-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. sepsis; lung injury; mitogen-activated protein kinases; mitochondria; mitophagy; biogenesis SEPSIS, A SYSTEMIC INFLAMMATORY reaction to infection, is the leading cause of death globally. The incidence of sepsis worldwide is 18 million every year with 30% mortality. The economic impact of sepsis is substantial with costs of up to $50,000/patient and $17 billion annually in United States alone (30, 43). Death from sepsis occurs due to multiorgan failure, and biological therapies do not exist. Prevailing theories attribute multiple organ failure in sepsis to an uncontrolled inflammatory response, apoptosis, or disorders in the coagulation (38). Unfortunately, therapies against these responses, such as anti-inflammatory agents and activated protein C, have been unsuccessful. Since current medical care remains purely supportive, there is an urgent need to develop targeted therapies.Mitochondria are mediators of inflammatory responses (19, 54) and become dysfunctional in sepsis and lung injury (4, 6), suggesting they are involved in the observed pathology. Mitochondria are essential hubs of innate immune signaling and inflammation in sepsis (48, 49) and are also major sites of reactive oxygen species (ROS) production in the cells. Mitochondria are constantly exposed to ROS, and hence ongoing biogenesis and turnover are needed to maintain a functional network. Dysfunctional mitochondria are removed through selective degradation via autophagy by the lysosomal mach...

High levels of inspired oxygen, hyperoxia, are frequently used in patients with acute respiratory failure. Hyperoxia can exacerbate acute respiratory failure, which has high mortality and no specific therapies. We identified a novel roles for PINK1 (PTEN-induced putative kinase 1), a mitochondrial protein, and the cytosolic innate immune protein, NLRP3, in the lung and endothelium. We generated double knockouts (PINK1−/−/NLRP3−/−) as well as cell-targeted PINK1 silencing and lung-targeted overexpression constructs to specifically show that PINK1 mediates cytoprotection in wild type (WT) and NLRP3−/− mice. The ability to resist hyperoxia is proportional to PINK1 expression – PINK1−/− mice were the most susceptible, WT mice, which induced PINK1 after hyperoxia, had intermediate susceptibility and NLRP3−/− mice, which had high basal and hyperoxia-induced PINK1, were the least susceptible. Genetic deletion of PINK1 or PINK1 silencing in the lung endothelium increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis and oxidant generation.

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 and need to be studied. We sought to determine the role of MAP kinase kinase 3 (MKK3), a kinase of the p38 group in the pathogenesis of sepsis. We used a murine intraperitoneal lipopolysaccharide (LPS) model of systemic inflammation to mimic sepsis. Lung injury parameters were assessed in lung tissue and bronchoalveolar lavage. Primary lung endothelial cells were cultured and assessed for mediators of inflammation and injury such as ICAM-1, AP-1, NF-κB and mitochondrial ROS. Our studies demonstrate that MKK3 deficiency confers virtually complete protection against organ injury after intraperitoneal LPS. Specifically, MKK3 −/− mice were protected against acute lung injury, as assessed by reduced inflammation, mitochondrial reactive oxygen species (ROS) generation, endothelial injury and ICAM-1 expression after LPS. 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.

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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 less cytokines than wild type (WT) after 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−/− BMDM did not show significant increase in cellular reactive oxygen species (ROS) production as well as mitochondrial superoxide (MitoSOX) 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), were lower in MKK3−/− BMDMs. p-38 and JNK activation were 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 like sepsis.

This data article reports changes in the phospho and total proteome of MKK3 knock out (MKK3−/−) mouse embryonic fibroblasts (MEFs). The dataset generated highlights the changes at protein level which can be helpful for understanding targets of the MAP kinase signaling pathway. Data was collected after TiO2-based phosphopeptide enrichment of whole cell lysate at baseline condition with bottom-up SILAC-based LC MS/MS quantitative mass spectrometry. We report all the proteins and peptides identified and quantified in MKK3−/− and WT MEFs. The altered pathways in MKK3−/− MEFs were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) and Ingenuity Pathway Analysis (IPA) and are presented as a table and graph, respectively. The data reported here is related to the published work [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.