Anja Jaeschke

Tuberous sclerosis complex tumor suppressor–mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Role of MLK3 in the regulation of mitogen-activated protein kinase signaling cascades

ABSTRACT Mixed-lineage protein kinase 3 (MLK3) is a member of the mitogen-activated protein (MAP) kinase kinase kinase group that has been implicated in multiple signaling cascades, including the NF-κB pathway and the extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways. Here, we examined the effect of targeted disruption of the murine Mlk3 gene. Mlk3 −/− mice were found to be viable and healthy. Primary embryonic fibroblasts prepared from these mice exhibited no major signaling defects. However, we did find that MLK3 deficiency caused a selective reduction in tumor necrosis factor (TNF)-stimulated JNK activation. Together, these data demonstrate that MLK3 contributes to the TNF signaling pathway that activates JNK.

Critical Role for Mixed-Lineage Kinase 3 in Acetaminophen-Induced Hepatotoxicity

c-Jun NH2-terminal kinase (JNK) activation plays a major role in acetaminophen (APAP)-induced hepatotoxicity. However, the exact mechanism of APAP-induced JNK activation is incompletely understood. It has been established that apoptosis signal-regulating kinase 1 (ASK1) regulates the late phase of APAP-induced JNK activation, but the mitogen-activated protein kinase kinase kinase that mediates the initial phase of APAP-induced JNK activation has not been identified. Oxidative stress produced during APAP metabolism causes JNK activation, which promotes mitochondrial dysfunction and results in the amplification of oxidative stress. Therefore, inhibition of the initial phase of JNK activation may be key to protection against APAP-induced liver injury. The goal of this study was to determine whether mixed-lineage kinase 3 (MLK3) mediates the initial, ASK1-independent phase of APAP-induced JNK activation and thus promotes drug-induced hepatotoxicity. We found that MLK3 was activated by oxidative stress and was required for JNK activation in response to oxidative stress. Loss of MLK3 attenuated APAP-induced JNK activation and hepatocyte death in vitro, independent of receptor-interacting protein 1. Moreover, JNK and glycogen synthase kinase 3β activation was significantly attenuated, and Mcl-1 degradation was inhibited in APAP-treated MLK3-knockout mice. Furthermore, we showed that loss of MLK3 increased expression of glutamate cysteine ligase, accelerated hepatic GSH recovery, and decreased production of reactive oxygen species after APAP treatment. MLK3-deficient mice were significantly protected from APAP-induced liver injury, compared with wild-type mice. Together, these studies establish a novel role for MLK3 in APAP-induced JNK activation and hepatotoxicity, and they suggest MLK3 as a possible target in the treatment of APAP-induced liver injury.

Cdc42 and Rac1 are major contributors to the saturated fatty acid-stimulated JNK pathway in hepatocytes

BACKGROUND & AIMS Saturated free fatty acid (SFA)-stimulated c-Jun NH(2)-terminal kinase (JNK) activation is associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the effects of SFA are incompletely understood. The goal of this study was to determine the molecular mechanisms by which SFA induce JNK activation in hepatocytes. METHODS We used siRNA-mediated knockdown in Hepa1c1c7 and AML12 cell lines, as well as primary mouse hepatocytes for these studies. RESULTS The current model for JNK activation by SFA involves endoplasmic reticulum (ER) stress, which induces JNK activation through an inositol requiring enzyme 1 (IRE1α) Apoptosis Regulating Kinase 1 (ASK1)-dependent mechanism. Here, we find that SFA-induced JNK activation is not inhibited in the absence of IRE1α and ASK1. Instead we show that activation of the small GTP-binding proteins Cdc42 and Rac1 is required for SFA-stimulated MLK3-dependent activation of JNK in hepatocytes. In addition, we demonstrate that SFA-induced cell death in hepatocytes is independent of IRE1α, but dependent on Cdc42, Rac1, and MLK3. CONCLUSIONS Our results demonstrate that Cdc42 and Rac1, rather than ER stress, are important components of a SFA-stimulated signaling pathway that regulates MLK3-dependent activation of JNK in hepatocytes.

Chronic dietary n-3 PUFA intervention improves dyslipidaemia and subsequent cardiovascular complications in the JCR:LA- cp rat model of the metabolic syndrome.

There is increasing interest in the potential chronic beneficial effects of dietary n-3 PUFA on the metabolic syndrome (MetS) and associated cardiovascular complications. We have recently established that increased dietary n-3 PUFA has a profound acute benefit on fasting lipids and the postprandial pro-inflammatory response in the JCR:LA-cp rat, a model of the MetS. However, it is unclear to what extent chronic dietary n-3 PUFA intervention can modulate the progression of end-stage metabolic and vascular complications. The present study aimed to determine the chronic effects of dietary n-3 PUFA supplementation on fasting and non-fasting dyslipidaemia, insulin resistance and vascular complications in the JCR:LA-cp rodent model. JCR:LA-cp rats were fed an isoenergetic lipid-balanced diet supplemented with 5 % n-3 PUFA (w/w) of the total fat (fish oil-derived EPA/DHA) for 16 weeks. Fasting and non-fasting (postprandial) plasma lipid profile was assessed. Hepatic and adipose tissue was probed for the expression of lipogenic proteins (acyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP-1)), while the activity of Jun N-terminal kinase (JNK) was assessed via Western blot to target phosphorylated JNK protein in primary enterocytes. The frequency of myocardial lesions was assessed by haematoxylin and eosin staining. Increased dietary n-3 PUFA improved both the fasting and postprandial lipid profiles (TAG, cholesterol and apoB48) in the JCR:LA-cp rat, potentially via the down-regulation of the hepatic or adipose tissue expression of lipogenic enzymes (ACC, FAS and SREBP-1). Rats fed the 5 % n-3 PUFA diet had lower (58·2 %; P < 0·01) enterocytic phosphorylated JNK protein and secreted less cholesterol (30 %; P < 0·05) into mesenteric lymph compared with the control. The chronic metabolic benefits of dietary n-3 PUFA may underlie the potential to reduce vascular complications during the MetS, including the observed reduction in the frequency (approximately 80 %) of late-stage 3 myocardial lesions.

Apolipoprotein E4 Impairs Macrophage Efferocytosis and Potentiates Apoptosis by Accelerating Endoplasmic Reticulum Stress

Background: Apolipoprotein E4 (apoE4) is associated with inflammatory metabolic diseases. Results: Human APOE4 gene replacement mice displayed elevated tissue inflammation. APOE4 macrophages showed impaired efferocytosis, increased apoptosis, and endoplasmic reticulum stress. Conclusion: ApoE4 structural abnormalities induce ER stress to promote inflammation. Significance: Reducing ER stress and/or apoE4 structure correctors may reduce inflammatory metabolic disease risk in human apoE4 subjects. Apolipoprotein (apo) E4 is a major genetic risk factor for a wide spectrum of inflammatory metabolic diseases, including atherosclerosis, diabetes, and Alzheimer disease. This study compared diet-induced adipose tissue inflammation as well as functional properties of macrophages isolated from human APOE3 and APOE4 mice to identify the mechanism responsible for the association between apoE4 and inflammatory metabolic diseases. The initial study confirmed previous reports that APOE4 gene replacement mice were less sensitive than APOE3 mice to diet-induced body weight gain but exhibited hyperinsulinemia, and their adipose tissues were similarly inflamed as those in APOE3 mice. Peritoneal macrophages isolated from APOE4 mice were defective in efferocytosis compared with APOE3 macrophages. Increased cell death was also observed in APOE4 macrophages when stimulated with LPS or oxidized LDL. Western blot analysis of cell lysates revealed that APOE4 macrophages displayed elevated JNK phosphorylation indicative of cell stress even under basal culturing conditions. Significantly higher cell stress due mainly to potentiation of endoplasmic reticulum (ER) stress signaling was also observed in APOE4 macrophages after LPS and oxidized LDL activation. The defect in efferocytosis and elevated apoptosis sensitivity of APOE4 macrophages was ameliorated by treatment with the ER chaperone tauroursodeoxycholic acid. Taken together, these results showed that apoE4 expression causes macrophage dysfunction and promotes apoptosis via ER stress induction. The reduction of ER stress in macrophages may be a viable option to reduce inflammation and inflammation-related metabolic disorders associated with the apoE4 polymorphism.

Mixed lineage kinase 3 deficient mice are protected against the high fat high carbohydrate diet-induced steatohepatitis

C‐Jun N‐terminal kinase (JNK) activation is pivotal in the development of nonalcoholic steatohepatitis (NASH). Mixed lineage kinase 3 (MLK) 3 is one of the mitogen activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in the liver. Despite this concept, the role of MLK3 in modulating liver injury during nutrient excess has not been explored. Our aim was to determine if MLK3 deficient mice were protected against high fat high carbohydrate (HFHC) diet‐induced NASH.

MLK3 promotes metabolic dysfunction induced by saturated fatty acid-enriched diet

Saturated fatty acids activate the c-Jun NH₂-terminal kinase (JNK) pathway, resulting in chronic low-grade inflammation and the development of insulin resistance. Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in response to saturated fatty acids in vitro; however, the exact mechanism for diet-induced JNK activation in vivo is not known. Here, we have used MLK3-deficient mice to examine the role of MLK3 in a saturated-fat diet model of obesity. MLK3-KO mice fed a high-fat diet enriched in medium-chain saturated fatty acids for 16 wk had decreased body fat compared with wild-type (WT) mice due to increased energy expenditure independently of food consumption and physical activity. Moreover, MLK3 deficiency attenuated palmitate-induced JNK activation and M1 polarization in bone marrow-derived macrophages in vitro, and obesity induced JNK activation, macrophage infiltration into adipose tissue, and expression of proinflammatory cytokines in vivo. In addition, loss of MLK3 improved insulin resistance and decreased hepatic steatosis. Together, these data demonstrate that MLK3 promotes saturated fatty acid-induced JNK activation in vivo and diet-induced metabolic dysfunction.

Multiparity leads to obesity and inflammation in mothers and obesity in male offspring

Multiparity is an independent risk factor for obesity in parous females. In addition to being a health issue for the mother, offspring of multiparous females may also be at risk for obesity later in life. The aim of the current study was to establish a mouse model that mimics the human pathology of multiparity and determine the effects of multiparity-induced obesity (MIO) on offspring in adulthood. C57BL/6 mice were mated and studied when primiparous (1st pregnancy) or multiparous (4th pregnancy). Dams became obese with multiparity, an effect that was independent of the age of the dam. Multiparous dams also had increased markers of inflammation (JNK activation, cytokine expression) in adipose tissue and liver that was greater than inflammation in nulliparous females made obese with a high-fat diet. Placental inflammation was prevalent in multiparous vs. primiparous dams as well. Male offspring of the multiparous dams developed increased adiposity by 24 wk of age relative to the progeny of primiparous dams, although food consumption was similar in both groups. Lipid metabolism was altered in liver and fat in that mRNA levels of regulatory genes (PGC-1α) as well as metabolic genes (CPT I) and Akt phosphorylation were decreased in offspring of multiparous dams. Thus, in mice, as in humans, multiparity increases adiposity and is associated with hepatic and placental inflammation and abnormal glucose tolerance. Importantly, MIO leads to increased body fat and metabolic dysfunction in the offspring, suggesting a role in the propagation of obesity.

Mixed-Lineage Kinase 3 Deficiency Promotes Neointima Formation Through Increased Activation of the RhoA Pathway in Vascular Smooth Muscle Cells

Objective—Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms. Approach and Results—Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated. Conclusions—We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.