Regulation of SIGNALLING PATHWAYS INVOLVING NUCLEAR FACTOR KAPPA B

National Institute Of Allergy And Infectious Diseases

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Abstract

Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a P50 DNA binding protein and a P65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. While studying a rare clinical condition of immunodeficiency, We have also discovered the first germline mutation in CARD11, a protein that forms a vital signaling link between the antigen receptor in both B and T lymphocytes, in one family with congenital lymphoid hyperplasia first reported in The New England Journal of Medicine in 1971 as well as in a child adopted from China in a second family. The affected family members exhibit excessive accumulation and defective differentiation of B lymphocytes but not T lymphocytes. The dominant missense mutations identified will constitutively activate NF-kappaB in both B and T cells contributing to downstream proliferation in B cells. However,it causes apparent non-responsivenes or anergy in T cells resulting in poor IL-2 production and proliferation. Thus, we have identified the underlying genetic cause of this hereditary B cell disorder and have uncovered a potential molecular explanation for why CARD11 mutations may predispose to B but not T lymphoid malignancies. This can be understood in terms of the 2 signal model in which T lymphocytes require antigen receptor(signal 1) as well as costimulatory (signal 2)both required for T cell proliferation, whereas the provision solely of signal 1 leads to poor responsiveness or anergy. We observed this phenomenon in our patients, since E127G CARD11 causes internal constitutive activation of NF-kappaB, an important feature component of signal 1, in the absence of a concomitant signal 2. In contrast, B cell proliferation can be triggered by BCR crosslinking alone, which is mimicked by mutant CARD11-driven NF-kappaB activity. We posit that a chronic TCR-like signal 1 provided through mutant CARD11 can be converted to a proliferative signal for the patients T cells in vivo when proper costimulation (signal 2) is provided by professional antigen presenting cells. Defects in T cell help to B cells, related to T cell hyporesponsiveness, may partly explain the paucity of germinal centers and autoimmune manifestations in these patients. On the other hand, deficiencies in T cell-independent humoral responses to polysaccharide antigens also point to intrinsic defects in B cell signaling and effector function with E127G CARD11 present. Our discovery of a germline gain-of-function mutation in CARD11 illuminates how antigen receptor signaling is regulated differently in B and T cells, even though the proximal signaling machinery is nearly identical. This surprising difference has not been revealed by somatic CARD11 mutations in diffuse large cell B cell lymphoma, in which only B cells harbor the mutation and can potentially explain the preponderance of B cell rather than T-cell lymphomas associated with activating mutations in this gene. Our molecular analysis of this autosomal dominant lymphoproliferative disorder, which may represent a novel precursor state for B cell malignancies like B-chronic lymphocytic leukemia, reveals how selective dysregulation of NF-kapppaB via CARD11 may predispose to selective proliferation and differentiation arrest in B cells, but defective proliferation and function of T cells. By re-examining the molecular basis of a rare genetic disorder first reported for decades ago, our discovery may open new avenues to treat this lymphoproliferative disease and prevent development of lymphoma by agents that can block NF-kB. Matt Biancalana, a biophysics student in the laboratory recombinantly expressed and purified to homogeneity the human versions of the subunits of the NF-kapppaB complex. He studied the subunit interactions between the p50 and p65 species and found that there was a dramatic preference of the p50/p65 Rel homology domain (RHD) heterodimer compared to either the p50 or p65 RHD. he concluded that the relative levels of Rel proteins present in a cell may be a means for modulating the populations of various Rel dimer species, in addition to well-characterized mechanisms like phosphorylation or cellular localization (e.g. export from or import into the nucleus). His studies of the DNA-binding properties of these RHD complexes revealed their sequence-specificity, and yielded insights into the mechanism by which the p50 and p65 NF-κB subunits recognize specific DNA elements. These results promise to provide insights into mammalian gene expression and given the importance of NF-kapppaB for the proper induction and transcription for genes involved in immunity,they may shed light on specific gene regulation in the healthy and diseased immune system. We have also investigated the role of microRNAs (miRNAs)in these gene regulatory processes. In particular, we have studied the effects of the deficiency of Dicer whichis essential for embryonic development and embryonic stem cell (ESC) proliferation and pluripotency. We found that Dicer and ESC-specific miRNAs, belonging to the miR-290 family, are important in silencing differentiation genes, such as the Hox genes, by regulating a pathway that coordinates proper Polycomb localization. Introduction of mature miR-291 into Dicer-deficient ESCs restores Polycomb-mediated silencing of Hox genes. This effect is mediated through a molecular cascade involving the methyltransferases Ash1l and Whsc1, whose expression is regulated by miR-290. Knockdown of Ash1l and Whsc1 partially prevented aberrant Hox gene expression in Dicer-deficient ESCs. Collectively, our data revealed that targets of ESC-specific miRNAs control Polycomb accumulation and thereby function to inhibit precocious ESC differentiation.