Xingbin Ai, Ph.D. - US grants

DESCRIPTION (provided by applicant) The objective of this proposal is to characterize the signaling roles of two candidate heparan sulfate proteoglycan (HSPG) modifying enzymes, quail glucosamine 6-O-sulfatase 1 (QSulf1) and QSulf2 in developing neural tube. Cell surface HSPGs play important roles in modulating the distribution and/or activity of signaling molecules such as Wnts, Shh, FGFs and BMPs. QSulf1, the first known extracellular matrix G6-sulfatase, is required for somite MyoD activation through its enzymatic activity to regulate Wnt pathway. QSulf2 is the second member of the family with unknown function. QSulf1 and QSulf2 have overlapping and discrete expression domains in embryonic neural tube. The proposal will test the hypothesis that QSulf1 and QSulf2 have unique functions in extracellular signaling pathways and the functional distinction is controlled by their divergent hydrophilic domains. First, we will characterize the expression pattern of QSulf1 and QSulf2 during embryogenesis by whole mount in situ hybridization combined with immunohistochemistry. Results from this experiment will serve as the initial steps for the functional analysis of QSulfs in developing neural tube. Second, to define QSulf function in the neural tube, we will manipulate the expression of each gene both by electroporation-mediated overexpression and antisense inhibition in chick embryos at stage 12. Molecular markers for neural tube patterning and neural crest cells will be characterized by immunohistochemistry. Results from these experiments will provide information in signaling roles of QSulfs in embryonic neural tube. Third, co-immunoprecipitation and yeast two-hybrid screen will be conducted to identify the binding proteins for the divergent hydrophilic domains of QSulfs that are hypothesized to control the localized functions of QSuIf1 or QSulf2. This study will provide insights on the mechanisms for localized QSulf function in the embryo.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Sulfs are a family of extracellular endosulfatases that modify heparan sulfate (HS) chains at cell surfaces and in extracellular matrices. They release sulfate groups at the 6-O-position of a subset of glucosamine residues in HS chains. The activity of Sulf enzymes serves to potentiate activities of growth factors including GDNF, BMP, Shh, and Wnt and to reduce activities of FGF2, HP-EGF, HGF, and TGF-[unreadable]. As a result of these activities and depending on the context, Sulfs have been observed to serve as oncogenic effectors and as tumor suppressors. Sulf activities appear to induce changes in expression of biosynthetic enzymes, resulting in changes in expressed HS structure that do not necessary reflect the direct Sulf enzymatic activity. As a result, the influences of Sulf activity on cellular phenotype are complex. The goals of this work are to (1) define the HS structural phenotype in mouse embryonic fibroblast cells as a function of Sulf enzyme knockout at both the HS disaccharide and oligosaccharide levels. These results will be compared against those obtained using recombinant Sulf enzymes on purified HS.

DESCRIPTION (provided by applicant): Aging is inevitably associated with diminished regeneration capacity of stem cells. We choose the skeletal muscle as a model system to study mechanisms that regulate the influence from aged environment on stem cell function. Aged skeletal muscle has reduced levels of FGF2 and elevated Wnts and TGF2, leading to decreased proliferation of resident stem cells (so called satellite cells) and increased fibrosis during regeneration. The bioavailability of FGF2, Wnts and TGF2 is regulated by sulfated heparan sulfate. This proposal investigates heparan sulfate-dependent mechanisms that regulate the transmission of age-related environmental signals to satellite cells during skeletal muscle regeneration. The first set of experiments focuses on roles of two extracellular heparan sulfate 6-O-endosulfatases (Sulfs) in differential regulation of the bioavailability of age-related signals. Sulfs enzymatically remodel heparan sulfate 6-O-sulfation, thereby Sulfs reduce Wnts and TGF2 binding to heparan sulfate, while disrupting FGF2 interaction with the receptor. Therefore, Sulfs are hypothesized to promote age-augmented Wnt and TGF2 signaling, while repressing age-reduced FGF2 signaling, leading to impaired function of satellite cells. This hypothesis will be tested by comparing the efficiency of myogenesis, fibrosis and age-related regeneration signaling in aged control, systemic and satellite cell-specific Sulf double mutant mice using a combination of in vivo regeneration and in vitro culture assays. The second set of experiments will test whether heparin, which is similar to heparan sulfate of Sulf-deficient mice in the structure and signaling function, will improve the efficiency of skeletal muscle regeneration in an aged environment. The results of these investigations are expected to lead to the discovery of Sulf- and heparan sulfate-dependent mechanisms that regulate signal communication between satellite cells and the aged muscle environment. Such knowledge may open new venues for prevention and therapy of impaired muscle regeneration by age. PUBLIC HEALTH RELEVANCE: Aging is inevitably associated with diminished capacity of stem cells to regenerate. In the skeletal muscle, age-related changes of environmental signals have a major impact on impaired function of resident stem cells, so-called satellite cells. Disruption of the communication between satellite cells and the aged muscle environment may lead to effective therapies for age-impaired skeletal muscle regeneration. This proposal investigates regulatory mechanisms during the transmission of age-related environmental signals into satellite cells. We have identified two Sulf enzymes that regulate the bioavailability of age-related signals in the regeneration environment and satellite cells. Proposed studies will test whether Sulfs are candidate regulators of the communication between satellite cells and the aged muscle environment. Our findings may open new venues for prevention and therapy of aging-related impairment of skeletal muscle regeneration.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparan sulfate (HS) chains on cell surfaces and in extracellular matrices are acted upon by enzymes and chemical species to alter their biological activities. Reactive nitrogen species (RNS) give rise to conditions for deaminative cleavage of HS chains in vascular tissue. It is likely that aldehyde-containing HS chains so liberated become reactive toward cellular proteins. Structural investigations to elucidate these mechanisms are the subject of this project. Our results using a mass spectrometry glycomics platform show the presence of a disaccharide of structure ([unreadable]HexA-GlcNH26S). These results are consistent with the presence of HS repeats corresponding to HexA-GlcNH26S that are not detectable using other methods. It is likely that disaccharides with free amino groups are difficult to detect using standard disaccharide analysis methods because its zwitterionic nature causes poor retention on ion exchange resins or weak pairing with ion-pairing agents. The presence of disaccharide units containing free glucosamine amino groups in mature HS chains seems to be related to the mechanisms of action of N-deacetylase/N-sulfotransferase enzymes and the availability of 3'-phosphoadenosine-5'-phosphosulfate. Our work shows that disaccharide units containing HexA-GlcNH26S are widely expressed in organ tissue. HS chains containing such disaccharide units would be susceptible to NO-mediated cleavage as has been described for glypican-1 cycling. Conditions for NO-mediated cleavage may exist in other biological processes. Glucosamine amino groups of HS are also known to react rapidly with oxidizing agents such as HOCl under conditions that are likely to be present at inflammation. It is clear that the HexA-GlcNH26S repeat is a widely expressed and functionally relevant HS sub-structure.

DESCRIPTION (provided by applicant): Asthma is a common disease with several effective treatments, including inhaled steroids and ¿-agonists. Despite this, a small subpopulation of patients has a severe unrelenting course associated with airway remodeling. A central feature of airway remodeling is alteration in bronchial smooth muscle (BSM) phenotype, which is classically characterized by expansion of cell number and size, and increased hyper- reactivity to specific and non-specific agonists. While fundamental to asthma pathogenesis and its clinical manifestations, a lack of knowledge regarding the basis for a deranged BSM phenotype is an ongoing, unresolved, issue in the field. This is manifested by the paucity of information regarding the molecular signals underlying bronchial hyper-reactivity and by the lack of treatments directed specifically at reversing the asthmatic BSM phenotype. One major contributing factor to this state-of-affairs is the lack of tools/methodologies that support the high fidelity isolation of pure BSM cells from asthmatic lungs for analysis. To overcome this, we developed a unique transgenic mouse in which BSM singly express a green fluorescent protein (hrGFP) whereas vascular smooth muscle express green (hrGFP) and red fluorescent proteins (dsRed); thereby providing for the first time a reliable methodology for separating each of these two smooth muscle cell populations from one another, and from other lung cells using flow cytometry. Using this unique mouse, our plan is to perform comprehensive mRNA and miRNA profiling of BSM RNA to test the following broad based hypothesis: 1) BSM express a distinct genetic signature and 2) alterations in this signature mediate asthmatic BSM phenotypes. Our plan is to use the profiling data to generate lists of complete and differentially expressed mRNAs and miRNAs in normal and asthmatic BSM. Relationships between deregulated miRNAs, mRNA expression, and the identity of active signaling pathways in asthmatic BSM will be examined by bioinformatic and functional studies. At the end of this work, we will have initiated a process to fill a marked knowledge void in the asthma field and will have established a foundation for a variety of future studies. PUBLIC HEALTH RELEVANCE: Asthma involves changes in the function and properties of the muscle that surrounds the bronchial tube. While central to the many of the symptoms associated with asthma including wheezing and shortness of breath, the molecular signals that cause changes in bronchial muscle are poorly understood. The objective of this proposal is to use several unique tools and methodologies that we developed to identify key molecular signals that underlay the change in bronchial smooth muscle in asthma.

DESCRIPTION (provided by applicant): Aging is inevitably associated with diminished regeneration capacity of stem cells. We choose the skeletal muscle as a model system to study mechanisms that regulate the influence from aged environment on stem cell function. Aged skeletal muscle has reduced levels of FGF2 and elevated Wnts and TGF2, leading to decreased proliferation of resident stem cells (so called satellite cells) and increased fibrosis during regeneration. The bioavailability of FGF2, Wnts and TGF2 is regulated by sulfated heparan sulfate. This proposal investigates heparan sulfate-dependent mechanisms that regulate the transmission of age-related environmental signals to satellite cells during skeletal muscle regeneration. The first set of experiments focuses on roles of two extracellular heparan sulfate 6-O-endosulfatases (Sulfs) in differential regulation of the bioavailability of age-related signals. Sulfs enzymatically remodel heparan sulfate 6-O-sulfation, thereby Sulfs reduce Wnts and TGF2 binding to heparan sulfate, while disrupting FGF2 interaction with the receptor. Therefore, Sulfs are hypothesized to promote age-augmented Wnt and TGF2 signaling, while repressing age-reduced FGF2 signaling, leading to impaired function of satellite cells. This hypothesis will be tested by comparing the efficiency of myogenesis, fibrosis and age-related regeneration signaling in aged control, systemic and satellite cell-specific Sulf double mutant mice using a combination of in vivo regeneration and in vitro culture assays. The second set of experiments will test whether heparin, which is similar to heparan sulfate of Sulf-deficient mice in the structure and signaling function, will improve the efficiency of skeletal muscle regeneration in an aged environment. The results of these investigations are expected to lead to the discovery of Sulf- and heparan sulfate-dependent mechanisms that regulate signal communication between satellite cells and the aged muscle environment. Such knowledge may open new venues for prevention and therapy of impaired muscle regeneration by age. PUBLIC HEALTH RELEVANCE: Aging is inevitably associated with diminished capacity of stem cells to regenerate. In the skeletal muscle, age-related changes of environmental signals have a major impact on impaired function of resident stem cells, so-called satellite cells. Disruption of the communication between satellite cells and the aged muscle environment may lead to effective therapies for age-impaired skeletal muscle regeneration. This proposal investigates regulatory mechanisms during the transmission of age-related environmental signals into satellite cells. We have identified two Sulf enzymes that regulate the bioavailability of age-related signals in the regeneration environment and satellite cells. Proposed studies will test whether Sulfs are candidate regulators of the communication between satellite cells and the aged muscle environment. Our findings may open new venues for prevention and therapy of aging-related impairment of skeletal muscle regeneration.

DESCRIPTION (provided by applicant): To what extent mesothelial-derived cells contribute to lung development and post-natal repair is an open and basic question for the field. The objectives of this grant are to address this fundamental issue and to assess the key role of the Wilm's tumor 1 transcription factor (WT1) in these events. Using mouse lines that carry WT1 alleles with a knock-in Cre recombinase and GFP genes, we generated preliminary data leading to 3 hypotheses that will be examined: 1) the fetal mesothelium contains progenitors for differentiated mesenchymal lung cells 2) WT1 controls the expression of key genes, such as hedgehog (Hh) pathway constituents that control mesothelial migration into the fetal lung, and 3) mesothelium-derived cells contribute to post-natal lung repair and re-growth. To summarize, we found that WT1 is selectively expressed in the lung mesothelium from E11.5 to E16 and is undetectable in the adult. We identified a similar temporal pattern of WT1 expression in the primate lung, suggesting a conserved mesothelial WT1 program across mammalian species. Lineage tracing showed that mesothelium-derived cells give rise to a substantial number of bronchial smooth muscle cells (BSM), along with other parenchymal lung cells whose identities will be established (Aim 1). We observed that WT1 expression coincides with mesothelial cell entry into the underlying lung and active Hh signaling. Mechanistically, we found that WT1 binds to the promoters of multiple Hh pathway genes in mesothelial cells, and that selective loss of mesothelial Hh signaling markedly attenuates entry into the underlying lung in association with diminished expression of EMT genes. These data point to a key role for WT1 in the fetal mesothelium, controlling pathways such as Hh signaling that are involved in migration and EMT, which will be further explored (Aim 2). Interestingly, preliminary data indicate that WT1 is reactivated during lung re-growth post-pneumonectomy whereas WT1 is not re-activated in inflammatory lung injuries, such as asthma and fibrosis. These findings suggest 2 models for how the mesothelium may contribute to lung remodeling in post-natal life. In model 1, the fetal WT1-regulated mesothelial program is re-activated. In model 2, parenchymal cells that arise from the fetal mesothelium in development contribute to repair. To what degree these models are involved in lung re- growth and remodeling in post-natal life will be further examined (Aim 3). We expect that completion of these studies will establish a firm foundation for future work in this new area of lung biology.

DESCRIPTION (provided by applicant): Aging is inevitably associated with diminished regeneration capacity of stem cells. We choose the skeletal muscle as a model system to study mechanisms that regulate the influence from aged environment on stem cell function. Aged skeletal muscle has reduced levels of FGF2 and elevated Wnts and TGF2, leading to decreased proliferation of resident stem cells (so called satellite cells) and increased fibrosis during regeneration. The bioavailability of FGF2, Wnts and TGF2 is regulated by sulfated heparan sulfate. This proposal investigates heparan sulfate-dependent mechanisms that regulate the transmission of age-related environmental signals to satellite cells during skeletal muscle regeneration. The first set of experiments focuses on roles of two extracellular heparan sulfate 6-O-endosulfatases (Sulfs) in differential regulation of the bioavailability of age-related signals. Sulfs enzymatically remodel heparan sulfate 6-O-sulfation, thereby Sulfs reduce Wnts and TGF2 binding to heparan sulfate, while disrupting FGF2 interaction with the receptor. Therefore, Sulfs are hypothesized to promote age-augmented Wnt and TGF2 signaling, while repressing age-reduced FGF2 signaling, leading to impaired function of satellite cells. This hypothesis will be tested by comparing the efficiency of myogenesis, fibrosis and age-related regeneration signaling in aged control, systemic and satellite cell-specific Sulf double mutant mice using a combination of in vivo regeneration and in vitro culture assays. The second set of experiments will test whether heparin, which is similar to heparan sulfate of Sulf-deficient mice in the structure and signaling function, will improve the efficiency of skeletal muscle regeneration in an aged environment. The results of these investigations are expected to lead to the discovery of Sulf- and heparan sulfate-dependent mechanisms that regulate signal communication between satellite cells and the aged muscle environment. Such knowledge may open new venues for prevention and therapy of impaired muscle regeneration by age.

ABSTRACT How pulmonary neuroendocrine cells (PNECs) become innervated and to what extent nerves regulate PNEC function in development and diseases are open and basic questions for the field. The objective of this proposal is to address these fundamental issues and to assess the key role of neurotrophin 4 (NT4) in these events. Using a NT4-/- mouse line and a neonatal mouse model of allergen exposure, we generated preliminary data leading to 3 hypotheses that will be studied in this proposal: 1) NT4 is required for PNEC innervation during development and for the increases in PNEC innervation following early life insults; 2) early life allergen exposure alters the function of sensory afferents and efferent nerves that innervate PNECs thereby causing deregulated ?-aminobutyric acid (GABA) secretion and long-term goblet cell metaplasia; 3) pulmonary mast cells are a candidate source of elevated NT4 levels following early life allergen exposure. To summarize, we found that NT4 was expressed by PNECs during postnatal development and acted as a trophic factor for the innervating nerves to establish connection. Allergen exposure to developing, postnatal lungs aberrantly elevated the levels of NT4. Under this pathological condition, we discovered that PNEC innervation was increased associated with prolonged goblet cell metaplasia. Notably, PNECs were the only cell source of GABA in lungs, a signal essential for allergen-induced goblet cell metaplasia in mouse models and associated with mucous overproduction in human asthmatics and smokers. In addition, NT4 was required for PNEC hyperinnervation and deregulated GABA secretion, consistent with the established paradigm in other neuroendocrine systems that nerves regulate endocrine secretion. These preliminary findings point to an essential role for NT4 in PNEC innervation during development and neuroplasticity following early life injury, which will be extensively characterized by comparing the pattern and degree of PNEC innervation between wild type and NT4-/- mice with and without allergen exposure using markers for different types of nerves (Aim 1). To connect NT4-induced PNEC hyperinnervation to prolonged goblet cell metaplasia following early life allergen exposure, proposed experiments in Aim 2 will assess functional changes in sensory afferents and efferent signals that induce GABA secretion from PNECs and their relationships to NT4. Lastly, given the central role of NT4 in aberrant PNEC innervation under pathological conditions, we examined NT4 expression in injured lungs. We found an enlarged, activated mast cell population expresses NT4 during early life allergen exposure. Whether pulmonary mast cells contribute to PNEC hyperinnervation by producing NT4 will be evaluated (Aim 3). To further enhance disease relevance, we will validate key findings from the mouse work in infant primate models of injury and human lung samples. Together, this proposal investigates complex interactions between nerves, PNECs, and inflammation during postnatal development and injury. Our findings indicate that the pathogenesis of chronic airway diseases, such as asthma, may involve disrupted developmental processes following early episodes of insults. We expect that completion of the proposed studies will provide fundamental knowledge about how the pulmonary neuroendocrine system forms and functions. Identification of the mechanisms along the nerve-PNEC axis underlying mucous overproduction may lay the foundation for the discovery of new treatment strategies.

PROJECT SUMMARY Allergic asthma is one of the most common, chronic airway diseases that often progresses from infancy and early childhood into adulthood. Current therapies are directed at antagonizing inflammation and bronchial constriction. Despite their widespread use, these therapies have no beneficial effect on slowing down the progression of allergic asthma. The central mediator of anamnestic allergic responses is allergen-specific, T helper 2 resident memory cells (Th2-TRMs). As such, targeting the establishment of allergen-specific, Th2-TRMs following early life exposure provides an opportunity to modulate and impede progressive allergic asthma. However, how Th2-TRMs are established in the early lung has never been studied. To address this critical issue in the pathogenesis of progressive allergic asthma, we have investigated the causal link between allergen exposure in early life and the long-term effect on airway inflammation. Our study focuses on the communication between sympathetic nerves and CD4+ T cells in the postnatal, developing lung. So far, our published and preliminary studies have identified a significant role of nerve-derived dopamine in susceptibility to allergic asthma in early life and anamnestic allergic responses in adults. We show that dopamine signals through a T cell-specific DRD4 receptor to promote Th2-TRM phenotypes by activating transcriptional factors and epigenetic modulators in Th2 cells. Interestingly, sympathetic nerves transition into an adrenergic phenotype with age. Therefore, nerve- derived dopamine operates in an age-related manner to promote Th2 memory. Given the critical role of dopamine in the establishment of Th2-TRMs in the early lung, we have investigated the postnatal development of sympathetic nerves. We found an age-related reduction in the levels of nerve growth factor (NGF) and brain- derived neurotrophic factor (BDNF) that was associated with the dopaminergic-to-adrenergic transition of sympathetic nerves. Empowered by these preliminary findings, we propose the central hypotheses: dopamine promotes the establishment of allergen-specific, Th2-TRMs in the early lung; the dopaminergic-to- adrenergic transition of sympathetic nerves is caused by age-related reduction in NGF and BDNF levels. These hypotheses will be tested by the following three specific aims. Aim 1 will define the specific role of dopamine in the abundance and the function of allergen-specific, Th2-TRMs following allergen exposure in early life. Aim 2 will identify functional mediators of dopamine signaling in Th2-TRM phenotypes. Aim 3 will determine the role of NGF and BDNF in sympathetic innervation and allergen-specific, Th2-TRMs in the lung. Of note, clinical studies and GWAS have reported positive correlation between the levels of NGF and BDNF and allergic asthma. Taken together, our proposed studies will provide insights into the establishment of Th2-TRMs in the early lung and identify molecular targets for the intervention of progressive asthma from childhood to adulthood.