Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase β (IKKβ)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.

Physiological processes that govern the normal functioning of mammalian cells are regulated by a myriad of signalling pathways. Mammalian mitogen-activated protein (MAP) kinases constitute one of the major signalling arms and have been broadly classified into four groups that include extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and ERK5. Each signalling cascade is governed by a wide array of external and cellular stimuli, which play a critical part in mammalian cells in the regulation of various key responses, such as mitogenic growth, differentiation, stress responses, as well as inflammation. This evolutionarily conserved MAP kinase signalling arm is also important for metabolic maintenance, which is tightly coordinated via complicated mechanisms that include the intricate interaction of scaffold proteins, recognition through cognate motifs, action of phosphatases, distinct subcellular localisation, and even post-translational modifications. Aberration in the signalling pathway itself or their regulation has been implicated in the disruption of metabolic homeostasis, which provides a pathophysiological foundation in the development of metabolic syndrome. Metabolic syndrome is an umbrella term that usually includes a group of closely associated metabolic diseases such as hyperglycaemia, hyperlipidaemia, and hypertension. These risk factors exacerbate the development of obesity, diabetes, atherosclerosis, cardiovascular diseases, and hepatic diseases, which have accounted for an increase in the worldwide morbidity and mortality rate. This review aims to summarise recent findings that have implicated MAP kinase signalling in the development of metabolic diseases, highlighting the potential therapeutic targets of this pathway to be investigated further for the attenuation of these diseases.

Modulating cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP₃R) Ca²⁺-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP₃R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca²⁺ concentration ([Ca²⁺]_ER) is poorly understood and controversial. We discovered that the InsP₃R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca²⁺]_ER. The widely-expressed Ca²⁺-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca²⁺]_ER inhibition of InsP₃R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP₃-mediated Ca²⁺ signaling events. Thus, [Ca²⁺]_ER is a major regulator of InsP₃R channel activity and InsP₃R-mediated [Ca²⁺]_i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca²⁺-binding protein, likely ANXA1.

Endothelial dysfunction, referring to a disturbance in the vascular homeostasis, has been implicated in many disease conditions including ischemic/reperfusion injury and atherosclerosis. Endothelial mitochondria have been increasingly recognized as a regulator of calcium homeostasis which has implications in the execution of diverse cellular events and energy production. The mitochondrial calcium uniporter complex through which calcium enters the mitochondria is composed of several proteins, including the pore-forming subunit MCU and its regulators MCUR1, MICU1, and MICU2. Mitochondrial calcium overload leads to opening of MPTP (mitochondrial permeability transition pore) and results in apoptotic cell death. Whereas, blockage of calcium entry into the mitochondria results in reduced ATP production thereby activates AMPK-mediated pro-survival autophagy. Here, we investigated the expression of mitochondrial calcium uniporter complex components (MCU, MCUR1, MICU1, and MICU2), induction of autophagy and apoptotic cell death in endothelial cells in response to oxygen-glucose deprivation. Human pulmonary microvascular endothelial cells (HPMVECs) were subjected to oxygen-glucose deprivation (OGD) at 3-h timepoints up to 12 h. Interestingly, except MCUR1 which was significantly downregulated, all other components of the uniporter (MCU, MICU1, and MICU2) remained unchanged. MCUR1 downregulation has been shown to activate AMPK mediated pro-survival autophagy. Similarly, MCUR1 downregulation in response to OGD resulted in AMPK phosphorylation and LC3 processing indicating the activation of pro-survival autophagy. Despite the activation of autophagy, OGD induced Caspase-mediated apoptotic cell death. Blockade of autophagy did not reduce OGD-induced apoptotic cell death whereas serum starvation conferred enough cellular and functional protection. In conclusion, the autophagic flux induced by MCUR1 downregulation in response to OGD is insufficient in protecting endothelial cells from undergoing apoptotic cell death and requires enhancement of autophagic flux by additional means such as serum starvation.

Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart’s function and its vulnerability to injury and disease.