SIGNALING FUNCTIONS OF THE NFkappaB1 GENE PRODUCTS IN MACROPHAGES AND T CELLS

Open Access
Author:
Chang, Mikyoung
Graduate Program:
Genetics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 26, 2008
Committee Members:
  • Shao Cong Sun, Committee Chair
  • Sarah Bronson, Committee Chair
  • David Joseph Spector, Committee Member
  • Christopher Charles Norbury, Committee Member
  • Jong Kak Yun, Committee Member
Keywords:
  • T cells
  • inflammation
  • macrophage
  • p105
  • IkappaBgamma
  • NFkappaB
Abstract:
The NF-kappaB family of transcription factors plays a pivotal role in the regulation of genes involved in diverse biological processes, including immune responses, inflammation, apoptosis, and oncogenesis. In mammalian systems, the NF-kappaB family includes five members, NF-kappaB1, NF-kappaB2, RelA, RelB and c-Rel, characterized by their structural homology in an evolutionary conserved domain, the Rel homology domain. The different NF-kappaB members form homo- or heterodimers that mediate transactivation of specific target genes. The NF-kappaB complexes are normally sequestered in the cytoplasm by a family of inhibitory proteins, IkappaBs, with IkappaBalpha being the prototypical IkappaB member that controls a so-called canononical pathway of NF-kappaB activation. Various stimuli such as proinflammatory cytokines, antigens, and TLR agonists, activate an IkappaB kinase (IKK) complex, which phosphorylates IkappaBalpha at specific serines. Phosphorylated IkappaBalpha is rapidly ubiquitinated and targeted to the 26S proteasome for degradation. These sequential signaling events lead to the liberation of NF-kappaB complexes, which concurrently move to the nucleus to regulate target gene expression by binding specific kappaB enhancers. Despite the extensive studies of the canonical NF-kappaB signaling pathway, many missing links exist for a comprehensive understanding of NF-kappaB activation. This thesis research focuses on the function of the nfkappaB1 gene that encodes two major proteins, the precursor protein p105 and its processing product, the mature NF-kappaB1 subunit p50. In addition to generating p50, p105 functions as an IkappaB-like molecule, although how p105 regulates NF-kappaB function is not well understood. At least in some cells, nfkappaB1 also produces a splicing variant mRNA that encodes the C-terminal portion of p105, known as IkappaBgamma based on its sequence homology to IkappaBalpha. The in vivo function of IkappaBgamma is also unclear, although it is generally thought to act similarly to p105. Studies in this dissertation were directed towards to elucidating the physiological role of the nfkappaB1 gene products, including p105, p50 and IkappaBgamma, in immune and inflammatory responses. To obtain genetic evidence, I utilized three mouse models, the nfkappaB1 knockout (-/-) mice that lack all of the nfkappaB1 gene products, the p50 knockin (KI) mice that express only p50, and the IkappaBgamma transgenic (Tg) mice that specifically express IkappaBgamma. The major findings are summarized below: 1. Aberrant activation of p50 causes T-cell dependent inflammation. Prior studies suggest that p50 is constitutively expressed in the nucleus of immune cells of the p50KI mice. This mouse model is thus useful for the study of the pathological consequences of deregulated p50 activation. Interestingly, the p50KI mice were found to spontaneously develop intestinal inflammation with typical features of human inflammatory bowel disease (IBD). This inflammatory disorder is mediated by T cells, since it can be induced by adoptive transfer of p50KI T cells into Rag1-/- recipients lacking lymphocytes. Furthermore, the IBD-like symptoms were also rescued when p50KI mice were crossed with the Rag1-/- mice. Thus, deregulated p50 activation leads to T-cell dependent intestinal inflammation. 2. NF-kappaB1 regulates the development of inflammatory Th17 cells. Recent studies have identified a subset of CD4+ T cells, Th17 cells, which produce proinflammatory cytokines, including IL-17, and mediate autoimmunity and inflammation. Interestingly, we found that the p50KI mice contain elevated frequency of Th17 cells in the spleen and mesenteric lymph nodes. High levels £eof Th17-specific cytokines were detected in the colons of p50KI mice, suggesting the infiltration of these inflammatory T cells to the site of inflamed tissues. Parallel biochemical analyses revealed that activation of CD4+ T cells under Th17 differentiation conditions leads to preferential activation of p50 homodimers. Interestingly, p50 synergizes with a known Th17-regulatory transcription factor, RORgammat, in the activation of IL-17 promoter. Consistently, p50 is recruited to the IL-17 promoter along with the induction of Th17 differentiation. Taken together, these findings suggest that NF-kappaB1 is involved in Th17 cell differentiation, which provides an important insight into the role of NF-kappaB in the development of inflammatory and autoimmune diseases. 3. IkappaBgamma is a specific inhibitor of p50 homodimer. To understand the physiological function of IkappaBgamma, we generated IkappaBgamma Tg mice and examined the role of IkappaBgamma in the regulation of different NF-kappaB members. In sharp contrast to IkappaBalpha, IkappaBgamma played a minimal role in regulating the inducible activation of typical NF-kappaB complexes, such as RelA- and c-Rel-containing dimers. Interestingly, we found that IkappaBgamma is essential for restricting the nuclear translocation of p50 homodimers. This finding is consistent with the constitutive nuclear translocation of p50 in p50KI cells, which lack IkappaBgamma or p105. We further demonstrated that the aberrant p50 activation in p50KI macrophages leads to either enhancement or repression of LPS-stimulated expression of pro-inflammatory genes. The induction of IL-12 and iNOS was greatly upregulated, whereas the induction of TNFalpha was suppressed in LPS-treated p50KI macrophages. These findings are in line with the findings that p50 associates with the co-activator protein Bcl3 in some, but not all, gene promoters. More importantly, these abnormal gene induction events were largely rescued by expression of IkappaBgamma. Similarly, IkappaBgamma also reverses the B-cell hyper-responsive phenotype of the p50KI mice. These results suggest that IkappaBgamma, and likely p105, functions as a specific inhibitor of p50 and regulates specific aspects of NF-kappaB function in the immune system. 4. Regulation of oncoprotein kinase Tpl2 by p105 and IƒÛBƒ×. Recent studies demonstrate that p105 regulates the stability and function of the oncoprotein kinase Tpl2, thereby mediating an interesting crosstalk with the ERK MAP kinase signaling pathway. In vitro studies suggest that the C-terminal portion of p105, equivalent to IkappaBgamma, is both required and sufficient for stabilizing Tpl2. Surprisingly, our studies using the IkappaBgamma Tg mice revealed that IkappaBgamma is insufficient for stabilizing Tpl2 or regulating ERK signaling in macrophages in vivo. These findings suggest that stabilization of Tpl2 by p105 may also require N-terminal sequences of p105. Moreover, since IkappaBgamma does not rescue the Tpl2 deficiency in p50KI mice, the phenotype of the IkappaBgammaTg/p50KI mice is mainly due to the regulation of NF-kappaB. In summary, the results presented in this dissertation demonstrate a novel function of NF-kappaB1 p50 in regulating T-cell differentiation and T-cell mediated inflammation and establish IkappaBgamma and NF-kappaB1 p105 as specific inhibitors of the p50 homodimers. These findings provide new insights into the mechanism of NF-kappaB regulation and highlight the complexity of the NF-kappaB signaling pathway. Based on these findings, it is tempting to propose that different IkappaB proteins may play a specific role in the regulation of distinct aspects of NF-kappaB function. This information is important for rational design of anti-inflammatory therapies based on inhibition of specific axis of NF-kappaB signaling.