ROLE OFCYLD, A DEUBIQUITINATING ENZYME, IN MAST CELL FUNCTION AND SPERMATOGENESIS
Open Access
- Author:
- Wright, Ato Obrumah
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 23, 2008
- Committee Members:
- Shao Cong Sun, Committee Chair/Co-Chair
Todd Schell, Committee Chair/Co-Chair
Robert Harold Bonneau, Committee Member
Neil David Christensen, Committee Member
Diane M Thiboutot, Committee Member - Keywords:
- deubiquitination
K63
DUB
K48
Mast cells
FceRI
BMMC
degranulation
cytokines
type I allergy
allergy
IgE
tyrosine kinases
anaphylaxis
spermatogenesis
bcl-2
male infertility
germ cells
TNF-alpha
TRAF
LOH
loss of heterozygosity
ubiquitination
apoptosis
cylindromatosis
NF-kB
CYLD
RIP
Receptor interacting protein
caspase
IKK - Abstract:
- Ubiquitination is a highly versatile post-translational modification of proteins by the covalent attachment of ubiquitin monomers. Ubiquitination controls a wide range of cellular events. Ubiquitin is ubiquitously expressed and highly conserved in eukaryotes. Ubiquitin can be appended as a monomer and also as an isopeptide-linked polymer via lysine 48 (K48) or lysine 63 (K63). Monoubiquitination is involved in biological processes such as endocytosis, DNA repair, and viral budding, while polyubiquitination mediates processes such as proteosomal degradation and signal transduction mechanisms, as K48 and K63-linked chains, respectively. Ubiquitination is reversible because of the existence of deubiquitinating enzymes (DUBs); cysteine proteases that digest ubiquitin chains. One such DUB is the recently identified tumor suppressor, cylindromatosis (CYLD). CYLD was originally identified as a tumor suppressor that is mutated in patients with benign head and neck tumors known as cylindromas. Cylindromas originate from hair follicles, usually in the head and neck region. Mutations associated with disease often occur in the C-terminal region, which usually encompass the DUB domain. CYLD is an important molecule in several signal transduction pathways, with the most prominent being the NF-kB activation pathway. As a DUB enzyme, CYLD’s involvement in these signaling pathways has implications in cell survival, inflammation, lymphocyte activation, thymocyte development, as well as cell cycle progression. Ubiquitination plays an important role in signal transduction pathways that are vital for efficient progression of spermatogenesis as well as mast cell development and function. In this thesis, we explore the role of CYLD as a DUB enzyme in apoptosis-mediated germ cell development and male infertility, as well as the role of CYLD in mast cell development and activation via the FceRI. The major findings are summarized in the following: 1. Tumor suppressor CYLD is required for FceRI signaling in mast cells. Mast cells have an important role in the body’s defense against bacteria and parasitic infections, as well as mediating allergic responses. Mast cells develop under the influence of various cytokines with the most important being interleukin-3 (IL-3). Chapter II of this thesis, focuses on the role of CYLD in the negative regulation of IL-3-mediated mast cell survival and proliferation and the positive regulation of FceRI-mediated BMMC degranulation and cytokine production. Activation of mast cells via the crosslinking of IgE on the FceRI culminates in the release of preformed inflammatory mediators such as serotonin and histamine, from granules, as well as the de novo synthesis and release of cytokines, such as tumor necrosis factor (TNF) -a and IL-6. The engagement of the FceRI elicits mast cell function through the activation of kinases, including proximal Src and Syk kinases and down stream MAP-kinases, as well as transcription factors such as NF-kB. Our findings suggest a critical role for CYLD in FceRI signaling. Although CYLD is dispensable for the development of bone marrow-derived mast cells (BMMCs) in vitro, the CYLD knockout (CYLD-/-) mast cells are largely defective in FceRImediated cytokine induction and degranulation. These functional defects of CYLD-/- mast cells are associated with severe attenuation in the activation of receptor proximal tyrosine kinases and the transcription factor NF-kB. Thus, CYLD positively regulates mast cell function in a manner that is dependent on NF-kB. 2. Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD. Chapter III focuses on the role of CYLD in the regulation of apoptotic mechanisms that are critical for spermatogenesis and male fertility. Spermatogenesis originates with the proliferation of the primordial male stem cell, spermatogonia, and its differentiation into spermatocytes. Spermatocytes undergo meiosis to become haploid round and elongating spermatids which subsequently mature into spermatozoa. Apoptotic cell death is an important mechanism that is believed to be important in the initial stages of germ cell development in order to establish a proper balance between germ cells and supporting Sertoli cells required for spermatogenesis progression. We demonstrate in this section that genetic deficiency of CYLD attenuates the early germ cell apoptosis and causes impaired spermatogenesis in mice leading to infertility. This loss of CYLD results in increased NF-kB activation with concomitant aberrant expression of NF-kB dependent, anti-apoptotic target genes. We further demonstrate that this phenomenon is dependent on the ability of CYLD to directly bind and deubiquitinate RIP1, an important positive regulator of the NF-kB pathway. These findings establish CYLD as a pivotal DUB that regulates germ cell apoptosis and spermatogenesis and suggests an essential role for CYLD in controlling the RIP1/NF-kB signaling axis in testis.