Multicellular organisms initiate adaptive responses when oxygen (O(2)) availability decreases, but the underlying mechanism of O(2) sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1 alpha and HIF-2 alpha and that an increase in reactive oxygen species (ROS) links this complex to HIF-alpha stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1 alpha stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O(2) sensors and signal hypoxic HIF-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.

Adaptive immune responses are tailored to different types of pathogens through differentiation of naïve CD4 T cells into functionally distinct subsets of effector T cells (TH1, TH2, and TH17) defined by expression of key transcription factors (TFs)1. Regulatory T (Treg) cells comprise a distinct anti-inflammatory lineage specified by the X-linked TF Foxp32, 3. Paradoxically, some activated Treg cells express the aforementioned effector CD4 T cell TFs, which have been suggested to endow Treg cells with enhanced suppressive capacity4, 5, 6. Whether expression of these factors in Treg cells—akin to effector T cells—is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here, we demonstrate that in Treg cells expression of the TH1-associated TF T-bet, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss-of-function or elimination of T-bet-expressing Treg cells—but not of T-bet in Treg cells—resulted in severe TH1 autoimmunity. Conversely, following depletion of T-bet-negative Treg cells, remaining T-bet+ cells specifically inhibited TH1 and CD8 T cell activation in agreement with their co-localization with T-bet+ effector T cells. These results suggest an essential immunosuppressive function for T-bet+ Treg cells and indicate that Treg cell functional heterogeneity is a critical feature of immune tolerance.

Transcriptional activation of the human interleukin-2 (IL-2) gene, like induction of the IL-2 receptor alpha (IL-2R alpha) gene and the type 1 human immunodeficiency virus (HIV-1), is shown to be modulated by a kappa B-like enhancer element. Mutation of a kappa B core sequence identified in the IL-2 promoter (-206 to -195) partially inhibits both mitogen- and HTLV-I Tax-mediated activation of this transcription unit and blocks the specific binding of two inducible cellular factors. These kappa B-specific proteins (80 to 90 and 50 to 55 kilodaltons) similarly interact with the functional kappa B enhancer present in the IL-2R alpha promoter. These data suggest that these kappa B-specific proteins have a role in the coordinate regulation of this growth factor-growth factor receptor gene system that controls T cell proliferation.

Zinc is a structural component of many regulatory molecules including transcription factors and signaling molecules. We report that two alternate signaling pathways of protein kinase C (PKC) activation involving either the lipid second messengers (diacylglycerol and its mimetics, the phorbol esters) or reactive oxygen converge at the zinc finger of the regulatory domain. They all trigger the release of zinc ions. An increase in intracellular free Zn 2؉ was observed by confocal microscopy in intact cells treated with phorbol ester or by mild oxidation. The source of liberated Zn 2؉ was traced to PKC and particularly the zinc finger domains. The activated form of native PKC␣ contained significantly less Zn 2؉ than the resting form. Furthermore, purified recombinant PKC protein fragments shed stoichiometric amounts of Zn 2؉ upon reaction with diacylglycerol, phorbol ester, or reactive oxygen in vitro. Our results offer new insight into the regulation of PKC. Far from cementing rigid structures, zinc actually is the linchpin that orchestrates dynamic changes in response to specific signals, allowing kinase activity to be turned on or off.Zinc is essential to the structure of numerous signaling proteins. Transcription factors and signaling molecules share zinc finger structures as a common motif, although they differ in composition and function (1). The C2H2 zinc finger is believed to provide rigidity to transcription factors for proper DNA binding capacity. By comparison, the structure/function relationship of the components in the C3H1 zinc fingers of signaling molecules is not fully understood. We report here that Zn 2ϩ ions of the cysteine-rich domain play a dynamic role in the function of protein kinase C.Protein kinase C (PKC) 1 isoforms function as central signal amplifiers. They are engaged by two alternate pathways. Most isoforms with the exception of atypical ones (2) are activated by the second messenger, diacylglycerol (3, 4), or its mimetics, the phorbol esters (5). Independent of this classic pathway, PKC is also controlled by a redox mechanism where oxidation converts the protein to the catalytically competent form (6 -8), whereas reduction reverses this process (9). Cofactors are required for enzyme regulation by both pathways. Calcium and phosphatidylserine are important for amplification of the diacylglycerol signal, whereas vitamin A, as we have shown recently, is needed for efficient redox activation of several PKC isoforms (9). The lipid binding sites, accommodating diacylglycerol or phorbol ester on one hand (10, 11) and retinol on the other (12) are located at the non-overlapping regions within the twin cysteine-rich structures (referred to as Cys domains) of the regulatory domains. These 50 amino acid-long highly homologous stretches contain 6 conserved cysteine and 2 conserved histidine residues, tetrahedrically coordinated by two Zn 2ϩ ions into a composite zinc finger (1,(13)(14)(15). We show that the diacylglycerol phorbol ester pathway and the alternate redox pathway converge at this zinc...

SUMMARYT cell receptor (TCR) signaling plays a key role in T cell fate determination. Precursor cells expressing TCRs within a certain low affinity range for self peptide-MHC complexes undergo positive selection and differentiate into naïve T cells expressing a highly diverse self-MHC restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for “self” are either eliminated through TCR agonist induced apoptosis (negative selection)1 or restrained by regulatory CD4+ T (Treg) cells, whose differentiation and function are controlled by the X-chromosome encoded transcription factor Foxp3 (review2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist driven TCR signals combined with interleukin-2 (IL-2) receptor signaling. In addition to Treg cells, TCR agonist-driven selection results in the generation of several other specialized T cell lineages like NKT and MAIT cells3. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs4-6, Here we explored whether a specialized mechanism enables agonist driven selection of Treg cells with a diverse TCR repertoire and its significance for self-tolerance. We found that intronic Foxp3 enhancer CNS3 acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to effectively control self-reactive T cells, especially when thymic negative selection was genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

The mammalian gut microbiota provides essential metabolites to the host and promotes the differentiation and accumulation of extrathymically generated regulatory T (pTreg) cells. To explore the impact of these cells on intestinal microbial communities, we assessed the composition of the microbiota in pTreg cell-deficient and -sufficient mice. pTreg cell deficiency led to heightened type 2 immune responses triggered by microbial exposure, which disrupted the niche of border-dwelling bacteria early during colonization. Moreover, impaired pTreg cell generation led to pervasive changes in metabolite profiles, altered features of the intestinal epithelium, and reduced body weight in the presence of commensal microbes. Absence of a single species of bacteria depleted in pTreg cell-deficient animals, Mucispirillum schaedleri, partially accounted for the sequelae of pTreg cell deficiency. These observations suggest that pTreg cells modulate the metabolic function of the intestinal microbiota by restraining immune defense mechanisms that may disrupt a particular bacterial niche.

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH2-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms α, δ, ζ, and μ, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKCα by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

The physiology of two metabolites of vitamin A is understood in substantial detail: retinaldehyde functions as the universal chromophore in the vertebrate and invertebrate eye; retinoic acid regulates a set of vertebrate transcription factors, the retinoic acid receptor superfamily. The third member of this retinoid triumvirate is retinol. While functioning as the precursor of retinaldehyde and retinoic acid, a growing body of evidence suggests a far more fundamental role for retinol in signal transduction. Here we show that retinol is essential for the metabolic fitness of mitochondria. When cells were deprived of retinol, respiration and ATP synthesis defaulted to basal levels. They recovered to significantly higher energy output as soon as retinol was restored to physiological concentration, without the need for metabolic conversion to other retinoids. Retinol emerged as an essential cofactor of protein kinase Cdelta (PKCdelta), without which this enzyme failed to be activated in mitochondria. Furthermore, retinol needed to physically bind PKCdelta, because mutation of the retinol binding site rendered PKCdelta unresponsive to Rol, while retaining responsiveness to phorbol ester. The PKCdelta/retinol complex signaled the pyruvate dehydrogenase complex for enhanced flux of pyruvate into the Krebs cycle. The baseline response was reduced in vitamin A-deficient lecithin:retinol acyl transferase-knockout mice, but this was corrected within 3 h by intraperitoneal injection of vitamin A; this suggests that vitamin A is physiologically important. These results illuminate a hitherto unsuspected role of vitamin A in mitochondrial bioenergetics of mammals, acting as a nutritional sensor. As such, retinol is of fundamental importance for energy homeostasis. The data provide a mechanistic explanation to the nearly 100-yr-old question of why vitamin A deficiency causes so many pathologies that are independent of retinoic acid action.