Keith E. Mostov, Ph.D / M.D. - US grants

Polymeric IgA and IgM (polymeric immunoglobulins, pIg) bind to a receptor on the basolateral surface of many types of epithelial cells, and are endocytosed, transported across the cell in vesicles, and exocytosed into external secretions. During transport, a portion of the receptor is cleaved off and released into the secretion with the pIg. This released fragment is called secretory component (SC). The goal of this research is to determine the molecula mechanism of transcellular transport of the pIg and the pIg receptor (pIgR). This is important for 3 reasons. First, transport of pIg into secretions is a major defense against infection. Second, failure to transport IgA may lead to IgA immune complex deposition, which occurs in Berger's disease, Henoch-Schonlein Purpurae, and possibly liver disease. Third, pIgR is an excellent model for studying general mechanisms of protein sorting and traffic, and can thus provide insights relevant to a broad range of biomedical problems. During the period of this proposal, several approaches will be used to study the mechanism of pIgR transport. First, it is known that inhibitors of acidification of endocytotic vesicles disrupt the transport of other endocytosed receptors and the effects of such inhibitors on pIgR transport will be investigated. Second, proteins that bind to the pIgR and may mediate its transport will be isolated and characterized. Third, the introns and exons in the pIgR gene will be located in order to help understand the structural and functional units of the pIgR molecule. Fourth, the pIgR gene will be introduced and expressed in mammalian cells. By altering the gene in vitro by deletions, mutagenesis, and recombination, the portion(s) of the pIgR molecule needed for transport can be determined.

Polymeric IgA and IgM (polymeric immunoglobulins, pIg) bind to a receptor on the basolateral surface of many types of epithelial cells and are endocytosed, transported across the cell in vesicles, and exocytosed into external secretions. In a related process, neonatal rats acquire immunity from their mothers by transporting milk IgG from the lumen of the small intestine to the basolateral surface of the intestinal cell and releasing it into the circulation. Transport of pIg and IgG are in opposite directions across the cell. The goal of the research is to determine the molecular mechanisms of transcellular transport of pIg and IgG. This is important for four reasons. First, transport of pIg into secretions is a major defense against infection. Second, failure to transport IgA may lead to IgA immune complex deposition, which occurs in several diseases. Third, intestinal transport of IgG in rats is a model for transplacental transport of IgG in humans, which is difficult to study. Fourth, transport of pIg and IgG is an excellent model for studying general mechanisms of protein sorting, and can thus provide insights relevant to a broad range of biomedical problems. Several approaches will be used. 1) The exons in the pIg receptor (pIg-R) gene will be located to understand the functional units of the protein. 2) The pIg-R has been expressed in polarized Madin Darby Canine Kidney (MDCK) cells, where it functions as in vivo. Its pathway will be studied by immunocytochemistry. 3) The cytoplasmic tail of pIg-R contains sorting signals that send pIg-R to the basolateral surface and causes endocytosis. This tail has been produced by expression in E. coli and its structure will be studied by X-ray diffraction. 4) The sorting signals in the tail will be studied by making various deletions as well as fusing it to other proteins and expressing these in MDCK cells. 5) Proteins that bind to this cytoplasmic tail will be isolated. 6) Cell-free assays for pIg-R transport will be developed. 7) The intestinal IgG receptor will be cloned and sequenced, and the cDNA will be expressed in MDCK cells to study the receptor's function.

Newborn humans rely on maternally-derived antibodies for protection against pathogens. IgG is transported across the placenta to the fetus. After birth lgA in the milk and colostrum protects the GI tract, but these antibodies are not specifically absorbed into the infants circulation. The purpose of this proposal is to develop a general method by which ingested antibodies can be absorbed across the intestine and into the circulation of the infant. Intestinal epithelial cells express the polymeric immunoglobulin receptor (pIgR), which transport dimeric IgA from the basolateral to the apical surface of the epithelial cell. At the apical surface the pIgR is proteolytically cleaved and the extracellular domain, known as secretory component (SC), is released into the intestinal lumen. Cleavage is not very rapid, so there is a pool of uncleaved pIgR at the apical surface. We have shown that ligands can bind to this pIgR, and are internalized and transcytosed to the basolateral surface. We plan to optimize this process for absorption of ingested antibodies across the intestinal epithelium and into the circulation. Our specific goals will be: 1). We will prepare antibodies against the extracellular, membrane- proximal "stalk" of the pIgR. These antibodies may prevent cleavage of the pIgR to SC and/or bind to the stalk that remains after cleavage. In either case, the antibodies should be efficiently taken up by the pIgR at the apical surface. Fab fragments of these antibodies will be used to study transcytosis from the apical to the basolateral surface. Fab fragments of antibodies of varying affinity will be used to optimize transcytosis. 2). These Fab-anti-stalk (Fab-AS) will be conjugated to Fab fragments of a model anti-pathogen antibody. Transcytosis of these conjugates will be analyzed. 3). These conjugates will also be linked to transferrin. The transferrin can bind to the transferrin receptor (TR) in endosomes and promote transcytosis to the basolateral surface. 4). For use in human recombinant antibodies against the stalk region of human pIgR will be selected by a phage display approach: Single chain Fy (scFv) fragments be genetically fused to scFv fragments of the anti-pathogen antibody and an anti-TR antibody.

DESCRIPTION (provided by applicant): The polymeric immunoglobulin receptor (plgR) is made by many mucosal epithelial cells ands transports the polymeric immunoglobulins, dimeric IgA (dlgA) and in some cases IgM, into external secretions, where the dlgA form the first adaptive immunologic defense against infection. Newly made plgR is first delivered to the basolateral surface of the epithelial cell, where it can bind dlgA. The plgR is then endocytosed and, via a series of endosomal compartments, is transcytosed to the apical surface. There the plgR is proteolytically cleaved and the extracellular, ligand binding domain is released together with the dlgA. This cleaved fragment is called secretory component and serves to protect the dlgA against degradation. Although the plgR is constitutively transcytosed in the absence of dlgA, binding of dlgA increases the rate of transcytosis. This allows the plgR to transport increased levels of dlgA, which may be produced during mucosal infection or inflammation. Binding of dlgA to plgR initiates a complex signaling cascade. One of the earliest steps is the activation of p62Yes, a Src-family non-receptor tyrosine kinase, which interacts indirectly with the plgR. We have identified the epidermal growth factor receptor (EGFR) as a major direct substrate of p62Yes, and EGFR is phosphorylated by p62Yes in response to dlgA binding. Here we will analyze how EGFR and p62Yes work together to control dlgA-stimulated transcytosis. Rab3b is a member of the Rab family of small GTPases that regulate membrane traffic. We have found that Rab3b binds directly to the cytoplasmic domain of plgR. Binding of dlgA to plgR causes Rab3b to hydrolyze its bound GTP and to dissociate from the plgR, thereby increasing plgR transcytosis. We will analyze how Rab3b controls plgR transcytosis. IgA nephropathy is the most common form of nephritis world-wide and a major cause of end stage renal disease. Nasopharyngeal carcinoma is caused by infection with Epstein-Barr virus. Entry of the virus can be promoted by binding of virus-specific dlgA, which binds to the plgR on nasopharyngeal cells. Both IgA nephropathy and nasopharyngeal carcinoma are associated with a single nucleotide polymorphism, which causes a point mutation in the plgR. We will analyze the effects of this point mutation on plgR trafficking and function in vitro system, using mutant plgR expressed in Madin-Darby canine kidney epithelial cells.

DESCRIPTION: (Adapted from the Applicant's Abstract): In immunocompromised and other severely ill patients, Pseudomonas aeruginosa (PA) causes an acute pneumonitis, which has an extremely high fatality rate. Improved methods for prevention and therapy are urgently needed. An in vitro model of PA infection using polarized Madin-Darby canine kidney (MDCK) cells or cultured lung epithelial cells mimics many important features of in vivo pneumonia. Live Pseudomonas organisms added to the apical surface of these cultures attach to and kill the cells. These foci of infection spread centripetally. The current proposal will study the host cell factors that determine susceptibility to Pseudomonas pneumonia using these in vitro model systems. There are four aims in this study: to investigate how modification of cell polarity affects virulence; to identify the receptors for PA and how their regulation affects bacterial interaction; to determine the pathway for internalization of PA organisms; and to examine the role of the actin cytoskeleton in interaction of PA with cells.

Cystic fibrosis is caused by mutation in the cystic fibrosis transmembrane conductance regulator (CFTR). In the lung the site of highest expression of CFTR is in the submucosal gland cells of the airways. These cells express high levels of the polymeric immunoglobulin receptor (pIgR), a receptor that endocytoses IgA at the basolateral surface and transcytoses IgA to the apical surface. We propose to use the pIgR to target vectors for expression CFTR to the submucosal gland cells. We will use monoclonal antibodies directed against the extracellular domain of the pIgR. Intact IgG or monovalant Fab' fragments will be coupled to various vectors. Two vector systems that will be tried are a liganded adenovirus system developed by David Curiel, and a amphiphillic polymer system developed by Frank Szoka. We will use the vectors to deliver and express either a trial reporter gene (beta-galactosidase) or the CFTR, driven by a cytomegalovirus vector. We will start by using Madin-Darby canine kidney cells that have been transfected with the rabbit pIgR. When grown on a permeable support, these, cells form a polarized monolayer that mimics an in vivo epithelial monolayer. If successful we will then use primary cultures of airway submucosal gland cells. Ultimately we will try an intact rodent model.

Delivery of genes to target cells by receptor-mediated endocytosis is a very appealing strategy for gene therapy. Many of the targets for gene therapy are epithelial cells that line a variety of important organs. For instance, airway cells are the main site of expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is the lesion in cystic fibrosis. Epithelial cells have an apical surface facing the lumen, and a basolateral surface facing underlying cells. Ideally, one would like to deliver genes through the apical or luminal surface of epithelial cells. In the lung, for instance, one could deliver the genes by introduction into the airways, via instillation or even aerosolization. However, the apical surface of most epithelial cells presents a formidable barrier to gene delivery. Most delivery methods, such as liposomes or viral vectors, work very poorly when applied to the apical surface of well polarized epithelial cells in culture or especially in vivo. The receptors that are usually used for receptor-mediated gene delivery are all located at the basolateral surface. The apical surface contains few receptors that are efficiently endocytosed. One receptor that has been used for basolateral gene delivery is the polymeric immunoglobulin receptor (pIgR), which is particularly abundant on the airway cells that express CFTR. Ordinarily, the extracellular domain of pIgR is cleaved off at the apical surface, which makes this receptor difficult to use for apical gene uptake. We have found a novel method to use the pIgR for effective apical gene uptake. In this pilot grant proposal we will test this in cultured airway epithelial cells and in the lungs of whole rats.

The poly-immunoglobulin receptor (pIgR) present in the epithelium throughout the body transports polymeric immunoglobulin (IgA and IgM) from their site of production into mucosal secretions by an active process called transcytosis. Although the receptor is constitutively transcytosed, it has been shown first in vitro using the pIgR-expressing MDCK epithelial cell line, and more recently in vivo, that upon ligand binding transcytosis of the pIgR is stimulated. In addition, we recently reported that in pIgR expressing MDCK cells dIgA binding to pIgR stimulates a PTK-dependent signaling pathway that controls dIgA stimulated pIgR transcytosis through the activation of the PLCg-1. In rodents pIgR is highly express in liver. After rat liver fractionation we isolated an endosomal fraction highly enriched in pIgR. Using this fraction we have succeeded to co immunoprecipitate and semi-purify a PTK activity together with the pIgR. This PTK activity is specifically associated to the pIgR but not to other receptors also enriched in this endosomal fraction, such as Tf-R, LDL-R nor ASGP-R. Injection of an excess of purified dIgA stimulates the PTK activity co-immunoprecipitated with pIgR. The biochemical characterization of the PTK activity associated to pIgR strongly suggested that it belonged to the src family. Immunoblot and co-immunoprecipitation experiments definitively proved that the PTK activity associated with pIgR and stimulated by dIgA was p62yes and not p60src although present in the fraction as well. Although p62yes knock-out mice have a comparable total amount of dIgA in the secretion estimated by ELISA (intestinal liquid, feces, vaginal liquid, saliva, bile liquid), the rate of IgA transcytosis, measured after injection in the blood of iodinated dIgA and collection of bile liquid, was cut down by almost 50%. Altogether these data revealed an unexpected role for p62yes in the efficient transport of IgA into the secretions and the mucosal immune response.

A major problem in drug delivery is the oral delivery of protein drugs. It was formerly believed that the principal problem was due to denaturation of the proteins by gastric acid and digestion by gastrointestinal proteases. However, encapsulation methods have been developed that can delivery proteins intact to the intestines. This has led to the realization that the major, unsolved problem is that intact proteins cannot cross the intact intestinal epithelium. Physiologically, proteins are transported across epithelial cells by transcytosis. The major transcytotic process in adult human intestine is the transport of IgA from the basolateral to he apical surface by the polymeric immunoglobulin receptor (pIgR). This receptor binds its ligand, IgA, at the basolateral surface of the epithelial cell, and then is endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. At the apical surface, the extracellular, ligand- binding domain of the pIgR is proteolytically cleaved off and is released together with the IgA into external secretions. This cleaved fragment of the pIgR is called secretory component (SC). We have previously shown that ligands that bind to SC can bind to uncleaved pIgR on the apical surface, and can be endocytosed and transcytosed to the basolateral surface. However, such a ligand will also bind to me large amount of SC that has been cleaved and is present in the luminal contents, and this will effectively compete for binding of the ligand to intact pIgR on the apical surface. We have developed a new class of ligand that binds pIgR but does not bind SC. These ligands are efficiently endocytosed at the apical surface and transcytosed to the basolateral surface in vivo and in vitro, i.e. in the opposite of the normal direction. We have shown that these ligands can be conjugated to a 150 kDa cargo protein, which can be transported from intestinal lumen into blood in rats. Our goal now is to improve this technology for eventual use for protein drug delivery clinically. In Specific Aim 1, we will analyze the exact route taken by molecules taking the apical to basolateral pathway, both in vivo and in vitro. In Specific Aim 2 we will maximize transcytosis by optimizing our new class of ligands. We will vary off rate, pH sensitivity, and the exact region of the pIgR to which they bind.

DESCRIPTION (provided by applicant): Exposed mucosal surfaces, such as the respiratory, gastrointestinal, and genitourinary surfaces, are lined primarily by a single layer of epithelial cells. This cell layer serves at least two primary functions in the mucosal immune system. First, it is a barrier to the entry of the >95% of infectious agents that enter through mucosal surfaces, as well as a barrier to allergens and other noxious agents. Mucosal infectious diseases include such high priority agents as AIDS and other sexually transmitted diseases, numerous opportunistic infections and emerging and re-emerging diseases, and bio-terrorist agents. Second, in response to these pathologic agents, inflammatory and immune cells are recruited and cross the epithelial barrier, following a chemotactic gradient. This Program Project presents a multidisciplinary and highly interactive approach to these problems. The Project and Core leaders combine a great deal of experience and diverse insights and techniques. Our experimental systems range from in vitro cell culture to genetically modified whole animals, though we focus on lung epithelium as an exemplary mucosal, and Pseudomonas aeruginosa as an exemplary mucosal pathogen. The integrity of the epithelial monolayer is essential to its mucosal immune function. The epithelial monolayer has sophisticated wound-healing mechanisms to maintain its integrity. Project 1 concentrates on the basic mechanisms of epithelial wound healing. Project 2 focuses on how wound healing is altered by P. aeruginosa and closely parallels Project 1. Projects 3 and 4 focus on the movement of inflammatory cells across the epithelial monolayer into the lumen. Project 3 considers the transmigration of the polymorphonuclear neutrophil, specifically the role of CD47 and the ligand for Mac-l. Project 4 focuses on the role of matrix metalloproteases (MMPs) in chemotaxis of inflammatory cells into the lumen. All four projects are supported by all three cores. Core A is administrative. Core B, Cell Isolation and Culture, provides primary lung epithelial cells for all projects. Core C provides Live Cell Multiphoton and Confocal Imaging, which will be vital to all projects. There is very extensive interaction and collaboration through out. For instance, Projects 1, 2 and 4 all utilize mice knocked-out for certain MMPs.

DESCRIPTION (provided by applicant): This is a proposal for a Zeiss LSM 510 Multiphoton and Meta Spectral Microscope for a facility to study processes in live cells. The investigators in this grant are a highly interactive group interested in a range of areas, including, neurobiology, membrane traffic, developmental biology, and host-pathogen interactions. Collectively, we have accomplished a great deal by localizing proteins in fixed cells. Our next general goal is to study movement of proteins, especially GFP labeled proteins, in live cells. We wish to study these processes in higher-order biological specimens, such as brain sections or developing organs in three-dimensional matrices. These particularly thick specimens cannot be adequately imaged by many techniques that are often used in live cells, such as deconvolution microscopy. Conventional confocal microscopy causes too much phototoxicity. The investigators in this grant are part of a group of 70 UCSF laboratories that are moving to the new Mission Bay Campus. Mission Bay is five miles away from the existing multiphoton microscopes located at the main UCSF Parnassus campus and the Mt. Zion Cancer Center, which prevents adequate access for Mission Bay labs, especially for long-term live cell studies. The existing multiphoton microscopes are also very heavily used, and are inadequate to meet the needs of the rapidly expanding university. Preliminary data confirms that the requested multiphoton microscope will greatly enhance our ability to image processes in live cells. A further advantage of the requested microscope is the newly developed Meta Spectral system. This new system greatly facilitates the use of fluorophores with closely overlapping spectra (such as different variants of GFP) and should significantly enhance the use of FRET.

DESCRIPTION (provided by applicant): The kidney consists mainly of polarized epithelial cells. Proper polarization of these epithelial cells is essential for normal kidney function and is deranged in several renal diseases, such as autosomal dominant polycystic kidney disease. The polarization of individual cells is coordinated to form multicellular structures. For instance, the cells that line the tubules of the nephron are all oriented such that their apical surfaces face the central lumen of the tubule, while their basal surface faces towards the periphery. We have found that the establishment of cell polarity and the coordinated orientation of that polarity can be experimentally separated. We use Madin-Darby canine kidney (MDCK) cells grown in three-dimensional collagen gels, where the MDCK cells form hollow cysts lined by a monolayer of polarized epithelial cells. Expression of a dominant negative (DN) form of the small GTPase Racl causes an inversion of the orientation of polarity, so that the apical surface now points toward the periphery of the cyst, rather than towards the central lumen. DN Racl also causes impaired assembly of laminin around the cyst. Addition of exogenous laminin rescues the orientation of cell polarity. We also found that inhibition of beta1 integrin, Cdc42, or atypical Protein Kinase C (aPKC); all result in inversion of orientation of polarity. This leads to a hypothesis of a pathway that controls orientation of polarity: Raclr-> beta1-> integrin-> laminin assembly-> laminin receptor (probably beta1 integrin) -> Cdc42-> aPKC/Par3/Par6 complex. The aPKC/Par3/Par6 complex is a conserved module that controls polarization in nearly all-metazoan cells. We will test most aspects of this model in this grant. We will test the hypothesis that laminin assembly is downstream of Racl and upstream of Cdc42. We will test the hypothesis that integrins act both upstream and downstream of laminin assembly. We will use a powerful collection of mutants in Cdc42, aPKC, Par3 and Par6 test the hypothesis that all of these proteins control orientation of polarity. This work will lead to important advances in understanding cell polarization and how the polarization of individual cells is coordinated to organize multicellular structures, such as tubules and cysts. This will be invaluable in understanding the pathogenesis of renal diseases where this polarity is deranged.

The kidney, like many epithelial organs, consists mainly of tubules lined by a monolayer of polarized epithelial cells. Though we have learned a great deal about the genes that control kidney development, we know much less about the mechanisms by which cells rearrange themselves into tubules. Weuse a simplified model system of Madin-Darbycanine kidney (MDCK) cells grown in a 3 dimensionalcollagen gel. Single MDCK cellsgrow to form hollow cysts lined by a monolayer of epithelial cells. When these cysts are stimulated with Hepatocyte Growth Factor (HGF), they form tubules over a period of ~3 days. This is a good model system for studying renal tubule formation, and can also provide informationon several pathophysiological processes, such as recovery from acute tubular necrosis, renal fibrosis, polycystickidney disease and perhaps renal regeneration from stem cells.We will study the signaling pathways by which cells respond to HGF. The kinetics of activation of ERKsuggest that it is activated by a MAPK cascadeinvolving B-Raf, rather than the better known Raf-1. We will test this hypothesis by studying activation of components of the B-Raf pathway and by using dominant negatives and RNAi. We will test the involvement of Rho family GTPases, Rho Kinase and lipid rafts in the early stages of tubulogenesis, especially the formation of extensions from the basolateral surface of cells in cysts. We will use time lapse confocal microscopy to observe the effects of perturbing these molecular components on extension formation.We will test the involvement of phosphatidyl inositol (3,4,5)P3 [PI(3,4,5)P3]in extension formation. We have found that exogenous PI(3,4,5)P3 induces extension formation and we will test if these extensions are identical to those produced by HGF. We will analyze possible effectors of PI(3,4,5)P3. Kidney diseases, including developmental defects, kidney injury and genetic conditions that form multiple cysts in the kidney, are major health problems. The kidney consists of many narrow tubes lined by cells. We are using a simple model system where kidney cells are grown in culture and produce such tubes, to study tube formation and how we can influence this to treat and prevent kidney diseases.

DESCRIPTION (provided by applicant): Overview of Program Project: This is a resubmission of a renewal of a Program Project Grant (PPG) application to study the mucosal barrier in infection and inflammation. Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal, mammary and urogenital tracts. Mucosal infections include bacterial and viral pneumonia, SARS, TB, AIDS and other sexually transmitted diseases, numerous opportunistic, emerging and re-emerging infections, and biological warfare/terrorist agents. Mucosal surfaces are lined by epithelial cells, usually in a monolayer. The epithelial cell layer is the principal barrier to entry of infectious agents, allergens and other noxious antigens. Fundamentally, the epithelial layer is the most basic component of the innate mucosal immune system. In this Program Project we focus on two broad and inter-related areas of how the epithelial layer performs its functions in mucosal immune protection, with a particular emphasis on pulmonary epithelium. Projects 1 (Mostov), 2 (Engel) and 3 (Rosen) study mechanisms of epithelial wound healing, the roles of sulfs (sulfatases which modify heparan sulfate proteoglycans) in epithelial response to injury and the interaction of polarized cells with a bacterial pathogen. Project 4 (Werb) is focused on how inflammatory cells cross the epithelial barrier to enter the lumen of the organ. Project 1 uses two novel three dimensional cell culture models of epithelial wound healing. Project 2 focuses on the opportunistic pathogen, Pseudomonas aeruginosa, as a model for epithelial pathogen interaction. Project 3 examines how sulfs participate in epithelial wound healing. Project 4 focuses on the role of proteases and sulfs in regulating inflammatory cell recruitment and migration in mucosal tissues. Core A supports the Program Project administratively. Core B (Matthay) provides primary human and rodent lung epithelial cells in monolayer cultures, three-dimensional models and lung slices, as well as functional studies in mice. Core C has an array of four microscopes suitable for three- and four-dimensional analysis of cell dynamics for in vitro studies as well as experiments in living, anesthetized mice. Most infectious agents enter the body through exposed mucosal surfaces. These surfaces are lined by epithelial cells, most commonly in a single layer. We are studying how this epithelial barrier protects us against infection. PROJECT 1: Epithelial wound healing in 3 dimensions (Mostov, K) Project 1 Description (provided by applicant): Greater than 95% of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal and genitourinary tracts. These include HIV, sexually transmitted diseases, numerous opportunistic infections, TB, many emerging and re-emerging infections, and biological warfare/terrorist agents, such as anthrax, Yersinia pestis and smallpox. Most mucosal surfaces are lined by a monolayer of polarized epithelial cells, which forms the principal barrier to entry by infectious agents. In essence, the epithelial layer can be considered the most basic component of the innate mucosal immune system. Some pathogens cross the epithelial layer by disrupting it. Other pathogens exploit disruptions in the monolayer, which can be caused by tissue injury secondary to inflammation, trauma, or may result from cell death or division within the monolayer. To maintain their function as a barrier to infection, epithelial tissues have developed efficient wound healing mechanisms. Wound healing is central to mucosal defense against infection. The epithelial barrier must be restored as quickly as possible, to minimize the opportunity for entry of infectious agents. Some infectious agents, such as Pseudomonas aeruginosa, not only exploit pre-existing wounds, but also impede the wound healing process. We are studying epithelial wound healing by growing epithelial cells in three-dimensional cultures of extracellular matrix, which causes the cells to more closely resemble in vivo conditions. In Aim 1 we are using a system of human primary lung alveolar type II cells, which form alveolar-like cysts, as a model to study response to acute lung injury/acute respiratory distress syndrome. In Aim 2, we are using a three-dimensional system of a well-differentiated human airway cell line, which forms cysts and tubules lined by pseudostratified epithelium, as a model to study airway response to injury. In Aim 3 we are studying the roles of matrix metalloproteinases and sulfatases (enzymes that remove 6-O-sulfate groups from heparan sulfate proteoglycans) in our three dimensional culture systems. This work will be in collaboration with Projects 2, 3 and 4, and supported by Cores B and C. Most infectious agents enter through the layer of cells that lines internal organs, such as the lung. Injuries to this cell layer make it much easier for pathogens to enter and we are studying how the cell layer heals itself.

This Administrative Core provides administrative support to ensure the proper functioning and coordination of the Program Project. The Core is Lead by Keith Mostov, M.D., Ph.D., who is Principal Investigator of the Program Project and is responsible for the overall administration of the Program Project. The Administrative Core provides an Administrative Assistant to assist Dr. Mostov in coordinating the administration of this large and complex Program Project. The Core also provides a Financial Analyst, to assist in the management of the budget and other financial matters.

Greater than 95% of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal and genitourinary tracts. These include HIV, sexually transmitted diseases, numerous opportunistic infections, TB, many emerging and re-emerging infections, and biological warfare/terrorist agents, such as anthrax, Yersinia pestis and small pox. Most mucosal surfaces are lined by a monolayer of polarized epithelial cells, which forms the principal barrier to entry by infectious agents. In essence, the epithelial layer can be considered the most basic component of the innate mucosal immune system. Some pathogens cross the epithelial layer by disrupting it. Other pathogens exploit disruptions in the monolayer, which can be caused by tissue injury secondary to inflammation, trauma, or may result from cell death or division within the monolayer. To maintain their function as a barrier to infection, epithelial tissues have developed efficient wound healing mechanisms. Wound healing is central to mucosal defense against infection. The epithelial barrier must be restored as quickly as possible, to minimize the opportunity for entry of infectious agents. Some infectious agents, such as Pseudomonas aeruginosa, not only exploit pre-existing wounds, but also impede the wound healing process. We are studying epithelial wound healing by growing epithelial cells in 3 dimensional cultures of extracellular matrix, which causes the cells to more closely resemble in vivo conditions. In Aim 1 we are using a system of human primary lung alveolar type II cells, which form alveolar-like cysts, as a model to study response to acute lung injury/acute respiratory distress syndrome. In Aim 2, we are using a three-dimensional system of a well-differentiated human airway cell line, which forms cysts and tubules lined by pseudostratified epithelium, as a model to study airway response to injury. In Aim 3 we are studying the roles of matrix metalloproteinases and sulfatases (enzymes that remove 6-O-sulfate groups from heparan sulfate proteoglycans) in our three dimensional culture systems. This work will be in collaboration with Projects 2, 3 and 4, and supported by Cores B and C. Most infectious agents enter through the layer of cells that lines internal organs, such as the lung. Injuries to this cell layer makes it much easier for pathogens to enter and we are studying how the cell layer heals itself.

DESCRIPTION (provided by applicant): The branching tree of intrahepatic bile ducts is lined by a monolayer of epithelial cells, known as cholangiocytes. During development hepatocytes and cholangiocytes arise from bipotential hepatoblast precursors. Certain hepatoblasts, which are destined to become cholangiocytes, form a monolayered ring surrounding the portal mesenchyme. This monolayer of biliary precursors becomes a double layered structure. Lumens then develop between the two layers. Eventually these structures remodel into a branching tree of intrahepatic bile ducts. We have developed a three dimensional culture system that recapitulates important events in early development. This enables us to study the these events with much greater mechanistic detail than would be possible in vivo. We use a stable mouse cell line termed Hepatic Progenitor cells Proliferating on Laminin, (HPPL), which is capable of differentiating into hepatocytes and cholangiocytes. We grow these as a monolayer on top of a layer of extracellular matrix. We then overlay the monolayer with a second layer of extracellular matrix. After overlay some of the HPPL cells move up out of the original monolayer and crawl on top of the original monolayer. These migrating cells eventually form a second monolayer on top of the first. Lumens then form between the two monolayers. This process closely resembles the early events in bile duct formation in vivo. However, migration of the cells out of the original monolayer requires phosphatidyl inositol 3- kinase (PI3K), the enzyme that generates phosphatidyl inositol 3,4,5-trisphosphate (PIP3), as well as the kinase Akt, which is downstream of PIP3. We will study how PI3K and PIP3 are involved in migration of cells out of the monolayer. We will examine the role of Akt, its effector GSK-3 and downstream processes during tubulogenesis. We will study how after migration the cells redifferentiate to form a second layer of polarized epithelial cells. We will test the role of the lipids PIP3 and , phosphatidyl inositol 4,5-bisphosphate in this process. Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases. PUBLIC HEALTH RELEVANCE: Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases.

DESCRIPTION (provided by applicant): Acute kidney injury (AKI) is an enormous medical problem, with a very high incidence and mortality rate in hospitalized patients, especially in the ICU. Treatment consists solely of supportive care and, in extreme cases, renal replacement. A major factor in our failure to find effective therapy is that our understanding of how the kidney recovers from AKI is very limited and so we have been handicapped in identification of possible approaches and drugs for therapy. We have developed an innovative new system to study the recovery from AKI, which will allow us to better analyze the molecular and cellular basis of this process from AKI. This will in turn allow us to identify, test and refine candidate therapies. A major target in AKI is the epithelium, especially proximal tubule cells. Severe insults produce dead cells, which are extruded into the tubular lumen. The focus of this grant is to understand how the tubule is subsequently repaired, with the long term goal of improving repair. We will test the hypothesis that phosphatidylinositols 3,4-bisphosphate (PIP2) and 3,4,5-trisphosphate (PIP3) play specific roles in wound healing. We will use live cell imaging to test the prediction that PIP3 controls both formation of the leading edge during cell spreading, as well as cell height during cell spreading and repolarization at the end of wound healing. We will also test the prediction that PIP2 controls the size of the apical plasma membrane and thereby influences cell shape and spreading. We will confirm this in an in vivo model of AKI. We predict that different isoforms of the enzyme that synthesizes PIP3 may have distinct functions, such as in controlling cell height or formation of the leading edge. When some cells in the tubule die, the surviving cells partially depolarize, dedifferentiate, migrate and proliferate to cover the denuded areas. Later, as the surviving cells make contact with their new neighbors, the cells stop migration and proliferation in the process of contact inhibition. We will test the roles of two signaling pathways, Ras-Raf-MEK-ERK cascade and the PAK-PIX complex, in control of differentiation, migration and contact inhibition. PUBLIC HEALTH RELEVANCE: Acute kidney injury is an enormous medical problem, for which there is no therapy. The absence of therapy is a result of our lack of understanding of how the kidney recovers from injury. We have developed an innovative cell culture model system to study the cellular and molecular mechanisms of recovery from acute kidney injury.

DESCRIPTION (provided by applicant): Many internal organs, such as the kidney, consist of hollow tubules and spheres lined by a layer of polarized epithelial cells. These cells have an apical plasma membrane domain facing the central lumen and a basolateral plasma membrane facing the underlying basement membrane. These two plasma membrane domains have completely different protein and lipid compositions, and trafficking of proteins and lipids to these distinct membrane surfaces is vital. We have found that the signaling lipid phosphatidyl inositol 3,4,5- trisphosphate (PIP3) is found only at the basolateral plasma membrane and is a key determinant of the identity and formation of this surface. In contrast phosphatidyl inositol 4,5-bisphosphate (PIP2) is concentrated at the apical plasma membrane, where it is a main determinant of this surface. We will test the hypothesis that PIP2 and PIP3 control the development of epithelial polarity, using live cell imaging. We will test the hypothesis that PIP3 is synthesized at the basolateral plasma membrane by a specific isoform(s) of phosphatidyl inositiol 3- kinase. We will test the respective roles of the lipid phosphatases PTEN and SHIP1/2 in preventing PIP3 from accumulating at the apical plasma membrane. We will test the hypothesis that gp135/podocalyxin and proteins with which it interacts are directly involved in formation of the apical surface. Gp135 is a negatively charged, transmembrane sialomucin, which binds via a PDZ motif at its C-terminus to the scaffolding protein NHERF1. We will mislocalize gp135 to the basolateral surface and see if NHERF1 and other proteins follow. Much of the PIP2 at the plasma membrane is synthesized by phosphatidyl inositol 4- phosphate 5-kinase (PI5K). The isoform PI5K1beta is found at the apical plasma membrane and interacts with NHERF1. We will test the involvement of PI5K1beta (as well as the alpha and gamma isoforms) in formation of the apical plasma membrane. We will also test if PTEN is recruited to the apical plasma membrane by its interaction with NHERF. Together, these experiments will help us understand the molecular mechanism of apical plasma membrane and lumen formation.