Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

The role of UV radiation in the development of myeloid malignancy in Rag2 knockout mice

Excessive exposure to the UV radiation present in sunlight can lead to the development of skin cancer in humans. Majority of the UV-induced skin tumors in immune-competent mice are highly antigenic in nature. Additionally, they exhibit a high frequency of mutations in the p53 gene, which arise very early in the course of UV radiation and most of them disappear before the development of skin tumors. ^ Initially, this study was to determine whether UV radiation induces skin tumors much earlier in immune deficient Rag2 knockout mice than in immune-competent mice, and if so, compare their antigenic properties and p53 mutation spectra. However, chronic UV irradiation (10 kJ/m2) induced myeloproliferative disease (MPD) as early as 4 weeks in Rag2 knockout mice instead of skin tumors. Conversely, unirradiated Rag2 knockout mice developed MPD at a low frequency, but the frequency increased with the animal's age. Although the UV-irradiated wild type mice (B6129) developed MPD, its frequency was lower and the occurrence much later than the Rag2 knockout mice. ^ This observation led to our new hypothesis that UV irradiation plays a role in the development of MPD in Rag2 knockout mice. After 4 weeks of UV radiation, both histopathology (myeloid:erythroid ratio, number of blast cells) and flow cytometry (mature myeloid, granulocytes and immature cells) demonstrated an increased number of mice affected with the disease in the UV-irradiated Rag2 knockout group than the other groups. ^ We also investigated the role of cytokines and absence of T and B cells in the development of MPD in the Rag2 knockout mice. Results indicated that IL-3 and IL-3Rα chain expression was upregulated in the spleens of the UV-irradiated Rag2 knockout mice (4 weeks). Reconstitution of the Rag2 knockout mice with T and B cells abrogated the UV-accelerated development of MPD. Both histopathology and flow cytometric analysis (mature myeloid cells, granulocytes) showed a decrease in the number of mice affected with the disease in the UV-irradiated, reconstituted group rather than any other group. In summary, this study provides the first experimental evidence that exposure to UV irradiation can lead to the development of MPD in immune deficient mice. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells.

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury.

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase-deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883-treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^