewal. ZBP-89 is an 89 kDa Kruppel-type zinc finger protein composed of 794 residues. During the past funding period, we demonstrated that ZBP-89 interacts with the tumor suppressor protein p53 to induce G1 arrest. We have recently found that ZBP-89 interacts with the tumor suppressor protein ataxia telangiectasia, mutated (ATM) in a butyrate specific manner. ATM modulates factors involved in both G1 and G2 cell arrest after DNA damage. ATM mediates cell cycle arrest through phosphorylation of p53 at Ser15. ZBP-89 is required for phosphorylation of p53 at Ser15. Therefore the specific aims of this proposal are 1) To dissect the interaction of ZBP-89 with ATM in response to butyrate. 2) To dissect the regulation of p53 activation by ZBP-89 in response to butyrate. 3) To dissect the mechanisms of p300 HAT activation in the regulation of ZBP-89. 4) To determine whether ZBP-89 exhibits tumor suppressor function. Site-direct mutations in various domains of ZBP-89 will be used to dissect the interactions with these cell cycle regulators. ZBP-89 interactions with chromatin will be studied using confocal microscopy, cell fractionation and ChIP assays. In this way, we will further the understanding of how butyrate inhibition of HDACs ultimately suppresses cell growth and prevents neoplastic transformation.

The purpose of the Molecular Biology core is to facilitate the application of the tools of molecular genetics[unreadable] to the study of gastrointestinal peptides and their physiological functions. In all aspects, the Molecular[unreadable] Biology Core functions to provide essential services and serves as an educational resource to all Center[unreadable] Investigators. Although basic molecular techniques are widely available to Center investigators through[unreadable] commercial sources, there remain specific services that are best provided by pooled resources and by highly[unreadable] trained personnel such as those that can be provided by a Center. During the prior funding period, three[unreadable] Core programs were provided: the Transgenic Animal Program, the Viral Vector Program and the Gene[unreadable] Expression Program. The Molecular Biology Core has added a Microarray Gene Chip Program and has[unreadable] essentially updated the Gene Expression Program to provide education and training in those techniques that[unreadable] can be performed in the investigators own laboratory after receiving some theoretical and practical training.[unreadable] Since investigator needs have likely moved beyond basic gene expression approaches to involve such[unreadable] techniques as chromatin structure and analysis, this primarily education program has been renamed the[unreadable] Molecular Techniques Training Program. Investigators needing assistance with basic molecular techniques,[unreadable] e.g., quantitative RT-PCR and more sophisticated techniques, e.g., chromatin immunoprecipitation assays[unreadable] (ChIP), can receive training from the Core Director (Dr. Merchant) and her associate (Dr. Bai).

The goals of the current application are to understand the role of the Sonic Hedgehog (Shh) ligand and components ofthe Hh signaling apparatus Glil and Gli2 in the homeostasis and subsequent translation of chronic gastritis to metaplasia, a preneoplastic lesion. It has been reported in human epidemiologic studies as well as mouse models that the development of tumors in the gastric corpus versus the antrum emerge ostensibly in response to different signals. As a result, we have focused our attention on cellular decisions that impact the emergence of corpus versus antral tumors. Studies completed during the prior funding period confirmed regional differences in the expression and function of the Shh ligand and the Hh signaling componets Glil-expressed in myeloid cell populations; and Gli2-expressed primarily in both antral mesenchyme and a hyperplastic.antral epithelium. In the antrum, primary cilia, an organelle linked to Gli2 function, and gastrin are important to normal gastric homeostasis, characterized by gastric acidity. We previously demonstrated that gastrin null mice develop antral tumors and recently reported an increase in epithelial Gli2 expression in these hyperplastic antrums. Moreover ectopic expression of Gli2 in the gastric epithelium suppresses gastrin expression and ultimately results in antral hyperplasia. In a mouse model of Helicobacter infection, we found that the corpus exhibits greater dependency on canonical Hh signaling than the antrum. In particular during /7e//co/)ac/er infection, the corpus acutely recruits Glil-expressing myeloid cells (<2 months) that appear to modify their surface markers in the chronically inflamed stomach to markers indicative of myeloid-derived suppressor cells (MDSCs). By 6 months, corpus metaplasia has emerged correlating with Gli1+-MDSCs their secretion of IL-lp. Thus we hypothesize that the contribution of Hh signaling to gastric homeostasis and hyperplasia differs according to their location in the stomach (corpus versus antrum). Aim 1 will examine the role of Hh signaling in antral homeostasis and in particular, its role in regulating gastrin and their relationship to primary cilia. Aim 2 will establish whether the proinflammatory cytokine IL-ip is sufficient to induce epithelial expression of Gli2 and antral hyperplasia. Crosstalk with the Notch signaling pathway will be explored in collaboration with Subproject #3. Aim 3 will test the hypothesis that the time lag between chronic gastritis and metaplasia in the corpus requires pathogen-related maturation of myeloid cells in the inflamed gastric environment. The role of Hh signaling through Glil will be compared to Hh signaling in the inflamed intestine in collaboration with Subproject #1. RELEVANCE (See instructions): This Subproject will define the functional differences in Hedgehog signaling components in gastric homeostasis that ultimately alters cellular decisions in the regional response to environmental stress, e.g., chronic inflammation and preneoplastic lesions such as metaplasia.

Abstract Chronic Helicobacter infection and the resulting inflammation induces gastric metaplasia in the corpus of WT mice but not in mice null for Gli1, demonstrating that this pre-neoplastic lesion requires the induction of canonical Hedgehog signaling. Moreover, we reported that a subset of myeloid cells expressing surface markers and T cell suppressor function indicative of myeloid-derived suppressor cells (MDSCs) express Schlafen4 (Slfn4), a direct target of the Gli1 transcription factor and a known myeloid differentiation factor. Since MDSCs are immature cells, collectively our studies demonstrate that maturation of this myeloid cell subpopulation requires Gli1 and produces proinflammatory cytokines creating a gastric microenvironment favorable for metaplasia and neoplastic transformation. More recently, we have analyzed the human homologs of Slfn4, which include SLFN5 and SLFN12L. We reported that peak expression of SLFN5 in gastric tissue occurred in human subjects with intestinal metaplasia who about a decade later developed gastric cancer. We therefore considered that the polarization of myeloid cells to MDSCs prior to neoplastic transformation might predict who is more likely to develop gastric cancer and as such could provide a therapeutic target to prevent future transformation. We used RNA-Seq and Nanostring microarrays to identify transcripts and microRNAs expressed in Slfn4+-MDSCs from the stomachs of a Helicobacter-infected mouse and found that miR130b co-localized with Slfn4+ cells in the metaplastic mouse stomach. A similar result was observed for SLFN12L in the metaplastic human stomach suggesting that miR130b might identify patients with gastric metaplasia. Our preliminary results demonstrated that Slfn4 and miR130b are required to exert T-cell suppression. In addition to type 1 interferon-regulated genes identified by RNA-Seq, these Slfn4+-MDSCs also expressed several tumor necrosis factor superfamily ligands (TNFsf) and the alarmin IL-1a. Therefore we will test the hypothesis that debris from damaged gastric epithelial cells activates Damage-activated molecular pattern (DAMP) signaling, production of IFNa and polarization to MDSCs, which contribute to a metaplastic phenotype. Aim 1, we will define how DAMP signals induce polarization of Slfn4+-MDSC. In Aim 2, we will define how Slfn4 contributes to MDSC function. In Aim 3, we will define the contribution of Slfn4+-MDSCs to Helicobacter-induced metaplasia. In Aim 1, we will use a combination of flow cytometry, T cell suppression assays and transfection studies to identify the cell populations producing IFNa and the gene targets responding to this DAMP-activated cytokine. In Aim 2, we will used mass spectrometry and pull down assays to identify Slfn4 and SLFN12L interacting proteins. In Aim 3, we will conditionally delete Slfn4 from Gli1- expressing cells to define their contribution to the metaplastic changes observed after Helicobacter infection. Completion of these aims will result in a better understanding of this MDSC subpopulation that can potentially be used as a biomarker and target of small molecule inhibitors.

Project Summary/Abstract. (30 lines) Our mission within the UA COM-T, MD-PhD Program is to develop a diverse pool of highly trained physician- scientists who have the aptitude to utilize clinical experience in developing biomedical hypotheses that integrate the research skills learned in our program. We intend to have all trainees independent, productive and rewarding physician-scientist careers. This program has established 6 objectives that include; 1) dual- degree completion rates at >97% with an appropriate time-to-degree (7.3 yrs on avg.), 2) the integration of research and clinical activities, 3) nurture a broad understanding of biomedical disciplines, 4) foster and encourage the development of good scientific premise, rigorous-research design, strong experimental methods with reproducibility/validation, as well as skills to analyze and interpret results/outcomes amid ethics and integrity, 5) build proficiency in initiating, conducting, interpreting, and presenting rigorous and reproducible biomedical research with increasing self-direction, while strengthen skills to teach and communicate, and 6) fortify diversity while advancing the knowledge, professional skills, and experiences required to identify and transition into productive careers in the biomedical research workforce that utilize the dual-degree training. UACOM-T MD-PhD Program offers a wide range of biomedical disciplines for trainees with 13 graduate programs available and over 60 faculty trained and excited to be mentors. Our rationale for an NIH-MSTP is the desperate need for the increase in Physician-Scientists in the state of AZ; a population that is rapidly growing and aging with multiple medical and research needs. AZ is the fourth most populous state for Hispanics and has the third largest Native American population in the US. UArizona is designated as both a Hispanic and American Indian/Alaska Native Serving Institution. UArizona has a strong understanding of the concerns of diverse individuals with behavioral health needs, excels in cultural competency, and has long recognized its unique mission and obligation to serve all of the diverse peoples and communities. UArizona has more than doubled its enrollment of URMs in the last 10 years, comprising 20% of all graduate students, placing UArizona at the top of all Research I, AAU-member institutions. Hence, the UA COM-T is a perfect institution to serve and uphold the goals and intentions of the MSTP by increasing the number of URMs as physician scientist. The current MD-PhD program has been funded entirely by support from the institution resulting in a small but overall very successful cohort of physician scientists with past trainees in academic positions at Stanford, Harvard, Yale, Vanderbilt, Baylor, OHSU, Scrips, etc. Our program has established a comprehensive 7-year training plan that reduces unnecessary redundancies, utilizes evidence based medicine training, integrates clinical and research over all 7 years while creating and encouraging an inclusive climate.