SUMMARYBAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

No abstract

The transcription factor NFκB is a regulator of inflammatory and adaptive immune responses, yet only IκBα has been shown to limit NFκB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in regulating B cell proliferation and survival. The loss of IκBε showed increased B cell proliferation and survival in response to both antigenic and innate stimulation. NFκB activity was elevated during late phase activation, but the dimer composition was stimulus-specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers were elevated also. The corresponding dimer-specific sequences were found in the promoters of hyper-activated genes. Using a mathematical model of the NFκB signaling system in B cells, we demonstrated that kinetic considerations of the IKK signaling input and IκBε’s interactions with RelA- and cRel-specific dimers could account for this stimulus-specificity. cRel is known to be the key regulator of B cell expansion. We found that RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling identified the inflammatory cytokine, interleukin 6 (IL-6) to be hyper-activated in IκBε−/− B cells. When the IL-6 receptor was blocked, LPS-responsive IκBε−/− B cell proliferation was specifically reduced to near wild type levels. Our results provide novel evidence of a critical role of immune-response functions for IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel and RelA-containing NFκB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative feedback regulators.

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

Key Points NF-κB family members RelB and cRel are coordinately activated by BAFF and provide distinct survival signals. In vivo and in vitro B-cell developmental defects are observed when both RelB and cRel are deleted.

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K 0.5 ( approximately 1 mM vs approximately 8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the k cat to various degrees (62-95% of the control activity). These activators relieved GK's inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose K D and the steady-state parameter K 0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric binding site which is present in the glucose-induced, active enzyme form of GK and absent in the inactive apoenzyme [Kamata et al. (2004) Structure 12, 429-438]. A mechanistic model that brings together the kinetic and structural data is proposed which allows qualitative and quantitative analysis of the glucose-dependent GK regulation by small molecules. The regulation of GK activation by glucose may have an important implication for the discovery and design of GK activators as potential antidiabetic agents.

No abstract

No abstract