Activation Of Mixed Lineage Kinase Research Articles

Activation of mixed lineage kinase 3 by fine particulate matter induces skin inflammation in human keratinocytes

Fine particulate matter (PM2.5) induces a range of diseases, including skin disorders, through inflammatory responses. In this study, we investigated the novel mechanisms by which PM2.5 causes skin inflammation in human keratinocytes HaCaT. We observed increased protein expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in PM2.5-treated HaCaT cells. To identify the pathways promoting the expression of these inflammatory proteins, we conducted a phospho-kinase antibody array and confirmed that the phosphorylation levels of JNK and p38 were increased by PM2.5-treated HaCaT cells. Further investigation of the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and upstream signals revealed that PM2.5 activated the MKK4/7-JNK-c-Jun and MKK3/6-p38-p70S6K signaling pathways, while the phosphorylation level of ERK1/2 remained unchanged. HaCaT cells treated with PM2.5 phosphorylated Mixed-lineage kinase 3 (MLK3), an upstream regulator of p38 and JNK. Furthermore, inhibition of ROS production by N-Acetylcysteine (NAC) treatment inhibited MLK3 phosphorylation. Taken together, ROS production induced by PM2.5 activated the MLK3 signaling pathway and induced skin inflammation.

MLK3 promotes prooncogenic signaling in hepatocellular carcinoma via TGFβ pathway.

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFβ signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFβ signaling. These findings reveal TGFβ playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFβ axis to be an ideal drug target for advanced HCC management.

Autophagy inhibitors 3-MA and BAF may attenuate hippocampal neuronal necroptosis after global cerebral ischemia-reperfusion injury in male rats by inhibiting the interaction of the RIP3/AIF/CypA complex.

Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.

MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our invitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function.

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3+/+) littermate controls. MLK3C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3+/+ but not MLK3C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.

MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

Osmotic and heat stress-dependent regulation of MLK4β and MLK3 by the CHIP E3 ligase in ovarian cancer cells

Mixed Lineage Kinase 3 (MLK3), a member of the MLK subfamily of protein kinases, is a mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) that activates MAPK signalling pathways and regulates cellular responses such as proliferation, invasion and apoptosis. MLK4β, another member of the MLK subfamily, is less extensively studied, and the regulation of MLK4β by stress stimuli is not known. In this study, the regulation of MLK4β and MLK3 by osmotic stress, thermostress and heat shock protein 90 (Hsp90) inhibition was investigated in ovarian cancer cells. MLK3 and MLK4β protein levels declined under conditions of prolonged osmotic stress, heat stress or exposure to the Hsp90 inhibitor geldanamycin (GA); and MLK3 protein declined faster than MLK4β. Similar to MLK3, the reduction in MLK4β protein in cells exposed to heat or osmotic stresses occurred via a mechanism that involves the E3 ligase, carboxy-terminus of Hsc70-interacting protein (CHIP). Both heat shock protein 70 (Hsp70) and CHIP overexpression led to polyubiquitination and a decrease in endogenous MLK4β protein, and MLK4β was ubiquitinated by CHIP in vitro. In untreated cells and cells exposed to osmotic and heat stresses for short time periods, small interfering RNA (siRNA) knockdown of MLK4β elevated the levels of activated MLK3, c-Jun N-terminal kinase (JNK) and p38 MAPKs. Furthermore, MLK3 binds to MLK4β, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4β-MLK3 binding is important for regulating MLK3 activity and MAPK signalling, and after prolonged periods of stress exposure, MLK4β and MLK3 proteins decline via CHIP-dependent degradation. These findings provide insight into how heat and osmotic stresses regulate MLK4β and MLK3, and reveal an important function for MLK4β in modulating MLK3 activity in stress responses.

Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.

Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage-associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone-marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10-neutralizing antisera. MLK3-deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild-type mice. During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.

Abstract 1967: Elucidation of signaling pathways that mediate gastrin-induced JNK activation and pGSK3β/Snail induction in gastric cancer cells

Abstract The gastrointestinal peptide hormone gastrin is known to regulate various cellular processes including proliferation, migration and metastasis in gastrointestinal cells. Our previous studies indicated that amidated gastrin (G-17) induced gastric cancer cell migration through two separate signaling axes. These revealed that inhibition of Glycogen Synthase Kinase-3β (GSK3β) was necessary to activate G17-induced migratory pathways in gastric cancer cells. Incubation of AGSE gastric cancer cells overexpressing CCK2 receptor (CCK2R) with G17 resulted in a dose and time dependent increase of GSK3βSer9 phosphorylation, indicative of an inhibition of the kinase. Treatment with G-17 was also associated with increased Snail expression, a downstream target of GSK3β and p21-activated protein kinases (PAKs). In a second pathway, G-17 also induced JNK phosphorylation through activation of Mixed Lineage Kinase 3 (MLK3). This axis also promotes gastric cancer cell migration via CCK2R. In the present study our goal was to determine whether PAKs, which are effectors for the Rho GTPases Cdc42 and Rac and are central players in cell proliferation, survival and motility, are involved in the G-17 effects on gastric cancer cells. Our studies showed that in AGSE cells when pretreated with a commercially available PAK4 inhibitor PF 3758309, G-17 was unable to phosphorylate GSK3β and G-17-induced Snail expression was markedly inhibited. G17-induced p-JNK expression was also significantly attenuated with PAK4 inhibitor pretreatment. The inhibitory effect was stronger when PAK4 inhibitor was used in combination with a pan-MLK inhibitor CEP11004. The involvement of PAK4 in G17-induced events were also confirmed by using the PAK4 siRNA. Knockdown of PAK4 inhibited G17-induced p-JNK expression which was more pronounced when both PAK4 and MLK3 were knocked down in AGSE gastric cancer cells. However, knocking down PAK4 alone does-not seem to inhibit G-17-induced p-GSK3β expression completely, which suggest the potential involvement of other PAK family members. Studies are currently underway to determine which PAK(s) is involved in mediating G17-pGSK3β-Snail axis and also to elucidate any crosstalk of PAK4 and MLK3 in this pathway. Elucidation of the signaling pathways that mediate G17-induced cellular events will be very important in designing therapeutic approaches in the future to restrict gastric cancer cell migration and metastasis. Citation Format: Aninda Basu, Goutam Sondarva, Sreevidya Santha, Ajay Rana, Basabi Rana. Elucidation of signaling pathways that mediate gastrin-induced JNK activation and pGSK3β/Snail induction in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2015-1967

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼ 15-20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2-) breast cancer tissues, whereas both HER2+ and HER2- tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser(674) by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.

232 Mixed Lineage Kinase 3 Mediates the Release of Proinflammatory Extracellular Vesicles in Nonalcoholic Steatohepatitis

Backgrounds and aims: We have recently demonstrated that genetic deletion of mixed lineage kinase 3 (MLK3) reduces inflammation in a murine model of NASH. However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are not fully elucidated. Our hypothesis is that MLK3 mediates the release of chemokine rich extracellular vehicles (EVs), which induce macrophage activation & trafficking to the liver in NASH. Methods: Two MLK3 inhibitors CLFB-1134 and URMC099 were kindly provided by Califia Bio, Inc. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with lysophosphatidylcholine (LPC), a toxic metabolite of saturated fatty acid with and without one of the MlK3 inhibitors. Released EVs were isolated by differential ultracentrifugation, quantified by nanoparticle tracking analysis, and employed for macrophage treatment. EVs protein contents were profiled by mass spectrometry (MSP). Results: LPC treatment of PMH & Huh7 cells induced a 3.6-fold and 140-fold increase in release of EVs, respectively, which was prevented by either genetic knock down or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs. CXCL10 was markedly enriched in EVs isolated from LPC treated PMH versus untreated cells, as assessed by immuno-gold electron microscopy and immunoblot analysis. Unexpectedly, either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EV (corrected for the number of EV secreted). Treatment of mouse bone marrow-derived macrophages with physiologically relevant concentrations of lipotoxic hepatocyte-derived EVs induced macrophage activation and chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antiserum. Finally, activating phospho-MLK3 expression was increased in liver biopsies of patients with fatty liver compared to the normal control. In Conclusion: during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes which are proinflammatory for macrophages. We speculate that these EVs mediate hepatic inflammation in vivo by inducing macrophages trafficking to the liver and that inhibition of MLK3-dependent EV release from hepatocytes could be salutary in human NASH.

233 Hepatic Overexpression of SIRT3 in Mice Heterozygous for Mitochondrial Trifunctional Protein Rescues Hepatic Steatosis and Improves Insulin Sensitivity

Backgrounds and aims: We have recently demonstrated that genetic deletion of mixed lineage kinase 3 (MLK3) reduces inflammation in a murine model of NASH. However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are not fully elucidated. Our hypothesis is that MLK3 mediates the release of chemokine rich extracellular vehicles (EVs), which induce macrophage activation & trafficking to the liver in NASH. Methods: Two MLK3 inhibitors CLFB-1134 and URMC099 were kindly provided by Califia Bio, Inc. Primary mouse hepatocytes (PMH) and Huh7 cells were treated with lysophosphatidylcholine (LPC), a toxic metabolite of saturated fatty acid with and without one of the MlK3 inhibitors. Released EVs were isolated by differential ultracentrifugation, quantified by nanoparticle tracking analysis, and employed for macrophage treatment. EVs protein contents were profiled by mass spectrometry (MSP). Results: LPC treatment of PMH & Huh7 cells induced a 3.6-fold and 140-fold increase in release of EVs, respectively, which was prevented by either genetic knock down or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs. CXCL10 was markedly enriched in EVs isolated from LPC treated PMH versus untreated cells, as assessed by immuno-gold electron microscopy and immunoblot analysis. Unexpectedly, either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EV (corrected for the number of EV secreted). Treatment of mouse bone marrow-derived macrophages with physiologically relevant concentrations of lipotoxic hepatocyte-derived EVs induced macrophage activation and chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antiserum. Finally, activating phospho-MLK3 expression was increased in liver biopsies of patients with fatty liver compared to the normal control. In Conclusion: during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes which are proinflammatory for macrophages. We speculate that these EVs mediate hepatic inflammation in vivo by inducing macrophages trafficking to the liver and that inhibition of MLK3-dependent EV release from hepatocytes could be salutary in human NASH.

Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction.

BackgroundUbiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14’s catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14’s catalytic activity is required for neuromuscular junction (NMJ) structure and function.ResultsMice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice.ConclusionsUSP14’s catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.Electronic supplementary materialThe online version of this article (doi:10.1186/1750-1326-10-3) contains supplementary material, which is available to authorized users.

Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival

Abstract Human Epidermal Growth Factor Receptor 2 (Her2) is a receptor tyrosine kinase that regulates cell growth and differentiation signaling pathways. The protein Her2 is significantly overexpressed in 20-30% of breast cancer. The available clinical data proves that Her2 amplification in breast cancer is associated with worst overall survival. Therefore, Her2 has become a very important candidate for drug targeting and currently Her2 positive patients are treated with humanized monoclonal antibody, Trastuzumab (Herceptin) and a small molecule, Lapatinib that blocks Her2 tyrosine kinase activity. It is reported that Her2 amplification leads to activation of multiple survival pathways, including PI3K-Akt. However, it is not clearly known how Trastuzumab promotes cell death and what pro-apoptotic pathways are activated downstream of Her2 blockage? It is also reported that patients on Trastuzumab develop subsequent resistance to the therapy. Previously we published that in breast cancer tumors, a pro-apoptotic kinase, Mixed Lineage Kinase-3 (MLK-3) was inhibited, specifically in estrogen receptor (ER) positive tumors. The kinase activity of MLK-3 was down regulated by estrogen and this prevented MLK-3-mediated cell death. Since Her2 serves as a drug target in breast cancer, therefore, we asked whether Her2 plays any role in regulating MLK-3 kinase activation and whether MLK-3 plays any role in Trastuzumab resistance. Our results showed that the endogenous MLK-3 kinase activity was up-regulated in SkBr3 cells upon treatment with Trastuzumab and Lapatinib. Whether the activation of MLK-3 upon Her2 blockage leads to cell death or survival is still not known and experiments are underway to investigate these aspects. We also observed that Her3 was the dimerization partner of Her2 in Trastuzumab- or Lapatinib- mediated MLK-3 activation. Not knowing any role of MLK-3 in Trastuzumab resistance, we used Trastuzumab sensitive and resistant BT474 cell model to investigate the role of MLK-3 in Trastuzumab resistance. Our data clearly showed that knockdown of MLK-3 caused spontaneous cell death in resistant cells. The Trastuzumab treatment further amplified cell death in MLK-3 knockdown resistant cells. The Akt1 activation in these cells was also diminished upon MLK-3 knockdown. Taken together, our data demonstrate that like ER, the Her2 also down regulates MLK-3 kinase activity and perhaps promote cell survival. Furthermore, paradoxically, the MLK-3 knockdown promoted cell death in Trastuzumab resistance BT474 cells, suggesting that MLK-3 could also serve as survival factor in resistant cells. Whether MLK-3 serves as a pro-apoptotic kinase or survival kinase in Trastuzumab mediated pathway needs to be extensively examined in near future. Citation Format: Subhasis Das, Gautam Sondarva, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Basabi Rana, Ajay Rana. Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2014-1829

Overexpression of C-terminal fragment of glutamate receptor 6 prevents neuronal injury in kainate-induced seizure via disassembly of GluR6-PSD-95-MLK3 signaling module.

Our previous study showed that when glutamate receptor (GluR)6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein (GluR6)-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 (MLK3) and c-Jun NH2-terminal kinase (JNK) is inhibited via GluR6-postsynaptic density protein 95 (PSD95). In the present study, we investigated whether the recombinant adenovirus (Ad) carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad-Glur6-9c on the phosphorylation of JNK, MLK3 and mitogen-activated kinase kinase 7 (MKK7) was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of JNK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was significantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.

Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway

Connective tissue growth factor (CTGF) plays an important role in lung fibrosis. In this study, we investigated the role of Rac1, mixed-lineage kinase 3 (MLK3), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CTGF-induced collagen I expression in human lung fibroblasts. CTGF caused concentration- and time-dependent increases in collagen I expression. CTGF-induced collagen I expression was inhibited by the dominant negative mutant (DN) of Rac1 (RacN17), MLK3DN, MLK3 inhibitor (K252a), JNK1DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). Treatment of cells with CTGF caused activation of Rac1, MLK3, JNK, and AP-1. The CTGF-induced increase in MLK3 phosphorylation was inhibited by RacN17. Treatment with RacN17 and the MLK3DN inhibited CTGF-induced JNK phosphorylation. CTGF caused increases in c-Jun phosphorylation and the recruitment of c-Jun and c-Fos to the collagen I promoter. Furthermore, stimulation of cells with the CTGF resulted in increases in AP-1-luciferase activity; this effect was inhibited by Rac1N17, MLK3DN, JNK1DN, and JNK2DN. Moreover, CTGF-induced α-smooth muscle actin (α-SMA) expression was inhibited by the procollagen I small interfering RNA (siRNA). These results suggest for the first time that CTGF acting through Rac1 activates the MLK3/JNK signaling pathway, which in turn initiates AP-1 activation and recruitment of c-Jun and c-Fos to the collagen I promoter and ultimately induces collagen I expression in human lung fibroblasts.

Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.