Proteomics data on MAP Kinase Kinase 3 knock out bone marrow derived macrophages exposed to cigarette smoke extract.

This data article reports changes in the phosphoproteome and total proteome of cigarette smoke extract (CSE) exposed WT and MAP Kinase Kinase 3 knock out (MKK3-/-) bone marrow derived macrophages (BMDM). The dataset generated is helpful for understanding the mechanism of CSE induced inflammation and the role of MAP kinase signaling pathway. The cellular proteins were labeled with isobaric tags for relative and absolute quantitation (iTRAQ®) reagents and analyzed by LC-MS/MS. The standard workflow module for iTRAQ® quantification within the Proteome Discoverer was utilized for the data analysis. Ingenuity Pathway Analysis (IPA) software and Reactome was used to identify enriched canonical pathways and molecular networks (Mannam et al., 2016) [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs.

MKK3 influences mitophagy and is involved in cigarette smoke-induced inflammation.

Cigarette smoking is the primary risk factor for COPD which is characterized by excessive inflammation and airflow obstruction of the lung. While inflammation is causally related to initiation and progression of COPD, the mitochondrial mechanisms that underlie the associated inflammatory responses are poorly understood. In this context, we have studied the role played by Mitogen activated protein (MAP) kinase kinase 3 (MKK3), a dual-specificity protein kinase, in cigarette smoke induced-inflammation and mitochondrial dysfunction. Serum pro-inflammatory cytokines were significantly elevated in WT but not in MKK3-/- mice exposed to Cigarette smoke (CS) for 2 months. To study the cellular mechanisms of inflammation, bone marrow derived macrophages (BMDMs), wild type (WT) and MKK3-/-, were exposed to cigarette smoke extract (CSE) and inflammatory cytokine production and mitochondrial function assessed. The levels of IL-1ß, IL-6, and TNFα were increased along with higher reactive oxygen species (ROS) and P-NFκB after CSE treatment in WT but not in MKK3-/- BMDMs. CSE treatment adversely affected basal mitochondrial respiration, ATP production, maximum respiratory capacity, and spare respiratory capacity in WT BMDMs only. Mitophagy, clearance of dysfunctional mitochondria, was up regulated in CS exposed WT mice lung tissue and CSE exposed WT BMDMs, respectively. The proteomic analysis of BMDMs by iTRAQ (isobaric tags for relative and absolute quantitation) showed up regulation of mitochondrial dysfunction associated proteins in WT and higher OXPHOS (Oxidative phosphorylation) and IL-10 signaling proteins in MKK3-/- BMDMs after CSE exposure, confirming the critical role of mitochondrial homeostasis. Interestingly, we found increased levels of p-MKK3 by immunohistochemistry in COPD patient lung tissues that could be responsible for insufficient mitophagy and disease progression. This study identifies MKK3 as a negative regulator of mitochondrial function and inflammatory responses to CS and suggests that MKK3 could be a therapeutic target.

Differential regulation of autophagy and mitophagy in pulmonary diseases.

Lysosomal-mediated degradation of intracellular lipids, proteins and organelles, known as autophagy, represents a inducible adaptive response to lung injury resulting from exposure to insults, such as hypoxia, microbes, inflammation, ischemia-reperfusion, pharmaceuticals (e.g., bleomycin), or inhaled xenobiotics (i.e., air pollution, cigarette smoke). This process clears damaged or toxic cellular constituents and facilitates cell survival in stressful environments. Autophagic degradation of dysfunctional or damaged mitochondria is termed mitophagy. Enhanced mitophagy is usually an early response to promote survival. However, overwhelming or prolonged mitochondrial damage can induce excessive/pathological levels of mitophagy, thereby promoting cell death and tissue injury. Autophagy/mitophagy is therefore an important modulator in human pulmonary diseases and a potential therapeutic target. This review article will summarize the most recent studies highlighting the role of autophagy/mitophagy and its molecular pathways involved in stress response in pulmonary pathologies.

Inducing mitophagy in diabetic platelets protects against severe oxidative stress.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in "prepackaging" the complex mitophagy machinery in a short-lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

MKK3 deletion improves mitochondrial quality.

Sepsis, a severe response to infection, leads to excessive inflammation and is the major cause of mortality in intensive care units. Mitochondria have been shown to influence the outcome of septic injury. We have previously shown that MAP kinase kinase 3 (MKK3)(-/-) mice are resistant to septic injury and MKK3(-/-) macrophages have improved mitochondrial function. In this study we examined processes that lead to improved mitochondrial quality in MKK3(-/-) mouse embryonic fibroblasts (MEFs) and specifically the role of mitophagy in mitochondrial health. MKK3(-/-) MEFs had lower inflammatory cytokine release and oxidant production after lipopolysaccharide (LPS) stimulation, confirming our earlier observations. MKK3(-/-) MEFs had better mitochondrial function as measured by mitochondrial membrane potential (MMP) and ATP, even after LPS treatment. We observed higher mitophagy in MKK3(-/-) MEFs compared to wild type (WT). Transmission electron microscopy studies showed longer and larger mitochondria in MKK3(-/-) MEFs, indicative of healthier mitochondria. We performed a SILAC (stable isotope labeling by/with amino acids in cell culture) study to assess differences in mitochondrial proteome between WT and MKK3(-/-) MEFs and observed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits. Further, inhibition of mitophagy by Mdivi1 led to loss in MMP and increased cytokine secretion after LPS treatment in MKK3(-/-) MEFs. In conclusion, this study demonstrates that MKK3 influences mitochondrial quality by affecting the expression of mitochondrial proteins, including TCA cycle enzymes, and mitophagy, which consequently regulates the inflammatory response. Based on our results, MKK3 could be a potential therapeutic target for inflammatory diseases like sepsis.

MKK3 mediates inflammatory response through modulation of mitochondrial function.

Mitochondria are increasingly recognized as drivers of inflammatory responses. MAP kinase kinase 3 (MKK3), a dual-specificity protein kinase, is activated in inflammation and in turn activates p38 MAP kinase signaling. Here we show that MKK3 influences mitochondrial function and acts as a critical mediator of inflammation. MKK3-deficient (MKK3(-/-)) mice and bone marrow-derived macrophages (BMDMs) secreted smaller amounts of cytokines than wild type (WT) after lipopolysaccharide (LPS) exposure. There was improved mitochondrial function, as measured by basal oxygen consumption rate, mitochondrial membrane potential, and ATP production, in MKK3(-/-) BMDMs. After LPS exposure, MKK3(-/-) BMDMs did not show a significant increase in cellular reactive oxygen species production or in mitochondrial superoxide compared to WT. Activation of two important inflammatory mediators, i.e., the nuclear translocation of NF-κB and caspase-1 activity (a key component of the inflammasome), was lower in MKK3(-/-) BMDMs. p38 and JNK activation was lower in MKK3(-/-) BMDMs compared to WT after exposure to LPS. Knockdown of MKK3 by siRNA in wild-type BMDMs improved mitochondrial membrane potential, reduced LPS-induced caspase-1 activation, and attenuated cytokine secretion. Our studies establish MKK3 as a regulator of mitochondrial function and inflammatory responses to LPS and suggest that MKK3 may be a therapeutic target in inflammatory disorders such as sepsis.

Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.

Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1-deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1-deficient mice. IL-1ß cleavage and secretion were impaired in HO-1-deficient macrophages, and CO-dependent processing of IL-1ß required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1ß inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.

Cathepsin E promotes pulmonary emphysema via mitochondrial fission.

Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

Macrophage migration inhibitory factor deficiency in chronic obstructive pulmonary disease.

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains poorly understood. Cellular senescence and apoptosis contribute to the development of COPD; however, crucial regulators of these underlying mechanisms remain unknown. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that antagonizes both apoptosis and premature senescence and may be important in the pathogenesis of COPD. This study examines the role of MIF in the pathogenesis of COPD. Mice deficient in MIF (Mif(-/-)) or the MIF receptor CD74 (Cd74(-/-)) and wild-type (WT) controls were aged for 6 mo. Both Mif(-/-) and Cd74(-/-) mice developed spontaneous emphysema by 6 mo of age compared with WT mice as measured by lung volume and chord length. This was associated with activation of the senescent pathway markers p53/21 and p16. Following exposure to cigarette smoke, Mif(-/-) mice were more susceptible to the development of COPD and apoptosis compared with WT mice. MIF plasma concentrations were measured in a cohort of 224 human participants. Within a subgroup of older current and former smokers (n = 72), MIF concentrations were significantly lower in those with COPD [8.8, 95%CI (6.7-11.0)] compared with those who did not exhibit COPD [12.7 ng/ml, 95%CI (10.6-14.8)]. Our results suggest that both MIF and the MIF receptor CD74 are required for maintenance of normal alveolar structure in mice and that decreases in MIF are associated with COPD in human subjects.

Endothelial MKK3 is a critical mediator of lethal murine endotoxemia and acute lung injury.

Sepsis is a leading cause of intensive care unit admissions, with high mortality and morbidity. Although outcomes have improved with better supportive care, specific therapies are limited. Endothelial activation and oxidant injury are key events in the pathogenesis of sepsis-induced lung injury. The signaling pathways leading to these events remain poorly defined. We sought to determine the role of MAPK kinase 3 (MKK3), a kinase of the p38 group, in the pathogenesis of sepsis. We used a murine i.p. LPS model of systemic inflammation to mimic sepsis. Lung injury parameters were assessed in lung tissue and bronchoalveolar lavage specimens. Primary lung endothelial cells were cultured and assessed for mediators of inflammation and injury, such as ICAM-1, AP-1, NF-κB, and mitochondrial reactive oxygen species. Our studies demonstrate that MKK3 deficiency confers virtually complete protection against organ injury after i.p. LPS. Specifically, MKK3(-/-) mice were protected against acute lung injury, as assessed by reduced inflammation, mitochondrial reactive oxygen species generation, endothelial injury, and ICAM-1 expression after LPS administration. Our results show that endothelial MKK3 is required for inflammatory cell recruitment to the lungs, mitochondrial oxidant-mediated AP-1, NF-κB activation, and ICAM-1 expression during LPS challenge. Collectively, these studies identify a novel role for MKK3 in lethal LPS responses and provide new therapeutic targets against sepsis and acute lung injury.