The SMYD3-MAP3K2 signaling axis promotes tumor aggressiveness and metastasis in prostate cancer.

Aberrant activation of Ras/Raf/mitogen-activated protein kinase (MAPK) signaling is frequently linked to metastatic prostate cancer (PCa); therefore, the characterization of modulators of this pathway is critical for defining therapeutic vulnerabilities for metastatic PCa. The lysine methyltransferase SET and MYND domain 3 (SMYD3) methylates MAPK kinase kinase 2 (MAP3K2) in some cancers, causing enhanced activation of MAPK signaling. In PCa, SMYD3 is frequently overexpressed and associated with disease severity; however, its molecular function in promoting tumorigenesis has not been defined. We demonstrate that SMYD3 critically regulates tumor-associated phenotypes via its methyltransferase activity in PCa cells and mouse xenograft models. SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition associated behaviors by altering the abundance of the intermediate filament vimentin. Furthermore, activation of the SMYD3-MAP3K2 signaling axis supports a positive feedback loop continually promoting high levels of SMYD3. Our data provide insight into signaling pathways involved in metastatic PCa and enhance understanding of mechanistic functions for SMYD3 to reveal potential therapeutic opportunities for PCa.

Regulation of a Novel CircTRRAP/miR-761/MAP3K2 CeRNA Cascade in Inflammation, Apoptosis, and Oxidative Stress in Human AC16 Cardiomyocytes under Hypoxia Conditions.

Emerging evidence uncovers the important involvement of circular RNAs (circRNAs) in the dysfunction of cardiomyocytes under hypoxia conditions. However, no studies proved whether circTRRAP (hsa_circ_0081241) can participate in cardiomyocyte injury evoked by hypoxia.A qRT-PCR or immunoblotting method was used to evaluate the expression of circTRRAP, miR-761, and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). The direct relationships of circTRRAP/miR-761 and miR-761/MAP3K2 were confirmed by RNA immunoprecipitation (RIP) assay, dual-luciferase reporter assay, and RNA pull-down assay. The effects of the circTRRAP/miR-761/MAP3K2 axis on cell functional behaviors were examined by 5-ethynyl-2'-deoxyuridine (EdU) assay, CCK-8 assay, and flow cytometry. The production levels of proinflammatory cytokines (IL-1ß, TNF-α, and IL-6) were evaluated by enzyme-linked immunosorbent assay.CircTRRAP and MAP3K2 were overexpressed but miR-761 was downregulated in AC16 cardiomyocytes under hypoxia and in the serum of patients with acute myocardial infarction. Silencing circTRRAP attenuated hypoxia-evoked inflammation, apoptosis, and oxidative stress in human AC16 cardiomyocytes. CircTRRAP targeted miR-761, and miR-761 directly targeted and suppressed MAP3K2. CircTRRAP involved the post-transcriptional regulation of MAP3K2 through miR-761, indicating its competing endogenous RNA (ceRNA) activity. Moreover, miR-761 inhibition abolished the effects of circTRRAP depletion in hypoxia-induced cell injury. MAP3K2 silencing phenocopied miR-761 increase in attenuating hypoxia-evoked cardiomyocyte inflammation, apoptosis, and oxidative stress.Our study demonstrates that circTRRAP can protect AC16 cardiomyocytes from hypoxia-evoked injury through the miR-761/MAP3K2 axis.

CircHSPG2 knockdown attenuates hypoxia-induced apoptosis, inflammation, and oxidative stress in human AC16 cardiomyocytes by regulating the miR-1184/MAP3K2 axis.

Circular RNAs (circRNAs) have been identified as vital regulators in cardiovascular diseases, including acute myocardial infarction (AMI). In this study, the function and mechanism of circRNA heparan sulfate proteoglycan 2 (circHSPG2) in hypoxia-induced injury in AC16 cardiomyocytes were investigated. AC16 cells were stimulated with hypoxia to establish an AMI cell model in vitro. Real-time quantitative PCR and western blot assays were performed to quantify the expression levels of circHSPG2, microRNA-1184 (miR-1184), and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). Counting Kit-8 (CCK-8) assay was used to measure cell viability. Flow cytometry was performed to detect cell cycle and apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of inflammatory factors. Dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were used to analyze the relationship between miR-1184 and circHSPG2 or MAP3K2. In AMI serum, circHSPG2 and MAP3K2 mRNA were highly expressed and miR-1184 was down-regulated. Hypoxia treatment elevated HIF1α expression and repressed cell growth and glycolysis. Moreover, hypoxia promoted cell apoptosis, inflammation, and oxidative stress in AC16 cells. Hypoxia-induced circHSPG2 expression in AC16 cells. CircHSPG2 knockdown alleviated hypoxia-induced AC16 cell injury. CircHSPG2 directly targeted miR-1184, and miR-1184 targeted and suppressed MAP3K2. Inhibition of miR-1184 or overexpression of MAP3K2 abolished the alleviated effect of circHSPG2 knockdown on hypoxia-induced AC16 cell injury. Overexpression of miR-1184 relieved hypoxia-induced impairment in AC16 cells by MAP3K2. CircHSPG2 could regulate MAP3K2 expression through miR-1184. CircHSPG2 knockdown protected AC16 cells from hypoxia-induced injury by regulating the miR-1184/MAP3K2 cascade.

E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2.

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-17 (IL-17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down-regulated gene 4-like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF-α, IL-1, and IL-17. NEDD4L modulates the degradation of mitogen-activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly-ubiquitination. In interleukin-17 receptor (IL-17R) signaling, Nedd4l knockdown or deficiency enhances IL-17-induced p38 and NF-κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2-dependent manner. We further show that IL-17-induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF-κB activation but also for NEDD4L-mediated MEKK2 degradation and the subsequent shutdown of IL-17R signaling. Importantly, Nedd4l-deficient mice show increased susceptibility to IL-17-induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL-17R signaling-dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL-17R signaling, which ameliorates the pathogenesis of IL-17-mediated autoimmune diseases.

miRNA-338-3p inhibits the migration, invasion and proliferation of human lung adenocarcinoma cells by targeting MAP3K2.

OBJECTIVE: This study aimed to investigate the effects of micro ribonucleic acid (miR)-338-3p on the migration, invasion and proliferation of lung adenocarcinoma (LUAD) cells. METHODS: Bioinformatics analysis was employed to evaluate the function and expression of related genes in lung cancer. Human A549 and NCI-H1299 cells cultured to logarithmic growth stage were assigned to negative control (NC) mimic group, miR-338-3p mimic group (miR-mimic group), NC inhibitor group and miR-338-3p inhibitor group (miR-inhibitor group) treated with or without MAP3K2 overexpression (OE)-lentivirus, or TBHQ or FR180204. Transwell assay, cell colony formation assay, Western blotting and cell-cycle analysis were carried out. RESULTS: Bioinformatics results manifested that miR-338 and MAP3K2 were involved in LUAD. The expression levels of MAP3K2, p-ERK1/2, MMP-2, MMP-3, MMP-9, cyclin A2 and cyclin D1 were increased after addition of miR-338-3p inhibitor, consistent with the raised amount of LUAD cells in migration and invasion experiments and number of colonies formed, as well as the cell cycle, but miR-338-3p mimic reversed these results. Moreover, MAP3K2 overexpression elevated the level of p-ERK1/2. Meanwhile, after treatment with TBHQ or FR180204, the influence of miR-338-3p inhibitor or mimic was also verified. CONCLUSIONS: MiR-338-3p overexpression can modulate the ERK1/2 signaling pathway by targeting MAP3K2, thus inhibiting the migration, invasion and proliferation of human LUAD cells.

MiR-20a-5p facilitates cartilage repair in osteoarthritis via suppressing mitogen-activated protein kinase kinase kinase 2.

Bone marrow mesenchymal stem cell (BMSC) chondrogenic differentiation contributes to the treatment of osteoarthritis (OA). Numerous studies have indicated that microRNAs (miRNAs) regulate the pathogenesis and development of multiple disorders, including OA. Nevertheless, the role of miR-20a-5p in OA remains obscure. Forty male C57BL/6 mice were divided into four groups and were surgically induced OA or underwent sham surgery in the presence or absence of miR-20a-5p. Flow cytometry was implemented to detect surface markers of BMSCs. Reverse transcription quantitative polymerase chain reaction revealed the upregulation of miR-20a-5p during BMSC chondrogenic differentiation. Western blotting displayed that miR-20a-5p inhibition decreased protein levels of cartilage matrix markers but enhanced those of catabolic and hypertrophic chondrocyte markers in BMSCs. Alcian blue staining, hematoxylin­eosin staining and micro-CT revealed that miR-20a-5p inhibition restrained chondrogenic differentiation and miR-20a-5p overexpression promoted the repair of damaged cartilage and subchondral bone, respectively. Luciferase reporter assay identified that mitogen activated protein kinase kinase kinase 2 (Map3k2) was a target of miR-20a-5p in BMSCs. Moreover, the expression of miR-20a-5p and Map3k2 was negatively correlated in murine cartilage tissues. Knocking down Map3k2 could rescue the suppressive influence of miR-20a-5p inhibition on chondrogenic differentiation of BMSCs. In conclusion, miR-20a-5p facilitates BMSC chondrogenic differentiation and contributes to cartilage repair in OA by suppressing Map3k2.

A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.

Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

CircRNA circBACH1 facilitates hepatitis B virus replication and hepatoma development by regulating the miR-200a-3p/MAP3K2 axis.

BACKGROUND: Hepatitis B virus (HBV) is a top contributor to hepatoma. Circular RNAs (circRNAs) have been elucidated to have a close connection with HBV-induced hepatoma. This study aimed to explore the role of circRNA BTB domain and CNC homolog 1 (circBACH1) in HBV replication and hepatoma progression, as well as the potential mechanistic pathway. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to assess the expression of circBACH1, microRNA (miR)-200a-3p, and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). HBV replication was determined by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assay. Cell viability and clonogenicity were detected via Cell Counting Kit-8 (CCK-8) assay and colony formation assay, respectively. Cell metastasis was examined by Transwell assay and wound healing assay. Annexing-V/PI staining was employed to monitor cell apoptosis using flow cytometry. Levels of MAP3K2, proliferation- and apoptosis-related proteins were analyzed by Western blotting. Target interaction between miR-200a-3p and circBACH1 or MAP3K2 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The role of circBACH1 in vivo was investigated by xenograft model assay. RESULTS: Expression of circBACH1 and MAP3K2 was increased, while miR-200a-3p expression was decreased in HCC tissues and HBV-transfected hepatoma cells. Depletion of circBACH1 or miR-200a-3p overexpression impeded HBV replication, proliferation, and metastasis in HBV-transfected hepatoma cells. CircBACH1 was able to regulate MAP3K2 expression by sponging miR-200a-3p. CircBACH1 regulated HBV replication and hepatoma progression through the miR-200a-3p/MAP3K2 pathway. Moreover, circBACH1 deficiency hampered tumor growth in vivo. CONCLUSION: CircBACH1 knockdown had inhibitory effects on HBV replication and hepatoma progression, at least partly by modulating the miR-200a-3p/MAP3K2 axis.

MicroRNA-379-5p targets MAP3K2 to reduce autophagy and alleviate neuronal injury following cerebral ischemia via the JNK/c-Jun signaling pathway.

MicroRNAs (miRNAs) are abundant in neurons and play key roles in the function and development of the nervous system. This study focuses on the function of miR-379-5p in neurological function recovery during ischemic stroke. The expression of miR-379-5p in the serum of patients with ischemic stroke was determined. Human cerebral cortical neuron cells (HCN-2) were subjected to oxygen/glucose deprivation (OGD) to mimic an ischemic stroke in vitro, whereas mice subjected to middle cerebral artery occlusion (MCAO) were used as an animal model. The serum of patients with ischemic stroke and OGD-treated HCN-2 cells displayed a poor expression of miR-379-5p. Upregulation of miR-379-5p reduced the OGD-induced cell damage and decreased the expression of the autophagy marker protein Beclin1 in cells. Rapamycin, an autophagy activator, blocked the protective functions of miR-379-5p. Further, miR-379-5p directly bound to MAP3K2. MAP3K2 activated the JNK/c-Jun signaling pathway and suppressed the neuroprotective events mediated by miR-379-5p. The in vitro results were reproduced in vivo, where upregulation of miR-379-5p reduced neurological impairment and infarct size in MCAO-induced mice. This study suggested that miR-379-5p showed a neuroprotective effect on ischemic stroke and reduced autophagy of neurons through the suppression of MAP3K2 and the JNK/c-Jun axis.

MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2.

γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.

miR­3613­3p/MAP3K2/p38/caspase­3 pathway regulates the heat­stress­induced apoptosis of endothelial cells.

Previous studies have identified microRNA (miRNA/miR)­3613­3p as a heat stress (HS)­related miRNA in endothelial cells that can lead to apoptosis. However, the mechanism underlying the miR­3613­3p­mediated apoptosis of HS­exposed endothelial cells remains unclear. In the present study, western blot analysis and reverse transcription­quantitative PCR were used to determine protein and miRNA expression levels, respectively. Annexin V­fluorescein isothiocyanate/propidium iodide staining, caspase­3 activity measurements and DNA fragmentation assays were performed to detect apoptosis. To evaluate whether mitogen­activated protein kinase kinase kinase 2 (MAP3K2) was a direct target of miR­3613­3p, a luciferase reporter assay was performed. In addition, transient transfection was used to carry out loss­ and gain­of­function experiments. The results revealed that miR­3613­3p expression was reduced in human umbilical vein endothelial cells (HUVECs) following HS, which led to apoptosis. Mechanistically, following HS, a decrease in miR­3613­3p binding to the 3'­untranslated region of MAP3K2 directly upregulated its expression, and the downstream p38 and caspase­3 pathways, thereby leading to apoptosis. Taken together, the results of the present study demonstrated that HS suppressed miR­3613­3p expression, which activated the MAP3K2/p38/caspase­3 pathway, leading to the apoptosis of HUVECs. In conclusion, the miR­3613­3p/MAP3K2/p38/caspase­3 pathway may serve an indispensable role in regulating the progression of apoptosis, indicating a regulatory role of miR­3613­3p in the pathophysiology of HS­exposed endothelial cells.

MicroRNA-335 inhibits the growth, chemo-sensitivity, and metastasis of human breast cancer cells by targeting MAP3K2.

The Editors of JBUON issue an Expression of Concern to 'MicroRNA-335 inhibits the growth, chemo-sensitivity, and metastasis of human breast cancer cells by targeting MAP3K2', by Yajue Wang, Feng Wen; JBUON 2020;25(2):666-674; PMID: 32521851. Following the publication of the above article, readers drew to our attention that part of the data was possibly unreliable. We sent emails to the authors with a request to provide the raw data to prove the originality, but received no reply. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.

LncRNA-ATB regulates epithelial-mesenchymal transition progression in pulmonary fibrosis via sponging miR-29b-2-5p and miR-34c-3p.

Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-ß1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-ß1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.

Pazopanib ameliorates acute lung injuries via inhibition of MAP3K2 and MAP3K3.

Acute lung injury (ALI) causes high mortality and lacks any pharmacological intervention. Here, we found that pazopanib ameliorated ALI manifestations and reduced mortality in mouse ALI models and reduced edema in human lung transplantation recipients. Pazopanib inhibits mitogen-activated protein kinase kinase kinase 2 (MAP3K2)- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 to increase reactive oxygen species (ROS) formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic mutation of p47phox Ser208 to alanine attenuated ALI manifestations and abrogates anti-ALI effects of pazopanib. This myeloid MAP3K2/MAP3K3-p47phox pathway acted via paracrine H2O2 to enhance pulmonary vasculature integrity and promote lung epithelial cell survival and proliferation, leading to increased pulmonary barrier function and resistance to ALI. Thus, pazopanib has the potential to be effective for treating ALI.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

Mass-spectrometry-based proteomic correlates of grade and stage reveal pathways and kinases associated with aggressive human cancers.

Proteomic signatures associated with clinical measures of more aggressive cancers could yield molecular clues as to disease drivers. Here, utilizing the Clinical Proteomic Tumor Analysis Consortium (CPTAC) mass-spectrometry-based proteomics datasets, we defined differentially expressed proteins and mRNAs associated with higher grade or higher stage, for each of seven cancer types (breast, colon, lung adenocarcinoma, clear cell renal, ovarian, uterine, and pediatric glioma), representing 794 patients. Widespread differential patterns of total proteins and phosphoproteins involved some common patterns shared between different cancer types. More proteins were associated with higher grade than higher stage. Most proteomic signatures predicted patient survival in independent transcriptomic datasets. The proteomic grade signatures, in particular, involved DNA copy number alterations. Pathways of interest were enriched within the grade-associated proteins across multiple cancer types, including pathways of altered metabolism, Warburg-like effects, and translation factors. Proteomic grade correlations identified protein kinases having functional impact in vitro in uterine endometrial cancer cells, including MAP3K2, MASTL, and TTK. The protein-level grade and stage associations for all proteins profiled-along with corresponding information on phosphorylation, pathways, mRNA expression, and copy alterations-represent a resource for identifying new potential targets. Proteomic analyses are often concordant with corresponding transcriptomic analyses, but with notable exceptions.

MEKK2 and MEKK3 orchestrate multiple signals to regulate Hippo pathway.

The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals via the regulation of cell proliferation and apoptosis. It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.

Upregulation of miR-335 reduces myocardial injury following myocardial infarction via targeting MAP3K2.

OBJECTIVE: Acute myocardial infarction (AMI) is a serious cardiovascular disease with a high incidence worldwide and the main cause of sudden cardiac death. The aim of this article was to study the protective role of miR-335 in myocardial infarction (MI) and the underlying molecular mechanism. MATERIALS AND METHODS: Thirty Sprague Dawley (SD) rats were randomly divided into sham group, MI + NC group and MI + agomiR-335 group. The expression of miR-335 in rat myocardium was detected by quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Western blot was performed to detect the expression of TNF-α, IL-6, IL-1ß, Caspase-3, Cleaved Caspase-3 (C-Caspase-3) and MAP3K2 in rat myocardium. On the 7th day of the establishment of the rat MI model, a high-resolution small animal ultrasound system was utilized to detect the cardiac function of the rats, and the heart tissues and blood samples of the rats were collected. The corresponding kits were purchased to detect the contents of LDH, CK-MB, MDA and SOD in rat serum, and HE staining was employed to observe the morphology of rat myocardial tissue. RESULTS: The expression of miR-335 in myocardial infarcted zones and border zones of MI rats decreased significantly. The upregulation of miR-335 remarkably inhibited myocardial inflammation and apoptosis after MI, thus improving the cardiac function of MI rats. Compared with the sham group, the MAP3K2 expression in the MI + NC group was observably increased, while the overexpression of miR-335 could inhibit the expression of this protein. Through the Luciferase reporter gene experiment, we found that miR-335 could directly target MAP3K2. CONCLUSIONS: The expression of miR-335 decreased in myocardial tissue after MI, and the overexpression of miR-335 reduced myocardial damage by inhibiting oxidative stress, inflammation, and apoptosis via targeting MAP3K2, thereby improving the cardiac function of MI rats.

Structural basis for antigen recognition by methylated lysine-specific antibodies.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.