Louise M. Carlson

Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation

I kappa B-alpha inhibits transcription factor NF-kappa B by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate I kappa B-alpha. This liberates NF-kappa B to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of NF-kappa B correlates with phosphorylation of I kappa B-alpha and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of I kappa B-alpha was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and NF-kappa B could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of NF-kappa B.

Critical Roles for the Bcl-3 Oncoprotein in T Cell–Mediated Immunity, Splenic Microarchitecture, and Germinal Center Reactions

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.

NF‐κB p50 and p52 Expression Is Not Required for RANK‐Expressing Osteoclast Progenitor Formation but Is Essential for RANK‐ and Cytokine‐Mediated Osteoclastogenesis

Expression of RANKL by stromal cells and of RANK and both NF‐κB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF‐κB signaling in this process, we used NF‐κB p50−/−;p52−/− double knockout (dKO) and wild‐type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony‐stimulating factor (M‐CSF). NF‐κB dKO splenocytes formed more colony‐forming unit granulocyte macrophage (CFU‐GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU‐GM colonies. RANKL increased the number of CFU‐GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence‐activated cell sorting (FACS) analysis of splenocytes from NF‐κB dKO mice revealed a two‐to threefold increase in the percentage of CD11b (Mac‐1) and RANK double‐positive cells compared with WT controls. Treatment of NF‐κB dKO splenocytes with interleukin (IL)‐1, TNF‐α, M‐CSF, GM‐CSF, and IL‐6 plus soluble IL‐6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF‐κB dKO or p50−/−;p52+/− (3/4KO) mice. Thus, NF‐κB p50 and p52 expression is not required for formation of RANK‐expressing osteoclast progenitors but is essential for RANK‐expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.

Sustained antibody responses depend on CD28 function in bone marrow-resident plasma cells.

CD28 signaling is essential for maintenance of long-term antigen-specific antibody production and for persistence of plasma cells in the bone marrow of mice.

CD28 Expressed on Malignant Plasma Cells Induces a Prosurvival and Immunosuppressive Microenvironment

Interactions between the malignant plasma cells of multiple myeloma and stromal cells within the bone marrow microenvironment are essential for myeloma cell survival, mirroring the same dependence of normal bone marrow-resident long-lived plasma cells on specific marrow niches. These interactions directly transduce prosurvival signals to the myeloma cells and also induce niche production of supportive soluble factors. However, despite their central importance, the specific molecular and cellular components involved remain poorly characterized. We now report that the prototypic T cell costimulatory receptor CD28 is overexpressed on myeloma cells during disease progression and in the poor-prognosis subgroups and plays a previously unrecognized role as a two-way molecular bridge to support myeloid stromal cells in the microenvironment. Engagement by CD28 to its ligand CD80/CD86 on stromal dendritic cell directly transduces a prosurvival signal to myeloma cell, protecting it against chemotherapy and growth factor withdrawal-induced death. Simultaneously, CD28-mediated ligation of CD80/CD86 induces the stromal dendritic cell to produce the prosurvival cytokine IL-6 (involving novel cross-talk with the Notch pathway) and the immunosuppressive enzyme IDO. These findings identify CD28 and CD80/CD86 as important molecular components of the interaction between myeloma cells and the bone marrow microenvironment, point to similar interaction for normal plasma cells, and suggest novel therapeutic strategies to target malignant and pathogenic (e.g., in allergy and autoimmunity) plasma cells.

Expression of Either NF-κB p50 or p52 in Osteoclast Precursors Is Required for IL-1-Induced Bone Resorption†

Interleukin (IL)‐1 is implicated in postmenopausal‐ and inflammation‐mediated bone loss. Its expression is regulated by NF‐κB and vice versa. To examine the role of NF‐κB p50 and p52 (they are required for osteoclast formation during embryonic development) in IL‐1‐induced resorption, we used various NF‐κB knockout (KO) mice, including p50−/− and p52−/− single KO, p50−/− and p52+/− (3/4KO), and p50−/− and p52−/− double KO (dKO) mice. IL‐1 increased blood calcium and bone resorption in wild‐type (wt), p50, and p52 single KO mice, but not in 3/4KO or dKO mice. Osteoclast formation was impaired in bone marrow cultures from 3/4KO compared with single KO and wt mice treated with IL‐1. IL‐1 receptor expression was similar in colony forming unit‐granulocyte macrophage (CFU‐GM) colony cells from wt and dKO mice. However, IL‐1 promoted CFU‐GM colony formation and survival as well as the formation, activity, and survival of osteoclasts generated from these colonies from wt mouse splenocytes, but not from dKO splenocytes. No difference in expression of the osteoclast regulatory cytokines, RANKL, and OPG, was observed in osteoblasts from wt and dKO mice. Thus, expression of either NF‐κB p50 or p52 is required in osteoclasts and their precursors, rather than osteoblasts, for IL‐1‐mediated bone resorption.

The signal response of IkappaB alpha is regulated by transferable N- and C-terminal domains.

IkappaB alpha retains the transcription factor NF-kappaB in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IkappaB alpha by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-kappaB translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IkappaB alpha to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IkappaB alpha is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IkappaB alpha is controlled by transferable N-terminal and C-terminal domains.

Regulation of RelB Expression during the Initiation of Dendritic Cell Differentiation

ABSTRACT The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CβII (PKCβII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCβII also downregulated constitutive expression of NF-κB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCβII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCβII-mediated regulation of transcriptional initiation and elongation.

Interferon regulatory factor-2 physically interacts with NF-κB in vitro and inhibits NF-κB induction of major histocompatibility class I and β2-microglobulin gene expression in transfected human neuroblastoma cells

Most neural cells constitutively lack major histocompatibility complex (MHC) class I and beta 2-microglobulin gene expression. Cytokines and viruses may, however, induce expression of these genes in some neural cells, and this correlates with factor binding to the NF-kappa B and interferon stimulated response elements of these genes. Here, we demonstrate that NF-kappa B is capable of inducing MHC class I and beta 2-microglobulin gene expression when transiently co-transfected into CHP-126 neuroblastomas, and that IRF-2 represses this induction. Interferon regulatory factor-2 (IRF-2) repression of MHC class I and beta 2-microglobulin gene expression in CHP-126 neuroblastomas may demonstrate a mechanism by which virus persists in neural cells. We show here that IRF-2 physically interacts in vitro with NF-kappa B. This interaction may contribute to the repression of the expression of these genes. Our demonstration that IRF family members, in addition to IRF-2, physically interact in vitro with NF-kappa B (p50 and p65), provides a general mechanism by which these transcription factors may, in concert, regulate the expression of a variety of genes involved in immune responses in the brain.

Novel regulation of CD80/CD86-induced phosphatidylinositol 3-kinase signaling by NOTCH1 protein in interleukin-6 and indoleamine 2,3-dioxygenase production by dendritic cells.

Background: Engagement of CD80/CD86 on dendritic cells by CD28 on T cells induces dendritic cell production of IL-6 and IDO. Results: The NOTCH pathway modulates activation of the PI3K pathway downstream of CD80/CD86 ligation and regulates IL-6 and IDO production. Conclusion: Cross-talk between NOTCH and PI3K pathways modulates dendritic cell production of IL-6 and IDO. Significance: Elucidating the molecular mechanism of NOTCH-PI3K cross-talk will have broad implications in human disease. Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K → AKT → NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.