T Cell Receptor-induced Nuclear Factor κB (NF-κB) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry.

T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca(2+) to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca(2+) has been determined. However, the role of Ca(2+) in controlling NF-κB signaling is poorly understood, and the source of Ca(2+) required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca(2+) via STIM1-dependent Ca(2+) release-activated Ca(2+)/Orai channels. We further show that Ca(2+) influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca(2+) is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca(2+)-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca(2+)-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca(2+) required for TCR-induced NF-κB activation and define a new distal Ca(2+)-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.

Negative feedback regulation of NF-κB-inducing kinase is proteasome-dependent but does not require cellular inhibitors of apoptosis.

Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism.

Stable reconstitution of IKK-deficient mouse embryonic fibroblasts.

Retroviral transduction is an invaluable technique in molecular biology used to express proteins encoded by nonviral genes in mammalian cells. A key feature of this technique is the ability to create cell lines that stably express the protein of interest and can be cultured long term. Here we describe a retroviral transduction procedure for mouse embryonic fibroblasts (MEFs) that uses Platinum-E cells to rapidly package high-titer, helper-free retrovirus. This technique is useful to study the role of key signaling kinases in the NF-κB signal transduction pathway.

Noncanonical NF-κB signaling is limited by classical NF-κB activity.

Precise regulation of nuclear factor κB (NF-κB) signaling is crucial for normal immune responses, and defective NF-κB activity underlies a range of immunodeficiencies. NF-κB is activated through two signaling cascades: the classical and noncanonical pathways. The classical pathway requires inhibitor of κB kinase ß (IKKß) and NF-κB essential modulator (NEMO), and hypomorphic mutations in the gene encoding NEMO (ikbkg) lead to inherited immunodeficiencies, collectively termed NEMO-ID. Noncanonical NF-κB activation requires NF-κB-inducing kinase (NIK) and IKKα, but not NEMO. We found that noncanonical NF-κB was basally active in peripheral blood mononuclear cells from NEMO-ID patients and that noncanonical NF-κB signaling was similarly enhanced in cell lines lacking functional NEMO. NIK, which normally undergoes constitutive degradation, was aberrantly present in resting NEMO-deficient cells, and regulation of its abundance was rescued by reconstitution with full-length NEMO, but not a mutant NEMO protein unable to physically associate with IKKα or IKKß. Binding of NEMO to IKKα was not required for ligand-dependent stabilization of NIK or noncanonical NF-κB signaling. Rather, an intact and functional IKK complex was essential to suppress basal NIK activity in unstimulated cells. Despite interacting with IKKα and IKKß to form an IKK complex, NEMO mutants associated with immunodeficiency failed to rescue classical NF-κB signaling or reverse the accumulation of NIK. Together, these findings identify a crucial role for classical NF-κB activity in the suppression of basal noncanonical NF-κB signaling.

A critical role for SOCS3 in innate resistance to Toxoplasma gondii.

The innate and adaptive immune responses that confer resistance to the intracellular pathogen Toxoplasma gondii critically depend on IL-12 production, which drives interferon-γ (IFN-γ) expression. Certain cytokines can activate STAT3 and limit IL-12 production to prevent infection-associated immune pathology, but T. gondii also directly activates STAT3 to evade host immunity. We show that suppressor of cytokine signaling molecule 3 (SOCS3), a target of STAT3 that limits signaling by the pleiotropic cytokine IL-6, is upregulated in response to infection but is dispensable for the immune-inhibitory effects of T. gondii. Unexpectedly, mice with targeted deletion of SOCS3 in macrophages and neutrophils have reduced IL-12 responses and succumb to toxoplasmosis. Anti-IL-6 administration or IL-12 treatment blocked disease susceptibility, suggesting that in the absence of SOCS3, macrophages are hypersensitive to the anti-inflammatory properties of IL-6. Thus, SOCS3 has a critical role in suppressing IL-6 signals and promoting immune responses to control T. gondii infection.

Complementary and alternative medicine use among health plan members. A cross-sectional survey.

UNLABELLED: CONTEXT. Many health plans have started to cover the cost of complementary and alternative medicine (CAM). National survey data indicate that CAM use is highly prevalent among adults. However, little is known about CAM use among health plan members. OBJECTIVE: To describe CAM users, the prevalence of CAM use, and how CAM use relates to utilization of conventional preventive services and health care satisfaction among health plan members. DESIGN: Cross-sectional mail survey in 1997. SETTING: Managed care organization in Minnesota. SAMPLE: Random sample of health plan members aged 40 and older stratified by number of chronic diseases; 4404 (86%) of the 5107 returned completed questionnaires. MEASURES: Use of CAM, patient characteristics (e.g., chronic diseases, health status), health behaviors (e.g., smoking, diet, exercise), and interaction with conventional health care (e.g., use of preventive services, having a primary care doctor, health care satisfaction). RESULTS: Overall, 42% reported the use of at least one CAM therapy; the most common were relaxation techniques (18%), massage (12%), herbal medicine (10%), and megavitamin therapy (9%). Perceived efficacy of CAM ranged from 76% (hypnosis) to 98% (energy healing). CAM users tended to be female, younger, better educated, and employed. Users of CAM reported more physical and emotional limitations, more pain, and more dysthymia but were not more likely to have a chronic condition. CAM users were slightly more likely to have a primary care provider (86% vs. 82% had chosen a primary care provider; P =0.014) and had more favorable health-related behaviors. CAM users and nonusers were equally likely to use conventional preventive services and were equally satisfied with their health plan. CONCLUSION: CAM use is highly prevalent among health plan members. CAM users report more physical and emotional limitations than do nonusers. CAM does not seem to be a substitute for conventional preventive health care.