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Mechanisms Regulating the Biological Activity of the Transcription Factor NFκB
Summary: Sankar Ghosh is interested in understanding the signal transduction pathways leading to the activation of transcription factor NFκB, particularly through the Toll/IL-1R pathway. His lab is also studying the mechanisms that regulate the activity of NFκB and exploring the role of NFκB in lymphocyte development and activation.
The transcription factor NFκB plays a critical role in the
inducible expression of a large number of genes, including cytokines
and adhesion molecules. In most cells, NFκ&B exists in an inactive;
form by being bound to inhibitory molecules known as IκBs and
remains in the cytoplasm as an NFκB-IκB complex. Treatment
of cells with different inducers, including lipopolysaccharide (LPS),
tumor necrosis factor (TNF), interleukin-1 (IL-1), and double-strand
RNA (dsRNA), results in phosphorylation, ubiquitination, and subsequent
degradation of the IκB proteins, allowing translocation of free
NFκB to the nucleus. The major goals of our research are to
understand the signal transduction pathways leading to the activation
of NFκB and to explore the mechanisms by which the activity of
NFκB is regulated in the context of lymphocyte development and
activation.
Activation of NFκB by various stimuli leads to the activation
of IKK, the IκB kinase that is responsible for specifically
phosphorylating IκB proteins, leading to their ubiquitination and
degradation. Determining how upstream signals activate the IKK is
therefore crucial for fuller understanding of how signals are
transduced to NFκB, as well as for the development of novel
strategies for blocking the activation of NFκB for use as
therapies for inflammatory diseases. The IκB kinase consists of
three subunits, two catalytic subunits known as IKKα (or IKK1)
and IKKβ (or IKK2), and a regulatory subunit named NEMO (or
IKKγ). The NEMO subunit is essential for IKK activation of the
IκB kinase, and its deletion in knockout mice completely
abolishes activation of NFκB. We recently determined the sites of
interaction between the IKK catalytic subunits and NEMO and localized
it to a small region of 6 amino acids in the carboxyl terminus of both
IKKα and IKKβ. A peptide containing the NEMO-binding domain
(NBD) disrupted the interaction of NEMO with the IKK complex in vitro,
and when fused to a cell-permeabilizing sequence could block NFκB
activation in vivo. More significantly, administration of the peptide
to animals leads to dramatic amelioration of inflammatory responses in
multiple mouse models of inflammation. Because the inhibitory peptides
have sequences that are unique for the IκB kinases, the
biological effects of the peptides provide strong support for the
hypothesis that inhibition of NFκB alone is likely to have
significant beneficial effect on treatment of inflammatory
diseases.
The studies on the NBD peptide reinforced the critical role of the
NEMO subunit in regulating the activity of the IκB kinase
complex. However, the mechanism by which NEMO functions in the IKK
complex has remained mysterious. We are systematically attempting to
follow changes in the NEMO protein following stimulation with
NFκB inducers, and these studies have led to the identification
of stimulus-dependent phosphorylation of NEMO that correlates with the
activation of the IKK complex. We have mapped the phosphorylation site
to a specific serine residue and are attempting to identify the kinase
responsible for this phosphorylation. We are also reconstituting
NEMO-deficient fibroblasts with mutant forms of NEMO to determine the
importance of phosphorylation in the activation of the IKK complex. We
believe that elucidation of the steps in the activation process will be
critical for further progress in understanding how different signals
are transduced to NFκB.
A major role for NFκB is to coordinate the expression of a
large number of inducible genes in the context of innate immunity.
Innate immunity is characterized by the nonspecific recognition and
response to pathogens through the recognition of microbial products
(termed pathogen-associated molecular patterns, or PAMPs). Recent
studies have also led to the recognition of a class of receptors, known
as the Toll receptors (TLRs), that are responsible for the recognition
of specific PAMPs such as bacterial lipopolysaccharide. TLRs represent
a large family of receptors characterized by multiple copies of
leucine-rich repeats (LRRs) in the extracellular domain and a
cytoplasmic Toll/IL-1R (TIR) motif. As their name suggests, TIR motifs
of TLRs exhibit significant homology to the intracellular signaling
domain of the type I IL-1 receptor (IL-1RI) and therefore TLRs are
thought to belong to the IL-1R superfamily. Although the IL-1R and TLRs
differ in their extracellular domains, the presence of the TIR domain
allows both receptors to activate similar intracellular signaling
pathways. We are therefore interested in characterizing the
TLR/IL-1RI–induced signal transduction pathways that lead to the
activation of NFκB. We have recently identified two novel
intermediates in these pathways, named ECSIT (evolutionarily conserved
signaling intermediate in Toll pathways) and X, and current efforts are
geared toward further characterizing the biological role of ECSIT. We
have generated mouse knockouts of both these genes and have found that
lack of ECSIT and X leads to early embryonic lethality. We are
attempting to understand the biological processes that are influenced
by these genes through the generation of conditional knockouts using
the cre-lox system. It has also been reported recently that signaling
from the Toll receptors can lead to apoptosis of immune cells, thus
leading to a resolution of inflammation following clearance of
infection. The mechanistic explanation for the choice between
activation or apoptosis through TLR signaling remains unclear, however,
and we are using a variety of biochemical and genetic approaches to
examine this issue.
We have recently demonstrated that activation of transcription
factor NFκB and pre–T cell receptor (pre-TCR) expression is
tightly correlated during thymocyte development. Expression of
components of the pre-TCR in a T cell line leads to the constitutive
activation of NFκB, implying a direct link between the pre-TCR
and signaling pathways leading to NFκB. Inhibition of NFκB
in isolated thymocytes in vitro results in spontaneous apoptosis of
cells expressing the pre-TCR, whereas inhibition of NFκB in
transgenic mice through expression of a mutated, super-repressor form
of IκBα leads to a loss of β-selected thymocytes. In
contrast, the forced activation of NFκB through expression of a
dominant active IκB kinase allows differentiation to proceed to
the CD4+ CD8+ stage in a
Rag1–/– mouse that cannot assemble the
pre-TCR. Therefore, our results suggested that signals emanating from
the pre-TCR are mediated at least in part by NFκB, which provided
a selective survival signal for developing thymocytes with productive
β-chain rearrangements. Consequently these studies demonstrated a
biological function for NFκB that is consistent with its
well-characterized function in promoting survival of developing
thymocytes. To our surprise, however, we observed that forced
activation of NFκB led to a dramatic loss of more mature T cells.
We are trying to understand the molecular basis for this unexpected
observation. Preliminary results suggest that hyperactivation of
NFκB leads to deletion of cells due to enhanced negative
selection. We believe that thymocytes regulate the balance between
positive and negative selection by determining their own activation
status and that artificial activation of NFκB is probably
interpreted as a signal for negative selection that leads to apoptosis
of those cells. We are using a variety of genetic systems to test this
hypothesis.
This research is also supported by grants from the National
Institutes of Health and the Sandler Foundation for Asthma
Research.
Last updated July 19, 2004
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