Histone oxidation as a new mechanism of metabolic control over gene expression.

The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior.

SOD2 acetylation on lysine 68 promotes stem cell reprogramming in breast cancer.

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.

Inorganic arsenic promotes luminal to basal transition and metastasis of breast cancer.

Inorganic arsenic (iAs/As2 O32- ) is an environmental toxicant found in watersheds around the world including in densely populated areas. iAs is a class I carcinogen known to target the skin, lungs, bladder, and digestive organs, but its role as a primary breast carcinogen remains controversial. Here, we examined a different possibility: that exposure to iAs promotes the transition of well-differentiated epithelial breast cancer cells characterized by estrogen and progesterone receptor expression (ER+/PR+), to more basal phenotypes characterized by active proliferation, and propensity to metastasis in vivo. Our results indicate two clear phenotypic responses to low-level iAs that depend on the duration of the exposure. Short-term pulses of iAs activate ER signaling, consistent with its reported pseudo-estrogen activity, but longer-term, chronic treatments for over 6 months suppresses both ER and PR expression and signaling. In fact, washout of these chronically exposed cells for up to 1 month failed to fully reverse the transcriptional and phenotypic effects of prolonged treatments, indicating durable changes in cellular physiologic identity. RNA-seq studies found that chronic iAs drives the transition toward more basal phenotypes characterized by impaired hormone receptor signaling despite the conservation of estrogen receptor expression. Because treatments for breast cancer patients are largely designed based on the detection of hormone receptor expression, our results suggest greater scrutiny of ER+ cancers in patients exposed to iAs, because these tumors may spawn more aggressive phenotypes than unexposed ER+ tumors, in particular, basal subtypes that tend to develop therapy resistance and metastasis.

The Akt1 isoform is required for optimal IFN-ß transcription through direct phosphorylation of ß-catenin.

IFN-ß is a critical antiviral cytokine that is capable of modulating the systemic immune response. The transcriptional induction of IFN-ß is a highly regulated process, involving the activation of pattern recognition receptors and their downstream signaling pathways. The Akt family of serine/threonine kinases includes three isoforms. The specific role for the individual Akt isoforms in pattern recognition and signaling remains unclear. In this article, we report that the TLR3-mediated expression of IFN-ß is blunted in cells that lack Akt1. The expression of IFN-ß-inducible genes such as CCL5 and CXCL10 was also reduced in Akt1-deficient cells; the induction of TNF-α and CXCL2, whose expression does not rely on IFN-ß, was not reduced in the absence of Akt1. Macrophages from Akt1(-/-) mice displayed deficient clearance of HSV-1 along with reduced IFN-ß expression. Our results demonstrate that Akt1 signals through ß-catenin by phosphorylation on Ser(552), a site that differs from the glycogen synthase kinase 3 ß phosphorylation site. Stimulation of a chemically activated version of Akt1, in the absence of other TLR3-dependent signaling, was sufficient for accumulation and phosphorylation of ß-catenin at Ser(552). Taken together, these results demonstrate that the Akt1 isoform is required for ß-catenin-mediated promotion of IFN-ß transcription downstream of TLR3 activation.

ROS production by mitochondria: function or dysfunction?

In eukaryotic cells, ATP generation is generally viewed as the primary function of mitochondria under normoxic conditions. Reactive oxygen species (ROS), in contrast, are regarded as the by-products of respiration, and are widely associated with dysfunction and disease. Important signaling functions have been demonstrated for mitochondrial ROS in recent years. Still, their chemical reactivity and capacity to elicit oxidative damage have reinforced the idea that ROS are the products of dysfunctional mitochondria that accumulate during disease. Several studies support a different model, however, by showing that: (1) limited oxygen availability results in mitochondria prioritizing ROS production over ATP, (2) ROS is an essential adaptive mitochondrial signal triggered by various important stressors, and (3) while mitochondria-independent ATP production can be easily engaged by most cells, there is no known replacement for ROS-driven redox signaling. Based on these observations and other evidence reviewed here, we highlight the role of ROS production as a major mitochondrial function involved in cellular adaptation and stress resistance. As such, we propose a rekindled view of ROS production as a primary mitochondrial function as essential to life as ATP production itself.

Metabolism and epigenetics: drivers of tumor cell plasticity and treatment outcomes.

Emerging evidence indicates that metabolism not only is a source of energy and biomaterials for cell division but also acts as a driver of cancer cell plasticity and treatment resistance. This is because metabolic changes lead to remodeling of chromatin and reprogramming of gene expression patterns, furthering tumor cell phenotypic transitions. Therefore, the crosstalk between metabolism and epigenetics seems to hold immense potential for the discovery of novel therapeutic targets for various aggressive tumors. Here, we highlight recent discoveries supporting the concept that the cooperation between metabolism and epigenetics enables cancer to overcome mounting treatment-induced pressures. We discuss how specific metabolites contribute to cancer cell resilience and provide perspective on how simultaneously targeting these key forces could produce synergistic therapeutic effects to improve treatment outcomes.

Histone H3.1 is a chromatin-embedded redox sensor triggered by tumor cells developing adaptive phenotypic plasticity and multidrug resistance.

Chromatin structure is regulated through posttranslational modifications of histone variants that modulate transcription. Although highly homologous, histone variants display unique amino acid sequences associated with specific functions. Abnormal incorporation of histone variants contributes to cancer initiation, therapy resistance, and metastasis. This study reports that, among its biologic functions, histone H3.1 serves as a chromatin redox sensor that is engaged by mitochondrial H2O2. In breast cancer cells, the oxidation of H3.1Cys96 promotes its eviction and replacement by H3.3 in specific promoters. We also report that this process facilitates the opening of silenced chromatin domains and transcriptional activation of epithelial-to-mesenchymal genes associated with cell plasticity. Scavenging nuclear H2O2 or amino acid substitution of H3.1(C96S) suppresses plasticity, restores sensitivity to chemotherapy, and induces remission of metastatic lesions. Hence, it appears that increased levels of H2O2 produced by mitochondria of breast cancer cells directly promote redox-regulated H3.1-dependent chromatin remodeling involved in chemoresistance and metastasis.

The multifaceted activities of AMPK in tumor progression--why the "one size fits all" definition does not fit at all?

AMP-activated kinase (AMPK) is a central cellular energetic biosensor and regulator of a broad array of cellular metabolic routes activated by nutrient deprivation, mitochondrial dysfunction, oxidative stress, and cytokines. The activation of AMPK maintains ATP levels in response to hypoxia, mitochondrial dysfunction, and shortage of essential metabolic fuels. Activated AMPK turns on energy sparing pathways and promotes antiapoptotic functions thereby permitting cells to survive extremely hostile conditions for prolonged periods of time. Cancer cells in solid tumors are generally subjected to such harsh conditions; however, they manage to efficiently survive and proliferate. This is likely due, in great part, to a peculiar form of metabolism that is heavily reliant on glycolysis and which promotes cancer cell adaptation and tumor progression. AMPK controls the influx and utilization of glucose by cancer cells and therefore has emerged as an attractive target to treat cancer. Investigations exploring this possibility demonstrated that activators or inhibitors of AMPK impact cancer cell viability and possibly cancer progression. For example, the AMPK activator metformin induces apoptosis in a variety of cancer cell lines and models. A major problem with many of the studies on metformin is that little effort has been invested in unraveling how metformin activates AMPK in the many contexts it has been tested. This is significant because many AMPK-independent effects of metformin have been documented. The notion that AMPK acts solely as a tumor suppressor also conflicts with findings that it confers resistance to nutrient deprivation, sustains NADPH levels in cancer cells, facilitates stress-induced gene transcription, promotes cell survival via antiapoptotic function upregulation, intermediates epithelial-to-mesenchymal transition, and increases malignant transformation. These are all recognized steps necessary for the successful evolution of tumors. This review highlights some of these findings and proposes that the role of AMPK in cancer should be reconsidered in light of the complex roles of AMPK under different metabolic conditions.

Prolonged warm ischemia time increases mitogen-activated protein kinase activity and decreases perfusate cytokine levels in ex vivo rat liver machine perfusion.

Introduction: Machine perfusion is increasingly being utilized in liver transplantation in lieu of traditional cold static organ preservation. Nevertheless, better understanding of the molecular mechanisms underlying the ischemia-reperfusion injury (IRI) during ex vivo perfusion is necessary to improve the viability of liver grafts after transplantation using machine perfusion technology. Since key cellular signaling pathways involved in hepatic IRI may allow a chance for designing a promising approach to improve the clinical outcomes from this technology, we determined how warm ischemia time (WIT) during procurement affects the activity of mitogen-activated protein kinase (MAPK) and perfusate concentration of cytokines in an ex vivo rat liver machine perfusion model. Methods: Male Sprague-Dawley rats underwent in situ hepatic ischemia with varying WIT (0, 10, 20, 30 min, n = 5 each), and subsequently 3 h of cold ischemia time and 2 h of machine perfusion prior to determining the degree of MAPK activation-phosphorylation and cytokine concentration in liver tissue and perfusates, respectively. Results: Our data revealed a strong correlation between incremental WIT and a series of liver injury markers, and that prolonged WIT increases ERK1/2 and p54 JNK phosphorylation during machine perfusion. Notably, specific cytokine levels (MCP-1, MIP-2, GRO/KC, IL-10, and IL-5) were inversely correlated with the phosphorylation levels of ERK1/2, p38 MAPK, and p46/p54 JNK. Discussion: These results suggest that MAPK activation, specifically ERK1/2 and p54 JNK phosphorylation, have potential as a biomarker for hepatic IRI pathophysiology during machine perfusion. Elucidation of their functional significance may lead to designing a novel strategy to increase the clinical benefit of machine perfusion.

SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes.

Suppressor of cytokine signaling-1 (SOCS1) exerts control over inflammation by targeting p65 nuclear factor-κB (NF-κB) for degradation in addition to its canonical role regulating cytokine signaling. We report here that SOCS1 does not operate on all p65 targets equally, instead localizing to a select subset of pro-inflammatory genes. Promoter-specific interactions of SOCS1 and p65 determine the subset of genes activated by NF-κB during systemic inflammation, with profound consequences for cytokine responses, immune cell mobilization, and tissue injury. Nitric oxide synthase-1 (NOS1)-derived nitric oxide (NO) is required and sufficient for the displacement of SOCS1 from chromatin, permitting full inflammatory transcription. Single-cell transcriptomic analysis of NOS1-deficient animals led to detection of a regulatory macrophage subset that exerts potent suppression on inflammatory cytokine responses and tissue remodeling. These results provide the first example of a redox-sensitive, gene-specific mechanism for converting macrophages from regulating inflammation to cells licensed to promote aggressive and potentially injurious inflammation.

Collagen-Specific HSP47+ Myofibroblasts and CD163+ Macrophages Identify Profibrotic Phenotypes in Deceased Hearts With SARS-CoV-2 Infections.

Background Cardiac fibrosis complicates SARS-CoV-2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress-inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection. Methods and Results Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47+ cells and collagen α 1(l) in hearts from people with SARS-CoV-2 infections. Because macrophages are also linked to inflammation, we measured CD163+ cells in the same tissues. We observed irregular groups of spindle-shaped HSP47+ and CD163+ cells as well as increased collagen α 1(I) deposition, each proximate to one another in "hot spots" of ≈40% of hearts after SARS-CoV-2 infection (HSP47+ P<0.05 versus nonfibrotics and P<0.001 versus controls). Because HSP47+ cells are consistent with myofibroblasts, subjects with hot spots are termed "profibrotic." The remaining 60% of subjects dying with COVID-19 without hot spots are referred to as "nonfibrotic." No control subject exhibited hot spots. Conclusions Colocalization of myofibroblasts, M2(CD163+) macrophages, and collagen α 1(l) may be the first evidence of a COVID-19-related "profibrotic phenotype" in human hearts in situ. The potential public health and diagnostic implications of these observations require follow-up to further define mechanisms of viral-mediated cardiac fibrosis.

Map kinase phosphatase 5 protects against sepsis-induced acute lung injury.

Mitogen-activated protein kinases (MAPKs) play a critical role in inflammation. Although activation of MAPK in inflammatory cells has been studied extensively, much less is known about the inactivation of these kinases. MAPK phosphatase 5 (MKP5) is a member of the dual-specificity phosphatase family that dephosphorylates activated MAPKs. Here we report that MKP5 protects sepsis-induced acute lung injury. Mice lacking MKP5 displayed severe lung tissue damage following LPS challenge, characterized with increased neutrophil infiltration and edema compared with wild-type (WT) controls. In response to LPS, MKP5-deficient macrophages produced significantly more inflammatory factors including inflammatory cytokines, nitric oxide, and superoxide. Phosphorylation of p38 MAPK, JNK, and ERK were enhanced in MKP5-deficient macrophages upon LPS stimulation. Adoptive transfer of MKP5-deficient macrophages led to more severe lung inflammation than transfer of WT macrophages, suggesting that MKP5-deficient macrophages directly contribute to acute lung injury. Taken together, these results suggest that MKP5 is crucial to homeostatic regulation of MAPK activation in inflammatory responses.

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies.

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.

Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.

Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

Nitric oxide in cellular adaptation and disease.

Nitric oxide synthases are the major sources of nitric oxide, a critical signaling molecule involved in a wide range of cellular and physiological processes. These enzymes comprise a family of genes that are highly conserved across all eukaryotes. The three family members found in mammals are important for inter- and intra-cellular signaling in tissues that include the nervous system, the vasculature, the gut, skeletal muscle, and the immune system, among others. We summarize major advances in the understanding of biochemical and tissue-specific roles of nitric oxide synthases, with a focus on how these mechanisms enable tissue adaptation and health or dysfunction and disease. We highlight the unique mechanisms and processes of neuronal nitric oxide synthase, or NOS1. This was the first of these enzymes discovered in mammals, and yet much remains to be understood about this highly conserved and complex gene. We provide examples of two areas that will likely be of increasing importance in nitric oxide biology. These include the mechanisms by which these critical enzymes promote adaptation or disease by 1) coordinating communication by diverse cell types within a tissue and 2) directing cellular differentiation/activation decisions processes.

Mitochondrial Superoxide Dismutase: What the Established, the Intriguing, and the Novel Reveal About a Key Cellular Redox Switch.

Significance: Reactive oxygen species (ROS) are now widely recognized as central mediators of cell signaling. Mitochondria are major sources of ROS. Recent Advances: It is now clear that mitochondrial ROS are essential to activate responses to cellular microenvironmental stressors. Mediators of these responses reside in large part in the cytosol. Critical Issues: The primary form of ROS produced by mitochondria is the superoxide radical anion. As a charged radical anion, superoxide is restricted in its capacity to diffuse and convey redox messages outside of mitochondria. In addition, superoxide is a reductant and not particularly efficient at oxidizing targets. Because there are many opportunities for superoxide to be neutralized in mitochondria, it is not completely clear how redox cues generated in mitochondria are converted into diffusible signals that produce transient oxidative modifications in the cytosol or nucleus. Future Directions: To efficiently intervene at the level of cellular redox signaling, it seems that understanding how the generation of superoxide radicals in mitochondria is coupled with the propagation of redox messages is essential. We propose that mitochondrial superoxide dismutase (SOD2) is a major system converting diffusion-restricted superoxide radicals derived from the electron transport chain into highly diffusible hydrogen peroxide (H2O2). This enables the coupling of metabolic changes resulting in increased superoxide to the production of H2O2, a diffusible secondary messenger. As such, to determine whether there are other systems coupling metabolic changes to redox messaging in mitochondria as well as how these systems are regulated is essential.

Acquired Immunity Is Not Essential for Radiation-Induced Heart Dysfunction but Exerts a Complex Impact on Injury.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG-/-)) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p < 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1.

The NF-κB pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-κB. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-κB transcriptional activity. As a result, NOS1(-/-) mice demonstrate reduced cytokine production, lung injury, and mortality when subjected to two different models of sepsis. Isolated NOS1(-/-) macrophages demonstrate similar defects in proinflammatory transcription on challenge with Gram-negative bacterial LPS. Consistently, we found that activated NOS1(-/-) macrophages contain increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells. NOS1-dependent S-nitrosation of SOCS1 impairs its binding to p65 and targets SOCS1 for proteolysis. Treatment of NOS1(-/-) cells with exogenous NO rescues both SOCS1 degradation and stabilization of p65 protein. Point mutation analysis demonstrated that both Cys147 and Cys179 on SOCS1 are required for its NO-dependent degradation. These findings demonstrate a fundamental role for NOS1-derived NO in regulating TLR4-mediated inflammatory gene transcription, as well as the intensity and duration of the resulting host immune response.