The Experts below are selected from a list of 3015 Experts worldwide ranked by ideXlab platform
Deborah N. Chadee - One of the best experts on this subject based on the ideXlab platform.
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The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3
Molecular and cellular biology, 2014Co-Authors: Natalya A. Blessing, April L. Brockman, Deborah N. ChadeeAbstract:Mixed-Lineage Kinase 3 (MLK3) activates mitogen-activated protein Kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion.
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Involvement of Mixed Lineage Kinase 3 in cancer.
Canadian journal of physiology and pharmacology, 2012Co-Authors: Deborah N. ChadeeAbstract:Mitogen-activated protein Kinase (MAPK) signaling pathways are composed of a phosphorelay signaling module where an activated MAP Kinase Kinase Kinase (MAP3K) phosphorylates and activates a MAPK Kinase (MAP2K) that in turn phosphorylates and activates a MAPK. The biological outcome of MAPK signaling is the regulation of cellular responses such as proliferation, differentiation, migration, and apoptosis. The MAP3K Mixed Lineage Kinase 3 (MLK3) phosphorylates MAP2Ks to activate multiple MAPK signaling pathways, and MLK3 also has functions in cell signaling that are independent of its Kinase activity. The recent elucidation of essential functions for MLK3 in tumour cell proliferation, migration, and invasion has drawn attention to the MLKs as potential therapeutic targets for cancer treatments. The mounting evidence that suggests a role for MLK3 in tumourigenesis and establishment of the malignant phenotype is the focus of this review.
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Mixed Lineage Kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells.
Experimental cell research, 2012Co-Authors: Yu Zhan, Widian F. Abi Saab, Nidhi Modi, Amanda M. Stewart, Jinsong Liu, Deborah N. ChadeeAbstract:Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein Kinase Kinase Kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.
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Regulation of Mixed Lineage Kinase 3 is required for Neurofibromatosis-2-mediated growth suppression in human cancer
Oncogene, 2011Co-Authors: Yu Zhan, Nidhi Modi, Amanda M. Stewart, R I Hieronimus, D H Gutmann, Deborah N. ChadeeAbstract:The Neurofibromatosis-2 ( NF2 ) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with Mixed Lineage Kinase 3 (MLK3), a mitogen-activated protein Kinase (MAPK) Kinase Kinase that is required for the proliferation of normal and neoplastic cells. In this study, we show that merlin inhibits MLK3 activity, as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin-mediated suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.
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Inhibition of Cdc42-mediated activation of Mixed Lineage Kinase 3 by the tumor suppressor protein merlin.
Small GTPases, 2010Co-Authors: Yu Zhan, Deborah N. ChadeeAbstract:Mammalian mitogen-activated protein Kinase (MAPK) signaling pathways respond to diverse extracellular signals and coordinate a range of cellular responses. Mixed Lineage Kinase 3 (MLK3) is a member of the Mixed Lineage Kinase family of MAPK Kinase Kinases (MAP3Ks) that functions to regulate multiple MAPK signaling pathways. Activated forms of the Rho GTP ases, Rac and Cdc42, interact with MLK3 through the Cdc42/Rac-interactive binding (CRIB) motif and promote MLK3 catalytic activity. Our recent findings demonstrate that merlin, the product of the neurofibromatosis type 2 (NF2) tumor suppressor gene, is a physiological inhibitor of MLK3. Our results suggest that merlin inhibits MLK3 activity by blocking the Cdc42-MLK3 interaction. In this commentary, the effect of merlin on Cdc42-mediated activation of MLK3 and MAPK signaling will be discussed.
Guang-yi Zhang - One of the best experts on this subject based on the ideXlab platform.
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S-nitrosylation of Mixed Lineage Kinase 3 contributes to its activation after cerebral ischemia.
The Journal of biological chemistry, 2011Co-Authors: Yan-yan Zong, Chang-cheng Sun, Dong-hai Liu, Tao Song, Guang-yi ZhangAbstract:Previous studies in our laboratory have shown that Mixed Lineage Kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-d-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein Kinase Kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.
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N-Acetylcysteine inhibit the translocation of Mixed Lineage Kinase-3 from cytosol to plasma membrane during transient brain ischemia in rat hippocampus.
Neuroscience letters, 2005Co-Authors: Dong-sheng Pei, Qiu-hua Guan, Ya-feng Sun, Qing-xiu Zhang, Guang-yi ZhangAbstract:Mixed Lineage Kinase-3 (MLK3) is a recently described member of the MLK subfamily of Ser/Thr protein Kinases that interacts with mitogen-activated protein Kinase (MAPK) pathways. In this study, we investigated the translocation of MLK3 during transient cerebral ischemia in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. Our data show that MLK3 can translocate from cytosolic fraction to the membrane fraction during ischemia and the increased MLK3 in the membrane fraction bind to postsynaptic density protein 95 (PSD-95). The antioxidant N-acetylcysteine (NAC) could inhibit the translocation of MLK3 from cytosolic fraction to the membrane fraction and decrease the interactions of MLK3 and PSD-95 in the membrane fraction. Consequently, these results indicate that reactive oxygen species (ROS) was closely associated with MLK3 translocation induced by transient global ischemia in rat hippocampus.
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N-Acetylcysteine inhibit the translocation of Mixed Lineage Kinase-3 from cytosol to plasma membrane during transient brain ischemia in rat hippocampus
Neuroscience Letters, 2005Co-Authors: Dong-sheng Pei, Qiu-hua Guan, Ya-feng Sun, Qing-xiu Zhang, Guang-yi ZhangAbstract:Mixed Lineage Kinase-3 (MLK3) is a recently described member of the MLK subfamily of Ser/Thr protein Kinases that interacts with mitogen-activated protein Kinase (MAPK) pathways. In this study, we investigated the translocation of MLK3 during transient cerebral ischemia in rat hippocampus. Transient brain ischernia was induced by the four-vessel occlusion in Sprague-Dawley rats. Our data show that MLK3 can translocate from cytosolic fraction to the membrane fraction during ischernia and the increased MLK3 in the membrane fraction bind to postsynaptic density protein 95 (PSD-95). The antioxidant N-acetylcysteine (NAC) could inhibit the translocation of MLK3 from cytosolic fraction to the membrane fraction and decrease the interactions of MLK3 and PSD-95 in the membrane fraction. Consequently, these results indicate that reactive oxygen species (ROS) was closely associated with MLK3 translocation induced by transient global ischernia in rat hippocampus. (C) 2005 Elsevier Ireland Ltd. All rights reserved.
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neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of Mixed Lineage Kinase 3 via nmda receptor mediated akt1 activation
Journal of Neurochemistry, 2005Co-Authors: Xiao Hui Yin, Quanguang Zhang, Bei Miao, Guang-yi ZhangAbstract:A number of works show that the mitogen-activated protein Kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of Mixed-Lineage Kinase 3 (MLK3), a MAPK Kinase Kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.
Ajay Rana - One of the best experts on this subject based on the ideXlab platform.
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The regulatory function of Mixed Lineage Kinase 3 in tumor and host immunity.
Pharmacology & therapeutics, 2020Co-Authors: Sandeep Kumar, Basabi Rana, Sunil Kumar Singh, Ajay RanaAbstract:Abstract Protein Kinases are the second most sought-after G-protein coupled receptors as drug targets because of their overexpression, mutations, and dysregulated catalytic activities in various pathological conditions. Till 2019, 48 protein Kinase inhibitors have received FDA approval for the treatment of multiple illnesses, of which the majority of them are indicated for different malignancies. One of the attractive sub-group of protein Kinases that has attracted attention for drug development is the family members of MAPKs that are recognized to play significant roles in different cancers. Several inhibitors have been developed against various MAPK members; however, none of them as monotherapy has shown sustainable efficacy. One of the MAPK members, called Mixed Lineage Kinase 3 (MLK3), has attracted considerable attention due to its role in inflammation and neurodegenerative diseases; however, its role in cancer is an emerging area that needs more investigation. Recent advances have shown that MLK3 plays a role in cancer cell survival, migration, drug resistance, cell death, and tumor immunity. This review describes how MLK3 regulates different MAPK pathways, cancer cell growth and survival, apoptosis, and host's immunity. We also discuss how MLK3 inhibitors can potentially be used along with immunotherapy for different malignancies.
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Correction: Mixed Lineage Kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated Kinase 1.
Oncogene, 2019Co-Authors: Subhasis Das, Rajakishore Mishra, Gautam Sondarva, Basabi Rana, Navin Viswakarma, Rakesh Sathish Nair, Hazem Abdelkarim, Vadim Gaponenko, Pradeep Sathyanarayana, Ajay RanaAbstract:Mixed Lineage Kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 Kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.
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Mixed Lineage Kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated Kinase 1
Oncogene, 2019Co-Authors: Subhasis Das, Rajakishore Mishra, Gautam Sondarva, Basabi Rana, Navin Viswakarma, Rakesh Sathish Nair, Hazem Abdelkarim, Vadim Gaponenko, Ajay RanaAbstract:Mixed Lineage Kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 Kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.
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Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival
Experimental and Molecular Therapeutics, 2014Co-Authors: Subhasis Das, Gautam Sondarva, Basabi Rana, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Ajay RanaAbstract: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
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Drosophila Mixed Lineage Kinase/slipper, a missing biochemical link in Drosophila JNK signaling
Biochimica et biophysica acta, 2003Co-Authors: Pradeep Sathyanarayana, Manoj K. Barthwal, Mary Ellen Lane, Summer F. Acevedo, Efthimios M. C. Skoulakis, Andreas Bergmann, Ajay RanaAbstract:Abstract Mixed Lineage Kinases (MLKs) belong to the family of mitogen activated protein Kinase Kinase Kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal Kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr) . However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila Mixed Lineage Kinase (dMLK) that is also known as slipper . The protein structure analysis of dMLK/ slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine–threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated Kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro Kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/ slipper.
Val S. Goodfellow - One of the best experts on this subject based on the ideXlab platform.
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The second generation Mixed Lineage Kinase-3 (MLK3) inhibitor CLFB-1134 protects against neurotoxin-induced nigral dopaminergic neuron loss.
Experimental neurology, 2019Co-Authors: Elizabeth M. Kline, Val S. Goodfellow, Harris A. Gelbard, Laura M. Butkovich, Joshua M. Bradner, Jianjun Chang, W. Michael Caudle, Malú G. TanseyAbstract:Dopaminergic neurons express Mixed Lineage Kinases which regulate the expression of cell death genes. In Parkinson's disease, cell death via apoptosis is prevalent, and previous work testing Mixed Lineage Kinase inhibitors in animal models suggested the inhibitors had some neuroprotective potential. CLFB-1134 is a new, brain-penetrant inhibitor specific for MLK3, tested here in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of dopaminergic depletion and nigral neuron death in mice. After ensuring that treatment with CLFB-1134 did not alter conversion of MPTP to MPP+, we demonstrated CLFB-1134's inhibition of MLK3 and neuroprotective efficacy. Specifically we evaluated the integrity of the nigrostriatal dopamine system following MPTP by assessing protein expression, high performance liquid chromatography, and immunohistology with stereology. We found that CLFB-1134 achieves protection of striatal dopaminergic terminals and nigral cell bodies when dosed simultaneously or following MPTP treatment. By preventing phosphorylation of JNK and other downstream targets of MLK3, CLFB-1134 protects against the neurotoxin MPTP. Inhibition of MLK3 may be a valid target for future work investigating treatment of Parkinson's disease.
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Mixed Lineage Kinase 3 mediates release of c x c motif ligand 10 bearing chemotactic extracellular vesicles from lipotoxic hepatocytes
Hepatology, 2016Co-Authors: Samar C Ibrahim, Val S. Goodfellow, Kyoko Tomita, Petra Hirsova, Steven F. Bronk, Stephen A Harrison, Nathan W Werneburg, Harmeet Malhi, Gregory J GoresAbstract: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. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages. (Hepatology 2016;63:731–744)
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The Mixed Lineage Kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy
Nanomedicine : nanotechnology biology and medicine, 2015Co-Authors: Gang Zhang, Dongwei Guo, Prasanta K. Dash, Mariluz Araínga, Jayme Wiederin, Nicole A. Haverland, Jaclyn S. Knibbe-hollinger, Andrea Martinez-skinner, Pawel Ciborowski, Val S. GoodfellowAbstract:During studies to extend the half-life of crystalline nanoformulated antiretroviral therapy (nanoART) the Mixed Lineage Kinase-3 inhibitor URMC-099, developed as an adjunctive neuroprotective agent was shown to facilitate antiviral responses. Long-acting ritonavir-boosted atazanavir (nanoATV/r) nanoformulations co-administered with URMC-099 reduced viral load and the numbers of HIV-1 infected CD4+ T-cells in lymphoid tissues more than either drug alone in infected humanized NOD/SCID/IL2Rγc-/- mice. The drug effects were associated with sustained ART depots. Proteomics analyses demonstrated that the antiretroviral responses were linked to affected phagolysosomal storage pathways leading to sequestration of nanoATV/r in Rab-associated recycling and late endosomes; sites associated with viral maturation. URMC-099 administered with nanoATV induced a dose-dependent reduction in HIV-1p24 and reverse transcriptase activity. This drug combination offers a unique chemical marriage for cell-based viral clearance. From the Clinical Editor: Although successful in combating HIV-1 infection, the next improvement in antiretroviral therapy (nanoART) would be to devise long acting therapy, such as intra-cellular depots. In this report, the authors described the use of nanoformulated antiretroviral therapy given together with the Mixed Lineage Kinase-3 inhibitor URMC-099, and showed that this combination not only prolonged drug half-life, but also had better efficacy. The findings are hoped to be translated into the clinical setting in the future.
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232 Mixed Lineage Kinase 3 mediates the release of proinflammatory extracellular vesicles in nonalcoholic steatohepatitis
Gastroenterology, 2015Co-Authors: Samar H. Ibrahim, Val S. Goodfellow, Petra Hirsova, Steven F. Bronk, Stephen A Harrison, Gregory J GoresAbstract: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.
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Abstract 315: The Role of Mixed Lineage Kinase 3 in Inflammatory Cell-Fibroblast Communication
Circulation Research, 2014Co-Authors: Emily M. Schulz, Val S. Goodfellow, Harris A. Gelbard, Stephen Dewhurst, Allison E Dixon, Michael S Burhans, Michelle L. Nieman, John N. Lorenz, Burns C BlaxallAbstract:Mixed Lineage Kinase 3 (MLK3) is a ubiquitously expressed pro-inflammatory, pro-apoptotic mitogen activated protein Kinase Kinase Kinase (MAP3K). MLK3 is a key regulator of the p38 and c-jun terminal Kinase (JNK) pathways and has been studied in cancer and neurodegenerative disease. Although the p38 and JNK pathways have been studied in the cardiac function and disease, little is known regarding the role of MLK3 in the heart. Studies in our laboratory have indicated that MLK3 RNA is highly expressed in macrophages, cardiomyocytes and cardiac fibroblasts, suggesting an important role in cardiac function. Recently published work has indicated that MLK3 plays an important role in inflammatory cell motility, leading us to hypothesize that MLK3 is involved in inflammatory cell-fibroblast communication during cardiac disease. Knockout (KO) or inhibition of MLK3 using the novel small molecule inhibitor URMC-099 does not significantly affect heart rate, mean arterial pressure, systolic pressure, minimum or maximum dp/dt compared to wild type (WT) controls as measured by invasive hemodynamics at 3 months of age. Echocardiographic analysis indicates that MLK3 KO or inhibition does not affect cardiac architecture or cardiac function, indicated by fractional shortening or ejection fraction. However, upon transaortic constriction (TAC), MLK3 KO and URMC-099 treatment results in decreases in Mac-3 positive staining at 3 and 7 days post-TAC as well as decreases in CD-45 staining 7 days post-TAC compared to WT TAC operated, vehicle treated controls, suggesting that MLK3 KO and drug treatment may attenuate the early inflammatory response after TAC. Studies examining the relationship between the MLK3 mediated inflammatory response and subsequent fibrosis and cardiac dysfunction post-TAC are currently ongoing.
Yu Zhan - One of the best experts on this subject based on the ideXlab platform.
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Mixed Lineage Kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells.
Experimental cell research, 2012Co-Authors: Yu Zhan, Widian F. Abi Saab, Nidhi Modi, Amanda M. Stewart, Jinsong Liu, Deborah N. ChadeeAbstract:Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein Kinase Kinase Kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.
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Regulation of Mixed Lineage Kinase 3 is required for Neurofibromatosis-2-mediated growth suppression in human cancer
Oncogene, 2011Co-Authors: Yu Zhan, Nidhi Modi, Amanda M. Stewart, R I Hieronimus, D H Gutmann, Deborah N. ChadeeAbstract:The Neurofibromatosis-2 ( NF2 ) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with Mixed Lineage Kinase 3 (MLK3), a mitogen-activated protein Kinase (MAPK) Kinase Kinase that is required for the proliferation of normal and neoplastic cells. In this study, we show that merlin inhibits MLK3 activity, as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated Kinase (ERK) and c-Jun N-terminal Kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin-mediated suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.
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Inhibition of Cdc42-mediated activation of Mixed Lineage Kinase 3 by the tumor suppressor protein merlin.
Small GTPases, 2010Co-Authors: Yu Zhan, Deborah N. ChadeeAbstract:Mammalian mitogen-activated protein Kinase (MAPK) signaling pathways respond to diverse extracellular signals and coordinate a range of cellular responses. Mixed Lineage Kinase 3 (MLK3) is a member of the Mixed Lineage Kinase family of MAPK Kinase Kinases (MAP3Ks) that functions to regulate multiple MAPK signaling pathways. Activated forms of the Rho GTP ases, Rac and Cdc42, interact with MLK3 through the Cdc42/Rac-interactive binding (CRIB) motif and promote MLK3 catalytic activity. Our recent findings demonstrate that merlin, the product of the neurofibromatosis type 2 (NF2) tumor suppressor gene, is a physiological inhibitor of MLK3. Our results suggest that merlin inhibits MLK3 activity by blocking the Cdc42-MLK3 interaction. In this commentary, the effect of merlin on Cdc42-mediated activation of MLK3 and MAPK signaling will be discussed.
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Mixed Lineage Kinase 3 negatively regulates IKK activity and enhances etoposide-induced cell death.
Biochimica et biophysica acta, 2009Co-Authors: Eric T. Cole, Yu Zhan, Amanda Korchnak, Widian F. Abi Saab, Brian P. Ashburner, Deborah N. ChadeeAbstract:Abstract Mixed Lineage Kinase 3 (MLK3) is a mitogen activated protein Kinase Kinase Kinase (MAP3K) that activates multiple MAPK signaling pathways. Nuclear factor kappa B (NF-κB) is a transcription factor that has important functions in inflammation, immunity and cell survival. We found that silencing mlk3 expression with RNA interference (RNAi) in SKOV3 human ovarian cancer epithelial cells and NIH-3T3 murine fibroblasts led to a reduction in the level of the inhibitor of kappa B alpha (IκBα) protein. In addition, we observed enhanced basal IκB Kinase (IKK) activity in HEK293 cells transiently transfected with MLK3 siRNA and in NIH3T3 cells stably expressing MLK3 shRNA (shMLK3). Furthermore, the basal level of NF-κB-dependent gene transcription was elevated in shMLK3 cells. Silencing mlk3 expression conferred resistance of cells to etoposide-induced apoptotic cell death and overexpression of wild type MLK3 (MLK3-WT) or Kinase-dead MLK3 (MLK3-KD) promoted apoptotic cell death and cleavage of poly (ADP-ribose) polymerase (PARP). Overexpression of MLK3-WT or MLK3-KD enhanced etoposide-induced apoptotic cell death and cleavage of PARP. These data suggest that MLK3 functions to limit IKK activity, and depleting MLK3 helps protect cells from etoposide-induced cell death through activation of IKK-dependent signaling.
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Cytokine-induced activation of Mixed Lineage Kinase 3 requires TRAF2 and TRAF6.
Cellular signalling, 2009Co-Authors: Amanda Korchnak, Yu Zhan, Michael T. Aguilar, Deborah N. ChadeeAbstract:Abstract Mixed Lineage Kinase 3 (MLK3) is a mitogen-activated protein Kinase Kinase Kinase (MAP3K) that activates multiple mitogen-activated protein Kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1β (IL-1β) stimulation rapidly activate MLK3 Kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1β stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 Kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1β.