Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool.

Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.

Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis.

The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

Mitochondrial division is requisite to RAS-induced transformation and targeted by oncogenic MAPK pathway inhibitors.

Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RAS(G12V)-mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin-related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RAS(G12V)-induced mitochondrial dysfunction and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1(S616) phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1(S616) phosphorylation status dichotomizes BRAF(WT) from BRAF(V600E)-positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with leverage in chemotherapeutic success.

GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells.

GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAFV600E), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAFV600E is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAFV600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

The BCL-2 family reunion.

B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.

Mitochondrial Fission in Human Diseases.

Mitochondria are an essential component of multicellular life - from primitive organisms, to highly complex entities like mammals. The importance of mitochondria is underlined by their plethora of well-characterized essential functions such as energy production through oxidative phosphorylation (OX-PHOS), calcium and reactive oxygen species (ROS) signaling, and regulation of apoptosis. In addition, novel roles and attributes of mitochondria are coming into focus through the recent years of mitochondrial research. In particular, over the past decade the study of mitochondrial shape and dynamics has achieved special significance, as they are found to impact mitochondrial function. Recent advances indicate that mitochondrial function and dynamics are inter-connected, and maintain the balance between health and disease at a cellular and an organismal level. For example, excessive mitochondrial division (fission) is associated with functional defects, and is implicated in multiple human diseases from neurodegenerative diseases to cancer. In this chapter we examine the recent literature on the mitochondrial dynamics-function relationship, and explore how it impacts on the development and progression of human diseases. We will also highlight the implications of therapeutic manipulation of mitochondrial dynamics in treating various human pathologies.

How do I kill thee? Let me count the ways: p53 regulates PARP-1 dependent necrosis.

Understanding the impact of the p53 tumor suppressor pathway on the regulation of genome integrity, cancer development, and cancer treatment has intrigued scientists and clinicians for decades. It appears that the p53 pathway is a central node for nearly all cell stress responses, including: gene expression, DNA repair, cell cycle arrest, metabolic adjustments, apoptosis, and senescence. In the past decade, it has become increasingly clear that p53 function is directly regulated by poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair signaling. Here, we will discuss the impact of PARP-1 on p53 function, along with a recently described novel role for the reciprocal regulation of p53 regulated, PARP-1 dependent necrosis following DNA damage.

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis.

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the antiapoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique among BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules BAX and BAK. Structural investigations using NMR spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the antiapoptotic BCL-2 repertoire to sensitize for death receptor-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

BAK/BAX activation and cytochrome c release assays using isolated mitochondria.

The mitochondrial pathway of apoptosis proceeds when the outer mitochondrial membrane (OMM) is compromised by the pro-apoptotic BCL-2 family members, BAK and BAX. Once activated, BAK and BAX form proteolipid pores in the OMM leading to mitochondrial outer membrane permeabilization (MOMP), and the release of inner membrane space proteins, such as cytochrome c, which promotes caspase activation. The use of isolated mitochondria has been instrumental to understanding the key interactions necessary to engage BAK and BAX activation, MOMP, and apoptosis. Furthermore, it is possible to biochemically define the relationships between BCL-2 family function and mitochondrial physiology using isolated systems. Our laboratory uses freshly isolated mitochondria from numerous sources to better understand BCL-2 family function and requirements for BAK and BAX activation. Here, we will discuss commonly used in vitro techniques to perform MOMP and cytochrome c release assays; and provide several key methodologies to implicate BAK and BAX activity in these processes.

BAK activation is necessary and sufficient to drive ceramide synthase-dependent ceramide accumulation following inhibition of BCL2-like proteins.

Determining mechanistic details about how drugs kill cancer cells is critical for predicting which cancers will respond to given therapeutic regimens and for identifying effective combinations of drugs that more potently kill cancer cells while sparing normal cells. The BCL2 family of proteins and bioactive sphingolipids are intricately linked during apoptotic cell death. In fact, many chemotherapeutic drugs are known to cause accumulation of the pro-apoptotic sphingolipid ceramide; however, the mechanism by which this occurs is not completely understood. In the present study we demonstrate that direct inhibition of anti-apoptotic BCL2 proteins with ABT-263 is sufficient to induce C(16)-ceramide synthesis in multiple cell lines, including human leukaemia and myeloma cells. ABT-263 activates CerS (ceramide synthase) activity only in cells expressing BAK or in cells capable of activating BAK. Importantly, recombinant BAK is sufficient to increase in vitro CerS activity in microsomes purified from Bak-KO (knockout) cells and activated BAK more potently activates CerS than inactive BAK. Likewise, ABT-263 addition to wild-type, but not Bak-deficient, microsomes increases CerS in vitro activity. Furthermore, we present a feed-forward model by which BAK activation of CerS by chemotherapeutic drugs leads to elevated ceramide levels that result in synergistic channel formation by ceramide (or one of its metabolites) and BAX/BAK.

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes.

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization.

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells.

Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we address how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. Contrary to expectations, we found that S1PR1 limits apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization are required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlight an unexpected effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggest both limitations and novel uses of this important class of drugs.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

BH3 domains other than Bim and Bid can directly activate Bax/Bak.

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.