Roger J. Davis - US grants

There is a close association between the expression of the receptor for transferrin and cellular growth. The cell surface number of transferrin receptors is acutely regulated by polypeptide mitogens. We propose to study the biochemical mechanisms by which platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) cause a rapid (less than 2 min) redistribution of intracellular transferrin receptors that results in the increased expression of the transferrin receptor at the cell surface. We will test the hypothesis that the mechanism of action of the growth factors is to alter the rate of exocytosis or endocytosis of the transferrin receptor by directly investigating the kinetics of the cycling of the transferrin receptor between cell surface and endosomal membrane compartments. We will also investigate the cell-cycle dependence of the growth factor regulation of the transferrin receptor by flow cytometry. A central issue that we address is whether covalent post-translational modification (phosphorylation, palmit ylation) of the transferrin receptor is relevant to the regulation of the transferrin receptor cycling by growth factors. Data is available from this laboratory which shows that EGF causes the phosphorylation of the transferrin receptor at a site that we have identified as serine-24. We propose to investigate this phosphorylation in detail. The effect of PDGF on transferrin receptor phosphorylation will also be examined. A critical element of our strategy to investigate the role of the phosphorylation of serine-24 is to examine whether serine-24 is essential for the regulation (by EGF and PDGF) of the expression of the transferrin receptor at the cell surface. We propose to introduce point mutations into the human transferrin receptor cDNA at codon-24 by oligonucleotide-directed mutagenesis. The wild-type and mutanttransferrin receptors will then be expressed in NIH 3T3 cells using a retrovirus vector. The regulation of the endogenous (murine) and introduced (human) transferrin receptors by EGF and PDGF will be investigated using species-specific monoclonal antibodies. We hope to learn fundamental information from the studies described in this proposal about (1) the biochemical mechanisms that regulate the cycling of the transferrin receptor, (2) the signalling mechanisms involved in the actions of PDGF and EGF, and (3) the relationship between the regulation of the expression of the transferrin receptor and cellular growth.

The overall goal of this research program is to understand the process of signal transduction by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin 1 (IL-1). The potent biological actions of these cytokines have been studied in detail. However, an understanding of the molecular basis of signal transduction has remained elusive. The long- term goal of this research is to define physiologically relevant signaling mechanisms employed by these cytokines. Recently, we identified a protein kinase that is markedly activated by treatment of cells with TNF or IL-1. This protein kinase, JNK, binds and phosphorylates the amino-terminal activation domain of the transcription factor c-Jun. The phosphorylation of c-Jun by JNK causes increased transcriptional activity and accounts, in part, for cytokine-stimulated gene expression mediated by AP-1. The mechanism of JNK activation by cytokines is mediated by dual phosphorylation on Thr and Tyr. A specific focus of this proposal is to define the functional significance and mechanism of activation of this protein kinase signal transduction pathway. Achievement of the goals of this proposal will increase understanding of signal transduction by cytokine receptors. This information represents a basis for the design of novel therapeutic strategies for the treatment of: 1) inflammation; and 2) proliferative diseases such as psoriasis and cancer. The Specific Aims of this proposal are to examine: 1. The JNK protein kinase cascade. 2. The activation of the JNK pathway by cytokine receptors. 3. The interaction of JNK with other proteins.

DESCRIPTION (Adapted from applicant's abstract): The overall goal of this research program is to define the signal transduction mechanisms that mediate the response of cells to inflammatory cytokines and environmental stress. A focus of the study is the c-Jun NH2-terminal kinase (JNK) group of MAP kinases, which are activated in response to cytokines and stress. Many of the components of the JNK protein kinase signal transduction pathway have been identified by molecular cloning and have been characterized in biochemical studies. However, an understanding of the functional significance of the JNK signaling pathway in vivo has remained elusive. Recently, the PI has constructed mice with germ-line mutations in the three gene that encode the JNK protein kinases (Jnk1, Jnk2 and Jnk3) and in both of the genes that encode the protein kinases that phosphorylate and activate JNK (Mkk4 and Mkk7). Comparison of cells with wild-type and mutant genotypes can therefore provide important information concerning the physiological function of the JNK signaling pathway. A specific focus of this proposal is to define the functional significance of the JNK signal transduction pathway. Achievement of the goals of this proposal will increase understanding of the MAP kinase signal transduction in vivo. The information represents a basis for the design of novel therapeutic strategies for the treatment of [1] inflammation, and [2] proliferative diseases such as psoriasis and cancer. The specific aims of this proposal are to examine the effects of the JNK signaling pathway on : 1. Apoptosis and cell survival; 2. Oncogenic transformation and 3. Gene expression.

The overall goal of this research program is to understand the mechanism of signal transduction mediated by MAP kinase scaffold complexes in mammalian cells. A focus of this study is the c-fun NH2-terminal kinase (JNK) group of MAP kinases. Many of the components of the JNK protein kinase cascade have been identified by molecular cloning and have been characterized in biochemical studies. However, an understanding of the molecular mechanism of signal transduction in vivo has remained elusive. The long-term goal of this research is to define physiologically relevant signaling mechanisms that are employed to regulate the JNK MAP kinase signaling cascade in vivo. Recently, we have identified JIP1 as a protein that functions as a scaffold for the assembly of components of the JNK MAPK signaling pathway. JIP1 binds selectively to JNK and also to a MAPKK (MKK7), to a MAPKKK (MLK protein kinases), and to a Ste20 protein kinase (HPK1). Binding to JIP1 facilitates JNK activation by this protein kinase signaling cascade. A specific focus of this proposal is to define the functional significance and mechanism of action of this JIP-mediated signal transduction pathway. Achievement of the goals of this proposal will increase understanding of the molecular mechanism of MAP kinase signal transduction in vivo. This information represents a basis for the design of novel therapeutic strategies for the treatment of: 1) inflammation; and 2) proliferative diseases such as psoriasis and cancer. The Specific Aims of this proposal are to examine: 1. The physiological role of JIP scaffold proteins. 2. The structure and dynamics of JIP scaffold complexes. 3. The mechanism of JIP-stimulated JNK activity.

DESCRIPTION (proved by applicant): This application is for partial funding for the 2001 FASEB conference on Protein Kinases and Phosphorylation. The meeting will be held in Snowmass, Colorado, Saturday July 24 to Thursday, July 29, 2001. This conference is generally considered to be the premiere bi-annual meeting on protein kinase research and brings together 43 speakers and more than two hundred research scientists. The conference will cover many aspects of protein kinase biochemistry, cell and physiological function, pharmacology, and target these enzymes inside the cell. The 2001 meeting will focus on emerging topics of kinase research and will center around the themes of: kinases/phosphatases involved in metabolism and disease, kinase/phosphatase regulatory mechanisms, MAP kinase signaling complexes, kinase/phosphatase structure-function analysis, small molecule inhibitors, imaging of phosphorylation events, kinases/phosphatase regulation of transcription, developmental fate, the cytoskeleton, and cell cycle regulation. The opening lecture will be given by Dr. John Scott. Dr. Scott has completed much of the pioneering work on the identification of protein modules such as SH2 and SH3 domains that govern the assembly of multienzyme signal transduction complexes. The keynote address will be given by Dr. Jack Dixon, who has conducted much of the early work on understanding the role of protein lipid phosphatases in normal and disease physiology.

DESCRIPTION (provided by applicant): The overall goal of this research program is to understand the role of the c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. Many of the components of the JNK protein kinase cascade have been identified by molecular cloning and have been characterized in biochemical studies. However, an understanding of the physiological role of the JNK signaling pathway has remained elusive. The long-term goal of this research is to define function of the JNK signaling pathway in the beta cell. Previous studies have demonstrated that JNK can phosphorylate and inhibit the function of insulin receptor substrate (IRS) proteins. In addition, the JIP scaffold proteins that co-ordinate the JNK protein kinase cascade have been found to be mutated in diabetic humans. We have constructed several mouse models with defects in the JNK signaling pathway, including mice with defects in the JIP scaffold proteins. These mouse models will be used to study the function of these proteins in beta cells Achievement of the goals of this proposal will increase understanding of signal transduction; mechanisms that contribute to normal beta cell function. This information may represent a basis for the design of novel therapeutic strategies for the treatment of diabetes. The Specific Aims of this proposal are to employ mouse models to: Define the role of JNK in beta cells. Define the role of JIP scaffold proteins in beta cells Define the function of diabetes-associated mutations in JIP1

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this research program is to understand the mechanism of signal transduction mediated by MAP kinases in mammalian cells. A focus of this study is the c-Jun NH2-terminal kinase (JNK) group of MAP kinases. Many of the components of the JNK protein kinase cascade have been identified by molecular cloning and have been characterized in biochemical studies. However, a complete understanding of the physiological function of JNK has remained elusive. The long-term goal of this research is to define the molecular mechanisms and physiological significance of JNK activation in neurons. A specific focus of our analysis will be to determine the role of JNK in neurodegeneration. [unreadable] Achievement of the goals of this proposal will increase understanding of the molecular mechanism of MAP kinase signal transduction in vivo. This information represents a basis for the design of novel therapeutic strategies for the treatment of neurodegenerative diseases, including stroke. [unreadable] The Specific Aims of this proposal are to examine: [unreadable] 1. The physiological role of JNK in neurons. [unreadable] 2. The mechanism of JNK-induced neurodegeneration. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this research program is to develop a general method that can be used to disrupt protein kinases selectively in the mouse prostate epithelium. This will be critical for understanding the role of protein kinases in the normal development of the prostate epithelium and also during malignant tumor progression. This understanding is essential if protein kinases are to be used as targets for the development of small molecule drugs for the treatment of diseases of the prostate epithelium, including cancer. The development of a successful strategy for disrupting protein kinases in the mouse prostate requires that two separate goals are met. First, the protein kinase must be selectively disrupted in the prostate epithelium. Second, the disruption must be temporally regulated since the exact timing of protein kinase loss- of-function will be very important for understanding the role of the protein kinase in both the development of the prostate and the function of the mature prostate gland. Similarly, temporal control of protein kinase disruption will be required for studies of tumor initiation and progression. The model system that we propose to examine involves the c-Jun NH2-terminal kinase (JTSIK) group of stress-activated MAP kinases. It is anticipated that the development of this specific model for JNK will provide information that will guide the application of the same method to other protein kinases The Specific Aims of this proposal are to examine: 1. Develop a method to selectively disrupt JNK in the murine prostate epithelium.. 2. Develop a general method that can be used to selectively and temporally regulate the function of JNK in the murine prostate epithelium. [unreadable] [unreadable] [unreadable]

The overall goal of this research program is to understand the mechanism of signal transduction mediated by MAP kinases in mammalian cells. A focus of this study is the c-Jun NH2-terminal kinase (JNK) group of MAP kinases. Many of the components of the JNK protein kinase cascade have been identified by molecular cloning and have been characterized in biochemical studies. However, a complete understanding of the physiological function of JNK has remained elusive. The long-term goal of this research is to define the molecular mechanisms and physiological significance of JNK activation caused by inflammatory cytokines. Achievement of the goals of this proposal will increase understanding of the molecular mechanism of MAP kinase signal transduction in vivo. This information represents a basis for the design of novel therapeutic strategies for the treatment of proliferative diseases, including cancer. The Specific Aims of thisproposal are to examine: 1. The physiological role of JNK in TNF signal transduction. 2. The mechanism of JNK-induced apoptosis.

The overall goal of this research program is to understand transplantation tolerance mediated by costimulation blockade. This form of transplantation tolerance is associated with the deletion of alloreactive CDS T cells. Importantly, the activafion of innate immunity by virus infection or exposure to Toll-like receptor agonists can prevent both alloreactive CDS T cell delefion and tolerance induction. The specific focus of this Study is to define the molecular mechanisms of CDS T cell apoptosis. We propose to examine the biochemical mechanism of CDS T cell death (Specific Aim 1). These Studies will provide the foundation for molecular studies of specific pathways of CD8 T cell death (Specific Aims 2 &3). It is established that members of the Bcl2 protein family act as critical regulators of CDS T cell death. Moreover, members ofthe stress-activated protein kinase family are implicated in the regulafion of CDS T cell death. We will examine these pathways during the induction of transplantation tolerance during co-sfimulation blockade. We will also examine CDB T cell death when transplantafion tolerance is disrupted by exposure to Toll-like receptor agonists and lymphocyfic choriomeningifis virus (LCMV) infection. These studies are fully integrated within the theme of the Program Project and depend upon collaborative studies with the other Projects. The Specific Aims of this proposal are to examine the: 1. Biochemical mechanism of CDS T cell death. 2. Role of stress-activated MAP kinases in CDS T cell death. 3. Role of Bcl2 familv proteins in CDB T cell death. We anticipate that the successful completion of these studies will provide important new insight into the understanding of transplantafion tolerance and that the new informafion we obtain will contribute to the design of therapies for the treatment of human disease.

DESCRIPTION (provided by applicant): Human obesity represents a serious world-wide health problem. One consequence of obesity is the development of insulin resistance, hyperglycemia, and metabolic syndrome that can lead to cell dysfunction and type 2 diabetes. It is therefore important that we gain an understanding of the physiology and pathophysiology of the development of obesity because this knowledge represents a basis for the design of potential therapeutic interventions. A significant challenge to understanding diet-induced obesity is the complexity of the signal transduction pathways that mediate the response. Indeed, these signaling pathways function within an interacting network. Here we propose to employ a systems biology approach to understanding the response to feeding a high fat diet by combining physiological analysis together with quantitative analysis of the signal transduction networks and the genomic response. This analysis requires the coordinated collaborative efforts of several laboratories with complementary expertise together with robust data analysis and computational modeling. We will focus our analysis on the liver. The overall goal of this research program is to understand the mechanism of the hepatic response to diet-induced obesity. Achievement of the goals of this proposal will increase understanding of the molecular response to obesity. We anticipate that the successful completion of this research program will lead to the identification of nodes in the signaling network that may represent a basis for the design of novel therapeutic strategies for the treatment of metabolic syndrome and type II diabetes. The Specific Aims of this proposal are to: 1. Examine the hepatic response to feeding a high fat diet. 2. Integrate data analysis using computational modeling. 3. Test predictions obtained from computational modeling. PUBLIC HEALTH RELEVANCE: Metabolic syndrome and type II diabetes are serious diseases that have a profound impact on the health of many Americans;it is essential that we develop new treatment options for these diseases. The purpose of this research proposal is to perform a systems biology analysis of the hepatic response to diet-induced obesity. We anticipate that this information will provide a basis for the design of novel therapeutic strategies for the treatment of metabolic syndrome and type II diabetes.

? DESCRIPTION (provided by applicant): Human obesity represents a serious world-wide health problem. One consequence of obesity is the development of metabolic syndrome, characterized by insulin resistance and hyperglycemia, that can lead to ? cell dysfunction and type 2 diabetes. It is therefore important that we gain an understanding of the physiology and pathophysiology of the development of obesity because this knowledge represents a basis for the design of potential therapeutic interventions. Recent studies have identified the cJun NH2-terminal kinase (JNK) signal transduction pathway as a mediator of metabolic stress responses. Feeding a high fat diet (HFD) causes increased JNK activity and promotes both obesity and insulin resistance. Studies using tissue-specific knockout mice demonstrate a central role for JNK in the regulation of energy expenditure and the development of obesity. In contrast, JNK in peripheral tissues can cause insulin resistance without changes in obesity. The mechanism that accounts for JNK- dependent insulin resistance caused by feeding a HFD has not been defined. We have identified the PPAR? pathway as a major target of hepatic JNK signaling that contributes to HFD- induced insulin resistance by regulating the expression of the hepatokine fibroblast growth factor 21 (FGF21). We have demonstrated that JNK activation caused by feeding a HFD potently suppresses PPAR? activity. Consequently, disruption of hepatic JNK activity causes increased hepatic PPAR? activity, increased amounts of FGF21 circulating in the blood, and improved glycemia in HFD-fed mice. Disruption of Fgf21 expression prevents the effects of JNK inhibition to cause improved glycemia. Based on these data, we propose that the PPAR?/FGF21 axis mediates the effects of hepatic JNK on insulin sensitivity. The overall goal of this research program is to test the hypothesis that the PPAR?/FGF21 axis contributes to metabolic stress signaling by hepatic JNK. We will examine the mechanism of JNK-mediated repression of PPAR? activity. We will also test the role of the PPAR? target gene Fgf21 as a mediator of JNK-regulated insulin resistance. Achievement of the goals of this proposal will increase understanding of the molecular response to obesity. We anticipate that the successful completion of this research program will lead to the identification of new mechanisms that contribute to the obesity response. This knowledge may represent a basis for the design of novel therapeutic strategies for the treatment of metabolic syndrome and type 2 diabetes.

Human obesity represents a serious world-wide health problem. One consequence of obesity is the development of metabolic syndrome, characterized by insulin resistance and hyperglycemia, that can lead to ? cell dysfunction and type 2 diabetes. It is therefore important that we gain an understanding of the physiology and pathophysiology of the development of obesity because this knowledge represents a basis for the design of potential therapeutic interventions. Recent studies have identified increased energy expenditure caused by adipose tissue thermogenesis as an important contributing factor that can limit obesity development. The sympathetic nervous system promotes adipose tissue thermogenesis by activating brown adipose tissue. The magnitude of this response can be increased by the presence of brown-like adipocytes in white adipose tissue depots. These brite/beige adipocytes are more common in sub-cutaneous adipose tissue compared with visceral adipose tissue, and their presence is strongly induced by exposure to cold. Control of beige/brite adipocytes ? for example, using pharmacological tools ? represents a potential therapeutic option for the treatment of obesity. Consequently, it is important that we gain an understanding of molecular mechanisms that contribute to adipose tissue thermogenesis. This knowledge is critical for identifying possible molecular targets that could be employed for therapeutic intervention. Significant progress has been achieved towards defining beige/brite cell development and function, including the role of signaling pathways and transcription factors. However, there are significant gaps in our knowledge. Recent studies in my laboratory have uncovered a role for alternative pre-mRNA splicing in the regulation of adipose tissue thermogenesis. We have identified widespread changes in alternative pre-mRNA splicing in white adipocytes following consumption of a high fat diet. Bioinformatic analysis identified NOVA binding sites in a large fraction of regulated adipocyte pre-mRNA splicing events. Indeed, we found that NOVA expression is regulated by diet-induced obesity in both rodents and humans. To test the role of NOVA proteins, we established Nova1LoxP/LoxP and Nova2LoxP/LoxP mice and studied the effect of NOVA-deficiency in adipocytes. We found that NOVA-deficiency caused ?browning? of white adipose depots, increased adipose tissue thermogenesis, and protection against diet-induced obesity and metabolic syndrome. These studies identify pre-mRNA splicing as a potential target for therapeutic intervention in obesity-induced metabolic syndrome. Importantly, previous studies have established pre-mRNA splicing as a pharmacologically tractable target for therapeutic intervention in diseases. The overall goal of this research program is to identify molecular mechanisms that account for the function of NOVA pre-mRNA splicing factors in adipocytes. Achievement of this goal will increase understanding of the molecular response to obesity. We anticipate that the successful completion of this research program will lead to the identification of new mechanisms that contribute to the obesity response. This knowledge may represent a basis for the design of novel therapeutic strategies for the treatment of metabolic syndrome and type 2 diabetes.

Project Summary Human obesity represents a serious world-wide health problem that is associated with metabolic syndrome and the development of non-alcoholic fatty liver disease (NAFLD) that can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). The estimated prevalence of NAFLD in the USA is approximately 25% of the population (1). Estimates for the prevalence of NASH are confounded by the limited availability of reliable non-invasive methods for diagnosis, but approximately 25% of patients with NAFLD exhibit NASH (1). The development of scarring (cirrhosis) and hepatic fibrosis contributes to the development of HCC, the most common liver cancer and the 3rd leading cause of cancer-related death in the USA (2, 3). The high incidence of NAFLD/NASH represents a major health problem because: 1) NASH is anticipated to become the leading indication for liver transplantation (4); and 2) NAFLD/NASH-associated HCC is the primary cause of obesity-related cancer death in the USA (4). Lifestyle interventions, including dietary calorie restriction and exercise, are key aspects of current therapy. However, there is an unmet need for effective pharmacotherapy. Several medications are currently under development, including approaches to reduce steatosis, correct intestinal dysbiosis, promote oxidative stress defense, and suppress fibrosis (5, 6). Recent studies have established that the oxidative stress-responsive protein kinase ASK1 (a member of the MAP3K group) is a promising drug target for the treatment of NASH (5, 6). The small molecule Selonsertib is a potent inhibitor of ASK1 protein kinase activity that causes reduced NASH-related hepatic fibrosis (7, 8), a key determinant of disease progression (4). Successful phase 2 trials of Selonsertib in NASH patients (7, 8) have led to phase 3 trials (STELLAR 3 and STELLAR 4) that are currently in progress (8). The mechanism that accounts for the beneficial effects of blocking ASK1 is unknown, but likely involves a reduction in the activation state of down-stream signaling pathways (e.g. stress-activated MAPK). ASK1 is expressed ubiquitously. Consequently, no information is available concerning the hepatic cell type that mediates the ASK1-promoted hallmarks of NASH, including hepatic fibrosis. It is possible that ASK1 plays a key role in an inflammatory response (e.g. in Kupffer cells and other immune cells) that drives hepatic fibrosis (9). Alternatively, ASK1 may play an important role in steatotic hepatocytes that promotes fibrosis (10). It is also possible that the key role of ASK1 may be in stellate cells that are directly involved in hepatic fibrosis (11). The relevant hepatic cell type that mediates the essential function of ASK1 in NASH has therefore not been defined. Moreover, the mechanism that mediates ASK1 regulation during the development of NASH is not understood. This proposal is designed to identify the mechanism of ASK1 signaling during NASH development. Completion of this study will provide important new information concerning the mechanism of action of drugs that inhibit ASK1. Two Specific Aims are proposed: 1) To identify the hepatic cell type that mediates the effects of ASK1 on hallmarks of NASH; and 2) To identify hepatic mechanisms that regulate ASK1 during NASH development. The overall goal of this research program is to identify molecular mechanisms that account for the function of ASK1 in NASH. Achievement of this goal will increase understanding of NASH development. We anticipate that the successful completion of this research program will lead to the identification of new molecular mechanisms. This knowledge may represent a basis for the design of novel therapeutic strategies for the treatment of NASH, including the rational design of combination therapies.