Animal Biotechnology & Biomedical Sciences Graduate Program Dissertations Collection

Characterization of the function and regulation of type-1 inositol 1,4,5-trisphosphate receptor in mouse oocytes and eggs

Zhang, Nan — Wed, 16 Jan 2013 12:25:23 PST

Given the indispensable role of Ca²⁺ release in fertilization, the type 1 inositol 1,4,5- trisphosphate receptor (IP₃R1) is a key regulatory molecule in mediating the cross-talk between cell cycle progress and the Ca²⁺ release machinery in mammalian oocytes. The studies in this thesis addressed several important regulatory aspects of the Ca²⁺ release activity at fertilization of mammalian eggs. In chapter I, we found that compromised functionality of IP₃R1 underlay the defective IP₃R1-mediated Ca²⁺ release in aging eggs. Further, our studies also indicated that modifications on the biochemical status of IP₃R1 by incubation with caffeine have positive effects on the Ca²⁺ release activity and developmental fate of aging mouse eggs. Thus, these results may help facilitate the invention of a cure that could delay/reverse many of age induced detrimental changes thereby restoring the fertilizability of aged eggs. For the next two chapters we mainly focus on the regulatory role of IP₃R1 as a significant participant in the early developmental event. Our completed results with the caspase-3 cleaved IP₃R1 indicated that the truncated IP₃R1 might not play a prominent role affecting the Ca²⁺ homeostasis during the aging process in mouse oocytes due to its rapid turnover rate. Regardless, we confirmed the leaky property of C-IP₃R1 in mouse oocytes and found a novel protective proteolysis pathway in mouse oocytes. Finally development of a system by which we could selectively overexpress IP₃R1 phosphorylation mutants allows us to probe important phosphorylation regulatory mechanisms underlying the [Ca²⁺]i oscillations in mammalian oocytes. Thus the experiments I have done provide valuable information regarding the extensive regulation of the PI pathway, regulation that in aged and non- aged MII eggs, results in a Ca²⁺ release program that is replete with information. ^

Characterization of the roles of Yy1 in early embryonic development in the mouse

Wallingford, Mary Catherine — Wed, 16 Jan 2013 12:25:14 PST

One of the many ways that the ubiquitously expressed Polycomb Group protein, Yin-Yang1 (Yy1), is believed to regulate gene expression is through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and the Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, Yy1 was deleted in the epiblast of mouse embryos using Sox2-Cre. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but the primitive streak fails to extend anteriorly. Migration through the streak is severely impaired, and streak descendants fail to downregulate E-Cadherin resulting in an aberrant accumulation of streak cells. Intriguingly, we find an accumulation of Nodal and a concomitant reduction of Nodal antagonists suggesting that YY1 is normally required for proper Nodal regulation. We have observed that definitive endoderm is specified but fails to properly delaminate into the outer layer and mutant embryos also fail to accumulate any axial midline structure. Although anterior neuroectoderm is clearly specified, mesoderm specification is severely restricted. Our results reveal critical requirements of YY1 in several important developmental processes, including epithelial to mesenchymal transition (EMT), Nodal regulation and PRC2 mediated H3K27Me3 of the inactive X-chromosome. ^ Despite the localization of Oct4 and Sox2 transcripts in the neuroectoderm of the Yy1 epiblast cKO and the presence of stable transcripts of both genes in the Yy1 RNAi knock down blastocyst, both proteins are void in these models and Oct4 protein is absent in the peri-implantion Yy1 KO mouse. We believe YY1 is required for stabilization of the Oct4/Sox2 pluripotency complex in vivo. We have identified two endogenous forms of YY1 and we believe these posttranslational modifications of YY1 permit the protein to perform the diverse activities it performs in vivo. For example, in addition to the roles in transcriptional regulation and protein complex stabilization, we have also observed a role in YY1 in epigenetic regulation in vivo, including deposition of histone 3 lysine 27 trimethylation (H3K27Me3) on the inactive X-chromosome in female embryos and a role in imprinted gene expression of the Dlk1/Dio3 locus. ^ Detailed analysis of the peri-implantation lethal Yy1 KO mouse in utero revealed unexpected novel developmental events. A large scale follow up examination of wildtype implantation primarily through analysis of immunohistochemical data and gene expression profiling at the cellular level. We analyzed expression patterns of important developmental genes including Oct4, Sox2, Nanog, Cdx2, Gata6 and Sox17, as well as markers of epithelial biogenesis including ZO1, E-Cadherin and Laminin. Interestingly we identified consistent variances in cell populations within the ICM as well as likely primitive endoderm progenitors that produce Laminin and first appear at the periphery of the ICM. We also identified a novel upregulation of Sox17 specifically at the site of implantation. With these data we compose a staging diagram of peri-implantation embryonic and maternal changes during the elusive window of development. ^ These results are the first to elucidate the role of YY1 during gastrulation and peri- implantation, providing potential in vivo targets of YY1 and highlighting the diversity of function of YY1 in the early embryo. Additionally we have been able to advance molecular knowledge of peri-implantation development, in order to provide a platform from which to analyze other peri-implantation lethal KO mice, as well as to aid biomedical understanding of implantation and implantation failure in mammals.^

The role of human NKG2d receptor-ligand function in tumor immunity and immune escape

Karimi, Mobin A — Wed, 16 Jan 2013 12:24:50 PST

NKG2D is a unique immunoreceptor of key significance to immune surveillance of tumor cells, and it represents an attractive candidate in the development of both molecular and cell-based immunotherapies. NKG2D is a stimulatory receptor expressed by human natural killer (NK) cells, cytokine induced killer/lymphokine activated killer (CIK/LAK) cells, γδ T cells and CD8⁺ &agr;β T cells. The NKG2D receptor plays a pivotal role in both innate and adaptive immunity, where it stimulates the cytotoxic function of (NK) cells and CD8⁺ T cells upon recognition of a diverse array of MHC-related ligands. In turn, these ligands are specifically induced under pathological conditions.Current research has established a prominent role for the NKG2D receptor in the modulation of tumor immunity by both NK cells and T cells. We discovered a novel genetic modifier that limits the effectiveness of NK/T cell immunotherapy in human cancer patients. Our objective of this study was to investigate how these genetic modifiers affect NK receptor-ligand interactions and ultimately tumor cell recognition, immunity, and immune evasion. ^ We characterized an alternate splice variant of human NKG2D that encodes a truncated receptor lacking the ligand-binding ectodomain. This truncated NKG2D variant (NKG2D^TR) does not activate cytotoxicity in a ligand-dependent manner, and its enforced expression inhibits killing mediated by the full-length NKG2D isoform (NKG2D^FL). Enforced expression of NKG2D^TR resulted in the retention of NKG2D^FL in intracellular compartments. The relative abundance of NKG2D^TR transcripts varies among PBMC and activated LAK/CIK cells and NK cells of diverse donors and inversely correlates with the killing capacity of expressing cells. Furthermore, specific shRNA-mediated knockdown of the endogenous NKG2D^TR isoform in human CIK cells and NK cells enhances endogenous NKG2D^FL-mediated cytotoxicity. Co-immunoprecipitation studies revealed that NKG2D^TR pairs with DAP10 and heterodimerizes with NKG2D^FL, suggesting that its dominant negative impact on cytotoxicity is due to the formation of heterodimeric NKG2D^TR/FL receptor complexes incapable of ligand binding. Thus, competitive interference via an alternatively spliced NKG2D variant constitutes a novel mechanism for regulation of NKG2D-mediated signaling and cytotoxicity in LAK/CIK cells and NK cells.^

Characterization of bovine gammadelta T cell WC1 coreceptors: Sequence, expression, and function

Chen, Chuang — Wed, 16 Jan 2013 12:24:33 PST

To better characterize the WC1 coreceptor family, by adapting Q-PCR for quantification, thirteen functional genes were found associated with ten different breeds of animals and the domain 1 sequence for an individual gene is highly conserved among breeds, with zero to three amino acids differences found per gene. Despite these differences, phylograms confirmed that the evolutionary divergence between individual WC1 genes was still greater than the divergence among animals for a particular gene. Analysis of the WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 forms. ^ We mapped the epitopes to particular SRCR domains and evaluated their distribution among WC1 molecules. We found that mAb CC15 is a pan-reactive anti-WC1 mAb recognizing an epitope in the closely related SRCR domains 2 and 7. Five other broadly-reactive anti-WC1 mAbs recognize epitopes in the related SRCR domains 4 and 9. Finally, the subpopulation-specific anti-WC1 mAbs were found to react with epitopes in the most variable WC1 domain, i.e. domain 1. ^ By applying real-time QPCR and quantification analysis of the serologically defined subpopulations of bovine γδ T cells, three subpopulations of WC1⁺ γδ T cells can be characterized by different expression patterns of thirteen WC1 genes. We for the first time determined the sites of tyrosine phosphorylation in both Type II (WC1-9) and Type III (WC1-11) cytoplasmic tail sequences in vivo. We found that tyrosine phosphorylation sites were different among WC1 cytoplasmic tails and observed that optimal levels of tyrosine phosphorylation and the time to reach the optimal level varied among them. WC1 cytoplasmic domain phosphorylation was also associated to its function in that three types of WC1 cytoplasmic tails resulted in WC1-mediated potentiation of the TCR-induced stimulation of Jurkat T cells at different levels. Co-crosslinking of TCR and the Type III (WC1-11) WC1 cytoplasmic tail induced the highest amount of Jurkat T cells IL-2 production, followed by co-crosslinking of TCR and the Type II (WC1-9) WC1 cytoplasmic tail. Similar findings were obtained in ex vivo primary bovine γδ T cells by using various anti-WC1 mAbs that reacted with WC1 molecules with different types of WC1 cytoplasmic tails.^

Molecular mechanisms regulating Complement Receptor 3-mediated phagocytosis of Borrelia burgdorferi

Hawley, Kelly Lynn — Wed, 12 Dec 2012 06:50:34 PST

The macrophage receptors that mediate phagocytosis of Borrelia burgdorferi, the Lyme disease spirochete, are unknown despite this cell type's importance in promoting pathogen clearance and inflammation-mediated tissue damage. We now demonstrate that the β2 integrin, Complement Receptor 3 (CR3), mediates the phagocytosis of opsonized and non-opsonized spirochetes by murine macrophages and human monocytes. Although, expression of the surface proteins, CspA and OspE, protects B. burgdorferi from complement-mediated phagocytosis, the versatility of CR3 counteracts the ability of B. burgdorferi to interfere with complement activation and complement-derived opsonins, thus minimizing the bacteria's anti-complement strategy. Interaction of the spirochete with the integrin is not sufficient to internalize B. burgdorferi; however, phagocytosis occurs when the GPI-anchored protein, CD14, is coexpressed in CHO-CR3 cells. CR3-mediated phagocytosis occurs independently of MyD88-induced or inside-out signals but requires the translocation of the integrin to cholesterol rich membrane microdomains. Interestingly, the absence of CR3 leads to marked increases in production of TNF in vitro and in vivo, in spite of reduced spirochetal uptake. Overall, our data establish CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. Macrophages are critical cellular components of the immune response to infectious agents. During infection with B. burgdorferi, macrophages infiltrate the cardiac tissue and induce the activation of invariant NKT cells, leading to the production of the protective cytokine IFNγ. The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen activated protein kinases, such as p38 MAP kinase. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response during infection with B. burgdorferi. The inhibition of p38 MAP kinase does not alter the ability of macrophages to phagocytose B. burgdorferi; however, inhibition of p38 during infection with B. burgdorferi results in increased carditis. Through the generation of transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, we demonstrate that this kinase regulates the production of the iNKT attracting chemokine, MCP-1 and the infiltration of these cells to the cardiac tissue during infection. Overall, the inhibition of p38 MAP kinase during infection with B. burgdorferi specifically in macrophages results in the deficient infiltration of iNKT cells and their diminished production of IFNγ, leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

Mechanisms of pathology in equine laminitis: Versican depletion from basal epithelial cells and suppressed canonical wnt signaling

Wang, Le — Mon, 24 Sep 2012 11:00:49 PDT

Laminitis is a crippling disease of horses resulting from faiure of the digital laminae, which suspend the distal pedal bone and hence the axial skeleton within the hoof capsule. This disease affects about 2% of all horses in US and there is no effective therapeutic agent. Failure of the laminae results from detachment of the epidermal and demal layers of the tissue which allows the distal phalanx to rotate and sink within the hoof capsule. These layers are joined at a basement membrane which is anchored to epithelial cells of the epidermal tissue by integrins. My studies focus on laminitis associated changes in this junctionl complex. The experimental model for my research is healthy horses administered a pro-laminitis gastric bolus of corn starch/wood flour gruel (CHO). Here, I have shown that: (i) In the laminae of healthy horses laminar basal epithelial cells are packed with the metalloproteinase ADAMTS-4 and its proteoglycan substrates versican and aggrecan. Suprabasal epithelial cells are also packed with aggrecan but not versican, making versican a specific basal epithelial cell differentiation antigen; (ii) A pro-laminitis bolus of starch gruel elevates ADAMTS-4 gene and protein expression in the laminae leading to the elevated cleavage of versican; (iii) in the laminae of horses with acute laminitis versican is depleted from the basal epithelial cells by elevated ADAMTS-4 cleavage and suppressed versican gene expression, which is significantly and positively correlated with a decline in β-catenin and integrin β4 gene expression, and (iv) decline in β-catenin gene and protein expression in laminitic laminae results from depressed canonical Wnt signaling, manifest as a reduced expression of positive regulators (Wnt 4, FzD4, LRP6, PP1, Akt2 and β-catenin) and the up-regulation of negative regulators (Axin1, CKIαand GSK3β). Taken together, the studies show that suppressed canonical Wnt signaling in laminitic horses results in loss of β-catenin and integrin β4, which respectively are required components of adherens junctions and hemidesmosomes, leading to the detachment of the laminar basal epithelial cells from each other and from the basement membrane and thus, destabilizing the epidermal:dermal junction. ^

The Roles of Notch1 and PKC-Θ in Immune Mediated Bone Marrow Failure

Roderick, Justine E. — Fri, 19 Aug 2011 13:51:18 PDT

We sought to evaluate the individual contributions of Notch1 and PKC-ζ to disease progression in a mouse model of immune-mediated bone marrow failure and to define a mechanism for their potential cellular cooperation. We transferred parental bulk splenocytes into F1-hybrid recipients to induce a robust immune-mediated bone marrow failure (BMF) that we could partially rescue by administering a pharmacological inhibitor of Notch activation. Transferring splenocytes from PKC--ζ-/- animals did not induce disease, and treating animals with a pharmacological inhibitor of PKC-ζ also provided full protection from disease. We found that inhibiting Notch1 resulted in PKC-ζ down-regulation, and blocking PKC-ζ reduced Notch1 activation, possibly within a positive feedback loop. Our data suggest that both Notch1 and PKC-ζ contribute to disease progression in our mouse model of immune-mediated bone marrow failure. Furthermore, additional findings from the lab demonstrated physical interactions between Notch1, members of the T cell signalosome and PKC-ζ that are essential to mediating full activation of T cells following signaling through the TCR and CD28. Notch1 and/or PKC-ζ may represent novel therapeutic targets in the treatment of bone marrow failure.

Innate immune responses to B. burgdorferi mediated by JNK1 and the cochaperone, methylation controlled DNAJ (MCJ)

Izadi, Hooman — Mon, 11 Apr 2011 09:40:19 PDT

The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).

Involvement of ion fluxes and cAMP pathways in sperm capacitation

Wertheimer, Eva — Mon, 22 Nov 2010 15:24:07 PST

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. It is well established that capacitation requires Na⁺, HCO₃^-, Ca²⁺ and a cholesterol acceptor; however, little is known about the function of Cl^- during this important process. In this study, it is shown that Cl^- is essential for capacitation and that Cl^- act upstream of the cAMP/PKA signaling pathway. To investigate the Cl^- transporters involved, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na⁺, K⁺, Clcotransporters (NKCC), inhibited all capacitation-associated events suggesting that these transporters may mediate Cl^- movements in sperm. Consistent with these results, western blots using anti NKCC1 antibodies showed the presence of this cotransporter in mature sperm.^ Other key issue in sperm capacitation that remains unsolved despite many years of research is the possibility that transmembrane adenylyl cyclases (tmACs) have a role in sperm physiology. If tmACs were involved in the increase of cAMP in sperm, a role of G proteins in these responses would be plausible. Uncertainties exist about the activation of sperm adenylyl cyclase(s) by heterotrimeric G proteins. In this regard, there is some debate as to whether Gα_s is present in spermatozoa. In this work, Gα_s was detected on membranes purified from mouse sperm by [³²P] ADP-ribosylation with cholera toxin followed by immunoprecipitation with a specific antibody. In addition, immunofluorescence studies identified a specific signal in the sperm acrosomal region. Moreover, forskolin was able to induce acrosome reaction after incubating sperm in capacitating conditions and a higher accumulation of cAMP was observed after incubating sperm with forskolin. In this context, the clarification of the role of the ACs and G proteins will constitute a pivotal step for further investigation on sperm capacitation. Forskolin induces acrosome reaction only on capacitated sperm. These data suggest that a tmAC might be involved in the acrosome reaction and that capacitation is needed to couple this signal to other pathways related to the exocytic process.^

Cooperative immunological and pharmacological control of SEB-induced T cell activation and subsequent pathology

Tilahun, Mulualem Enyew — Tue, 25 May 2010 11:01:32 PDT

Staphylococcal Enterotoxin B (SEB) is one of the potent exotoxins synthesized by Staphylococcus aureus that causes toxic shock, is a primary cause of food poisoning and is a Class B bioterrorism agent. SEB, a superantigen, mediates antigen-independent activation of a major subset of the T-cell population by crosslinking TCRs of T-cells with MHC class II molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. This excessive secretion of cytokines, some of which are inflammatory, causes immune dysregulation, systemic inflammation and disease. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin’s interaction with the TCR or MHC class II and provide protection against the debilitating effects of this superantigen. In a series of experiments, we derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of noncrossreactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically-acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human PBMCs, or splenocytes derived from BALB/c or HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T cell activation and cytokine production. When tested in vivo in HLA-DR3 transgenic mouse TSS model, the two chimeric antibodies acted synergistically and provided full protection against SEB-mediated TSS symptoms and lethality of SEB. Furthermore, combination of chimeric anti-SEB, an extracellular inhibitor of SEB, and pharmacological agents (γ-secretase inhibitors, rapamycin, or lovastatin), an inhibitor of intracellular pathways recruited by SEB, provided significant reduction of SEB-induced T cell activation in cultures of mouse splenocytes and human PBMCs. Combination of chimeric anti-SEB antibody and lovastatin also provided in vivo protection against lethal doses of SEB in HLA-DR3 transgenic TSS model. In this study, we have developed a pair of chimeric anti-SEB antibodies (for the first time) that neutralize SEB efficiently in vitro as well as in vivo. In addition, we demonstrated that in vivo protection against lethal doses of SEB can be achieved by a statin of proven safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. Both these findings have provided potential treatment options for diseases mediated by SEB, as there is no prophylaxis, or therapy against accidental or malicious exposure.^