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Stress-activated signaling pathways orchestrate cellular behaviors and fates. Studying the precise role(s) of stress-activated protein kinases is challenging, because stress conditions induce adaptation and impose selection pressure. To meet this challenge, we have applied an optogenetic system with a single plasmid to express light-activated p38α or its upstream activator, MKK6, in conjunction with live-cell fluorescence microscopy. In starved cells, decaging of constitutively active p38α or MKK6 by brief exposure to UV light elicits rapid p38-mediated signaling, release of cytochrome c from mitochondria, and apoptosis with different kinetics. In parallel, light activation of p38α also suppresses autophagosome formation, similarly to stimulation with growth factors that activate PI3K/Akt/mTORC1 signaling. Active MKK6 negatively regulates serum-induced ERK activity, which is p38-independent as previously reported. Here, we reproduce that result with the one plasmid system and show that although decaging active p38α does not reduce basal ERK activity in our cells, it can block growth factor-stimulated ERK signaling in serum-starved cells. These results clarify the roles of MKK6 and p38α in dynamic signaling programs, which act in concert to actuate apoptotic death while suppressing cell survival mechanisms., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Objective: Apurinic/apyrimidinic endonuclease 1 (APEX1), a key enzyme responsible for DNA base excision repair, has been linked to development and progression of cancers. In this work, we aimed to explore the role of APEX1 in hepatocellular carcinoma (HCC) and elucidate its molecular mechanism., Methods: The expression of APEX1 in HCC tissues and matched adjacent normal tissues ( n = 80 cases) was evaluated by immunohistochemistry. Web-based tools UALCAN and the Kaplan-Meier plotter were used to analyze the Cancer Genome Atlas database to compare expression of APEX1 mRNA to 5-year overall survival. APEX1 was stably silenced in two HCC cell lines, Hep 3B and Bel-7402, with shRNA technology. An in vivo tumorigenesis model was established by subcutaneously injecting sh-APEX1-transfected Bel-7402 cells into mice, and tumor growth was determined. We performed high-throughput transcriptome sequencing in sh-APEX1-treated HCC cells to identify the key KEGG signaling pathways induced by silencing of APEX1 ., Results: APEX1 was significantly upregulated and predicted poor clinical overall survival in HCC patients. Silencing APEX1 inhibited the proliferation of HCC cells in vivo and in vitro , and it repressed invasion and migration and increased apoptosis and the percentage of cells in G1. Differentially expressed genes upon APEX1 silencing included genes involved in TNF signaling. A positive correlation between the expression of APEX1 and MAP2K6 was noted, and overexpressing MAP2K6 overcame cancer-related phenotypes associated with APEX1 silencing., Conclusion: APEX1 enhances the malignant properties of HCC via MAP2K6 . APEX1 may represent a valuable prognostic biomarker and therapeutic target in HCC.

Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6 , CBL , RUNX3 , STAT1 , and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1 / JAK2 , which varied alongside the patient's viral load. Expression of MX1 , MX2 , ISG15 , and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1 / JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38β, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity., Competing Interests: RR, AM, EM, IN, AS, VM, FC, ER, ML, LL, LS, VB, DF, LJ, JJ, BG, GS No competing interests declared, (© 2022, Romero-Becerra et al.)

Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes., (© 2021. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Obesity is a state of abnormal fat accumulation caused by an energy imbalance potentially caused by changes in multiple factors. MEK6 engages in cell growth, such as inflammation and apoptosis, as one of the MAPK signaling pathways. The MEK6 gene was found to be related to RMR , a gene associated with obesity. Because only a few studies have investigated the correlation between MEK6 and obesity or the relevant mechanisms, we conducted an experiment using a Tg MEK6 model with MEK6 overexpression with non-Tg and chow diet as the control to determine changes in lipid metabolism in plasma, liver, and adipose tissue after a 15-week high-fat diet (HFD). MEK6 overexpression in the Tg MEK6 model significantly increased body weight and plasma triglyceride and total cholesterol levels. p38 activity declined in the liver and adipose tissues and lowered lipolysis, oxidation, and thermogenesis levels, contributing to decreased energy consumption. In the liver, lipid formation and accumulation increased, and in adipose, adipogenesis and hypertrophy increased. The adiponectin/leptin ratio significantly declined in plasma and adipose tissue of the Tg MEK6 group following MEK6 expression and the HFD, indicating the role of MEK6 expression in adipokine regulation. Plasma and bone-marrow-derived macrophages (BMDM) of the Tg MEK6 group increased MEK6 expression-dependent secretion of pro-inflammatory cytokines but decreased levels of anti-inflammatory cytokines, further exacerbating the results exhibited by the diet-induced obesity group. In conclusion, this study demonstrated the synergistic effect of MEK6 with HFD in fat accumulation by significantly inhibiting the mechanisms of lipolysis in the adipose and M2 associated cytokines secretion in the BMDM.

Objective: Osteoarthritis (OA) is a degenerative joint disease characterized by deterioration of articular cartilage functions. Previous studies have confirmed the role of circular RNAs (circRNAs) in OA, but the role of mechanical stress-related circRNA (circRNA-MSR) in OA is unknown., Design: The human chondrocytes C28/I2 were cultured and treated with lipopolysaccharide (LPS) to establish the OA model. The mRNA and protein levels were measured by qRT-PCR or Western blot. Cell viability was analyzed by MTT assay. Flow cytometry was carried out to detect cell apoptosis. The levels of TNF-α, IL-1β, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Pull-down assay was conducted to measure circRNA-MSR-related miRNA. Dual-luciferase reporter gene detection was performed to detect the target relationships between miR-643 and circRNA-MSR or Mitogen-activated protein kinase kinase 6 (MAP2K6). The RNA-fluorescence in situ hybridization (RNA-FISH) assay was conducted to verify the localization of circRNA-MSR and miR-643., Results: The expressions of circRNA-MSR were upregulated in LPS stimulated C28/I2 cells. Knockdown of circRNA-MSR can inhibit LPS-induced apoptosis, inflammatory response, and extracellular matrix (ECM) degradation, and promote cell C28/I2 cells proliferation. Moreover, circRNA-MSR directly targeted miR-643. RNA-FISH exhibited that circRNA-MSR may act as a competing endogenous RNA (ceRNA) of miR-643. Over-expression of miR-643 could alleviate LPS-induced C28/I2 chondrocyte injury and promote cell proliferation. Besides, miR-643 directly bound to MAP2K6 mRNA. MiR-643 inhibition or MAP2K6 overexpression can reverse the role of circRNA-MSR knockdown on LPS-treated chondrocytes., Conclusion: circRNA-MSR can upregulate MAP2K6 by targeting miR-643, thereby inhibiting cell proliferation and promoting apoptosis of C28/I2 cells.

Background: The mitogen-activated protein kinase (MAPK) pathway is highly associated with the progression and metastasis of various solid tumours. MAPK14, a core molecule of the MAPK pathway, plays vital roles in the colorectal cancer (CRC). Recent studies have shown that circRNAs can affect tumour progression by encoding peptides. However, little is known regarding the potential protein translated from circMAPK14 and whether it plays a role in the carcinogenesis of colorectal cancer., Methods: The RNA level and translatable potential of circMAPK14 in CRC was verified using qRT-PCR and public databases. RNase R digestion assay, qRT-PCR, sanger sequencing and FISH assays were utilised to verify the circular characteristics and subcellular localisation of circMAPK14. The suppressive role of circMAPK14 on the progression and metastasis of CRC was verified in vivo and in vitro. LC/MS analysis combined with western blotting demonstrated the presence and relative expression of circMAPK14-175aa. The underlying mechanism of circMAPK14-175aa action to inhibit CRC was identified by co-IP analysis. The binding of U2AF2 within the flanking introns of circMAPK14 was evaluated by RNA pull-down assay and RIP assay. Ultimately, luciferase reporter gene assays and ChIP assays confirmed that FOXC1 suppressed transcription of U2AF2 by binding to the U2AF2 promoter in the -400 bp to -100 bp region.　 RESULTS: We identified that hsa_circ_0131663 (termed circMAPK14) showed significantly decreased expression level in cells and tissue samples of CRC, and was primarily localised in the cytoplasm. A series of function experiments demonstrated that circMAPK14 influenced CRC progression and metastasis by encoding a peptide of 175 amino acids (termed circMAPK14-175aa). We also found that circMAPK14-175aa reduced nuclear translocation of MAPK14 by competitively binding to MKK6, thus facilitating ubiquitin-mediated degradation of FOXC1. Moreover, we described a positive feedback loop in CRC in which elevated FOXC1 expression was caused by reduced circMAPK14-175aa expression. This, in turn, decreased circMAPK14 biogenesis by suppressing U2AF2 transcription., Conclusion: In summary, we reported for the first time that circMAPK14 functioned as a tumour-suppressor by encoding circMAPK14-175aa, which blocked the progression and metastasis of colorectal cancer., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

To investigate whether the beiging process changes the interactive effects of salt and MEK6 gene on inflammatory adipogenesis, the salt treatment (NaCl 50 mM) and MEK6 transfection of Tg (+/+) cells were performed with white adipocytes (WAT) and beige-like-adipocytes (BLA). BLA induced by T3 were confirmed by UCP-1 expression and the MEK6 protein was 3.5 times higher in MEK6 transfected WAT than the control. The adipogenic genes, PPAR - γ and C/EBP - α , were 1.5 times more highly expressed in the salt-treated groups than the non-salt-treated groups, and adipogenesis was greatly increased in Tg (+/+) WAT compared to non-transfected Tg (-/-) . The adipogenesis induced by salt treatment and MEK6 transfection was significantly reduced in BLA. The inflammatory adipocytokines, TNF-α, IL-1β, and IL-6, were increased in the salt-treated Tg (+/+) WAT, but an anti-inflammation biomarker, the adiponectin/leptin ratio, was reduced in Tg (+/+) , to tenth of that in Tg (-/-) . However, the production of adipocytokines in WAT was strongly weakened in BLA, although a combination of salt and MEK6 transfection had the most significant effects on inflammation in both WAT and BLA. Oxygen consumption in mitochondria was maximized in salt-treated and MEK6 transfected WAT, but it was decreased by 50% in BLA. In conclusion, beiging controls the synergistic effects of salt and MEK6 on adipogenesis, inflammation, and energy expenditure.

Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding., (© 2021 The Protein Society.)

To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high-fat/high-sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Transdifferentiation of lung fibroblasts to myofibroblasts is a crucial pathophysiological process in pulmonary fibrosis. MicroRNA‑375 (miR‑375) was initially identified as a tumor‑suppressive factor, and its expression was negatively associated with the severity of lung cancer; however, its role and potential mechanism in myofibroblast transdifferentiation and pulmonary fibrosis remain unclear. In the present study, human lung fibroblasts were stimulated with transforming growth factor‑β (TGF‑β) to induce myofibroblast transdifferentiation. A mimic and inhibitor of miR‑375, and their negative controls, were used to overexpress or suppress miR‑375 in lung fibroblasts, respectively. The mRNA expression levels of fibrotic markers, and protein expression of α‑smooth muscle actin and periostin, were subsequently detected by reverse transcription‑quantitative PCR and western blotting, to assess myofibroblast transdifferentiation. miR‑375 was markedly upregulated in human lung fibroblasts after TGF‑β stimulation. The miR‑375 mimic alleviated, whereas the miR‑375 inhibitor aggravated TGF‑β‑dependent transdifferentiation of lung fibroblasts. Mechanistically, miR‑375 prevented myofibroblast transdifferentiation and collagen synthesis by blocking the P38 mitogen‑activated protein kinases (P38) pathway, and P38 suppression abrogated the deleterious effect of the miR‑375 inhibitor on myofibroblast transdifferentiation. Furthermore, the present study revealed that mitogen‑activated protein kinase kinase 6 was involved in P38 inactivation by miR‑375. In conclusion, miR‑375 was implicated in modulating TGF‑β‑dependent transdifferentiation of lung fibroblasts, and targeting miR‑375 expression may help to develop therapeutic approaches for treating pulmonary fibrosis.

The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal-regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Rahman et al.)

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1β (IL-1β) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1β-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1β specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Stroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown., Methods: Mice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B-I, LC3B-II, Beclin 1, and p62 expression., Results: Significantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B-I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719., Conclusions: Our results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation., (Copyright © 2019. Published by Elsevier Ltd.)

Reprogramming somatic cells to pluripotency by Oct4, Sox2, Klf4, and Myc represent a paradigm for cell fate determination. Here, we report a combination of Jdp2, Jhdm1b, Mkk6, Glis1, Nanog, Essrb, and Sall4 (7F) that reprogram mouse embryonic fibroblasts or MEFs to chimera competent induced pluripotent stem cells (iPSCs) efficiently. RNA sequencing (RNA-seq) and ATAC-seq reveal distinct mechanisms for 7F induction of pluripotency. Dropout experiments further reveal a highly cooperative process among 7F to dynamically close and open chromatin loci that encode a network of transcription factors to mediate reprogramming. These results establish an alternative paradigm for reprogramming that may be useful for analyzing cell fate control., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Apoptosis and senescence are two mutually exclusive cell fate programs that can be activated by stress. The factors that instruct cells to enter into senescence or apoptosis are not fully understood, but both programs can be regulated by the stress kinase p38α. Using an inducible system that specifically activates this pathway, we show that sustained p38α activation suffices to trigger massive autophagosome formation and to enhance the basal autophagic flux. This requires the concurrent effect of increased mitochondrial reactive oxygen species production and the phosphorylation of the ULK1 kinase on Ser-555 by p38α. Moreover, we demonstrate that macroautophagy induction by p38α signaling determines that cancer cells preferentially enter senescence instead of undergoing apoptosis. In agreement with these results, we present evidence that the induction of autophagy by p38α protects cancer cells from chemotherapy-induced apoptosis by promoting senescence. Our results identify a new mechanism of p38α-regulated basal autophagy that controls the fate of cancer cells in response to stress.

Mitogen-activated protein kinase kinase 6 (MKK6) is an essential component of the p38MAPK signaling pathway, which is involved in the modulation of inflammation, cell apoptosis and survival responses in mammals. However, the function of MKK6s in teleosts is still unclear. In this study, a fish MKK6 homolog (CiMKK6) was first identified from the grass carp (Ctenopharyngodon idella), a freshwater fish. CiMKK6 cDNA encodes a putative protein of 357 amino acids that contains conserved structural characteristics of the MKK6 family, including the S_TKc domain, SVAKT motif and DVD site. The deduced CiMKK6 protein exhibits high sequence homology with other reported fish MKK6s and shares the closest relationship with MKK6 from Danio rerio. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMKK6 mRNA was widely expressed in all tested tissues and stages of embryonic development. Additionally, the transcript levels of CiMKK6 in the intestine were significantly upregulated in response to bacterial muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) stimulation. Moreover, subcellular localization analysis indicated that CiMKK6 was distributed in both the cytoplasm and the nucleus of HEK293T cells. Finally, overexpression of CiMKK6 significantly enhanced the transcriptional activity of the AP-1 reporter gene in HEK293T cells. Overall, these findings may help better clarify the immune function of teleost MKK6s and provide new insight into the immune defense mechanisms of grass carp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. However, a major challenge is the large-scale production of phosphorylated proteins in vitro for these structural, functional, and dynamic studies. Here, we describe an efficient protocol used routinely in our laboratory for the production of phosphorylated proteins. We also describe the methods used for identifying, characterizing, and separating the resulting phosphorylated proteins for subsequent studies., (© 2019 Elsevier Inc. All rights reserved.)

Mitogen-activated protein kinase kinases (MKKs) are important components of the MAPK signaling pathways, which play a key role in responding to stress and inflammatory stimuli. Here, a new MKK gene, AccMKK6 , was identified and functionally analyzed in Apis cerana cerana Real-time quantitative PCR (qPCR) and Western blot analysis demonstrated that the AccMKK6 expression level was up-regulated by several environmental stresses. Moreover, the knockdown of AccMKK6 by RNA interference technology altered the expression levels of some antioxidant genes. In addition, the knockdown of AccMKK6 resulted in increased malonyldialdehyde (MDA) concentration and decreased antioxidant-related enzymes activity in honeybees. To explore the MAPK signaling pathways involved in AccMKK6 , we identified the transcription factor kayak in A. cerana cerana We analyzed the interactions of AccMKK6, Accp38b, and Acckayak using the yeast two-hybrid system. AccMKK6 and Acckayak showed similar expression profiles after several stress treatments. In addition, the expression level of Acckayak was significantly increased when AccMKK6 was silenced. Therefore, we speculate that AccMKK6 may be involved in the MAPK cascades, which play a crucial role in counteracting oxidative stress caused by external stimuli., (© 2018 The Author(s).)

Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Arabidopsis ( Arabidopsis thaliana ) MAP KINASE (MPK) proteins can function in multiple MAP kinase cascades and physiological processes. For instance, MPK4 functions in regulating development as well as in plant defense by participating in two independent MAP kinase cascades: the MEKK1-MKK1/MKK2-MPK4 cascade promotes basal resistance against pathogens and is guarded by the NB-LRR protein SUMM2, whereas the ANPs-MKK6-MPK4 cascade plays an essential role in cytokinesis. Here, we report a novel role for MKK6 in regulating plant immune responses. We found that MKK6 functions similarly to MKK1/MKK2 and works together with MEKK1 and MPK4 to prevent autoactivation of SUMM2-mediated defense responses. Interestingly, loss of MKK6 or ANP2/ANP3 results in constitutive activation of plant defense responses. The autoimmune phenotypes of mkk6 and anp2 anp3 mutant plants can be largely suppressed by a constitutively active mpk4 mutant. Further analysis showed that the constitutive defense response in anp2 anp3 is dependent on the defense regulators PAD4 and EDS1, but not on SUMM2, suggesting that the ANP2/ANP3-MKK6-MPK4 cascade may be guarded by a TIR-NB-LRR protein. Our study shows that MKK6 has multiple functions in plant defense responses in addition to cytokinesis., (© 2018 American Society of Plant Biologists. All rights reserved.)

Using Tandem Mass Tags (TMT) labeling and LC-MS/MS analysis of peptides from two cell lines (CNE2 and its radioresistant subclone CNE2-IR), we identified 754 proteins differentially expressed in CNE2-IR compared to CNE2. MAP2K6 was identified as a candidate radioresistance-related protein kinase. In vitro functional analysis revealed that over-expression of MAP2K6 significantly enhanced cell survival and colony formation following irradiation in NPC cells. Further, knockdown of MAP2K6 in radioresistant NPC cells led to decreased colony formation and increased apoptotic cells following irradiation. However, the effect of MAP2K6 in regulating the radioresistance in NPC cells did not seem to depend on p38/MAPK activity. Importantly, MAP2K6 might be required for leukemia inhibitory factor receptor (LIFR)-regulated radioresistance, as the expression levels of MAP2K6 affected LIFR/p70S6K signaling activation in NPC cells. Further, MAP2K6 kinase activity is required to activate LIFR/p70S6K signaling and to confer pro-survival effect on NPC cells. In conclusion, MAP2K6 might be an important regulator of LIFR-induced radioresistance in NPC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The important role of miR-422a in tumor has been reported in several studies. Recent research discovered that the expression of miR-422a was significantly decreased in colorectal cancer tissues, providing miR-422a as a tumor suppressor in CRC. However, the concrete mechanism of miR-422a regulating CRC cell is still unclear. In this study, we demonstrated that miR-422a could inhibit CRC cell growth and promote cell apoptosis via in vitro analyses. Moreover, computational methods were adopted to identify the targets of miR-422a. We found MAPKK6 was the direct target of miR-422a. Consequently, we further elucidated that miR-422a inhibited CRC cell growth and induced cell apoptosis by inhibiting p38/MAPK pathway. Besides that, we established the tumor xenograft model using nude mice and the inhibitory effects on tumor volumes and weights by miR-422a mimic transfection were also detected. Taken together, these findings demonstrated miR-422a exerted anti-cancer activities on CRC, which could be potentially used for CRC prognosis prediction and treatment., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

p38 signaling is broadly involved in controlling inflammation and stress-induced cell death; however, the mechanisms controlling its activity have seldom been studied. Here, we report that TRIM9 short isoform (TRIM9s) potentiates p38 signaling by stabilizing MKK6. Mechanistic studies revealed that TRIM9s promotes the K63-linked ubiquitination of MKK6 at Lys82, thus inhibiting the degradative K48-linked ubiquitination of MKK6 at the same lysine. MKK6 could also stabilize TRIM9s by promoting the phosphorylation of TRIM9s at Ser76/80 via p38, thereby blocking the ubiquitin-proteasome pathway. Further functional analyses showed that p38 signaling plays a critical role in suppressing glioblastoma progression. Co-reduction of MKK6 and TRIM9s is significantly associated with overall poor survival of glioblastoma patients. We identify a positive feedback loop in p38 signaling generated by MKK6-TRIM9s, which suppresses glioblastoma progression, and we provide insights into the mechanisms by which TRIM9s and MKK6 potentiate p38 signaling through mutual stabilization., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale : Excessive myocardial fibrosis is the main pathological process in the development of cardiac remodeling and heart failure; therefore, it is important to prevent excessive myocardial fibrosis. We determined that microRNA-378 (miR-378) is cardiac-enriched and highly repressed during cardiac remodeling. We therefore proposed that miR-378 has a critical role in regulation of cardiac fibrosis, and examined the effects of miR-378 on cardiac fibrosis after mechanical stress. Methods: Mechanical stress was respectively imposed on mice through a transverse aortic constriction (TAC) procedure and on cardiac fibroblasts by stretching silicon dishes. A chemically modified miR-378 mimic (Agomir) or an inhibitor (Antagomir) was administrated to mice by intravenous injection and to cells by direct addition to the culture medium. MiR-378 knockout mouse was constructed. Cardiac fibroblasts were cultured in the conditioned media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869. Quantitative real-time polymerase chain reaction analysis of gene and miRNA expression, Western blot analysis, immunochemistry and electron microscopy were performed to elucidate the mechanisms. Results : Mechanical stress induced significant increases in fibrotic responses, including myocardial fibrosis, fibroblast hyperplasia, and protein and gene expression of collagen and matrix metalloproteinases (MMPs) both in vivo and in vitro . All these fibrotic responses were attenuated by treatment with a chemically modified miR-378 mimic (Agomir) but were exaggerated by treatment with an inhibitor (Antagomir). MiR-378 knockout mouse models exhibited aggravated cardiac fibrosis after TAC. Media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869 enhanced the fibrotic responses of stimulated cardiac fibroblasts, confirming that miR-378 inhibits fibrosis in an extracellular vesicles-dependent secretory manner. Mechanistically, the miR-378-induced anti-fibrotic effects manifested partially through the suppression of p38 MAP kinase phosphorylation by targeting MKK6 in cardiac fibroblasts. Conclusions : miR-378 is secreted from cardiomyocytes following mechanical stress and acts as an inhibitor of excessive cardiac fibrosis through a paracrine mechanism., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The aim of the present work was to study the immune profiling of prostate epithelial cells by the expression of ASK-1/p38 and Raf-1/ERK MAP Kinases signaling pathways mediated by TRAF-6. Immunohistochemical and Western blot analyses for TRAF-6, ASK-1, MEK-6, p38, Raf-1, MEK-1, ERK-1, ERK-2 and PSA were carried out in 5 samples of normal prostate gland, 24 samples of BPH and 19 samples of PC. Immunoreaction to TRAF-6 was found in the cytoplasm of epithelial cells of BPH and tumor cells of PC samples. For patients with the profile (TRAF-6+), optical densities revealed a weak immunoexpression of ASK-1 in PC compared to BPH patients. Whereas, immunoexpression to Raf-1 was higher in PC than in BPH. According to the expression of ASK-1 and Raf-1, two main profiles were identified: (TRAF-6+, ASK-1+, Raf-1+) and (TRAF-6+, ASK-1+, RAF-1-) in both BPH and PC. In addition, ASK-1/p38 axis expression was increased in BPH. Raf-1/ERK signaling pathway was increased in PC samples. On the other hand, representing of individual signaling protein expression enclosing each of p38 and ERK MAP Kinases according to TRAF-6+ showed a qualitative behavior of ASK61/p38 and Raf-1/ERK signaling pathways and a dynamic expression of PSA associated with immune and inflammatory process. These findings suggest that prostate epithelial cell could able an immune and inflammatory setting., (Copyright © 2017. Published by Elsevier Taiwan.)

Arabidopsis mitogen-activated protein (MAP) kinase kinase 6 (MKK6) has previously been shown to play important roles in plant immunity and cytokinesis. Here we report that it is also involved in the negative regulation of anthocyanin biosynthesis in Arabidopsis. Elevated anthocyanin levels are observed in mkk6 knock out mutant plants. RNA-sequencing analysis revealed that the expression level of MYB75 (Myb transcription factor 75), which encodes a key positive regulator of anthocyanin biosynthesis, is up-regulated in mkk6 mutant plants, suggesting that increased MYB75 expression contributes to the elevated anthocyanin levels. In addition, suppression of high sucrose-induced anthocyanin production by pathogen associated molecular pattern-triggered immunity is also dependent on MKK6, further supporting that MKK6 is a key negative regulator of anthocyanin biosynthesis in Arabidopsis.

Background: The matrine-type alkaloids are bioactive components extracted from Sophora flavescens , which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine-type alkaloids on advanced glycation end products-induced reactive oxygen species-mediated endothelial apoptosis., Methods and Results: Rats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine-type alkaloids, p38 mitogen-activated protein kinase (MAPK) inhibitor, and small interference RNAs against p38 MAPK kinases MAPK kinase kinase (MKK)3 and MKK6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKKs/p38 MAPK, and expression levels of heme oxygenase/NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2-related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2-related factor 2 with antioxidant response element were also evaluated. Matrine-type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK3/6 and p38 MAPK, nuclear factor erythroid 2-related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine-type alkaloids in vivo and in vitro. MKK3/6 silencing impaired the effects of matrine-type alkaloids in vitro., Conclusions: Matrine-type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species-mediated apoptosis by targeting MKK3/6 and enhancing their phosphorylation., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Cancer cells often have faulty apoptotic pathways resulting in sustenance of survivability, tumour metastasis and resistance to anticancer drugs. Alternate strategies are sought to improve therapeutic efficacy and therefore HepG2 cells were treated with S-allyl-cysteine (SAC) and berberine (BER) to analyze their mechanistic impact upon necroptosis along with its interacting relationship to apoptosis. In the present study we observed that SAC and BER exposure reduced NFκβ nuclear translocation through adenylate cyclase-cAMP-protein kinaseA axis and eventually evaded c-FLIP inhibition. Effective RIP1 k63-polyubiquitination and persistent MKK3/MKK6 expression during drug treatment potentiated caspase8 activity via p53-DISC conformation. Resultant tBid associated lysosomal protease mediated AIF truncation induced DNA fragmentation and persuaded effector caspase mediated scramblase activation resulting induction of necroptosis in parallel to apoptotic events. SAC+BER effectively reduced Rb-phosphorylation resulting insignificant nuclear E2F presence led to ending of cell proliferation. Therefore necroptosis augmented the drug response and may be targeted alongside cell proliferation inhibition in formation of efficient therapeutics against liver cancer.

Adequate responses to environmental stresses are essential for cell survival. The regulation of cellular energetics that involves mitochondrial energy production and oxidative stress is central in the process of stress adaptation and response. The p38α signalling pathway plays a key role in the response to stress stimuli by orchestrating multiple cellular processes. However, prolonged activation of the p38α pathway results in impaired cell proliferation and can lead to cell death. Here we use a system to specifically activate p38α signalling and show that sustained activation of this pathway suffices to induce important metabolic changes, including high dependence on glucose for cell survival, increased consumption of glutamine, enhanced respiration rate and elevated production of mitochondrial reactive oxygen species (ROS). Moreover, we provide evidence that increased production of mitochondrial superoxide as a consequence of elevated mitochondria activity, contributes to the p38α reduced cell survival triggered by sustained p38α activation. We also show that the p38α-activated kinase MAPKAPK2 (MK2) plays an important role orchestrating the observed metabolic changes. Our results illustrate a new function of p38α signalling in the regulation of cellular metabolism, which may lead to cell death upon persistent activation of the pathway.

Senescent cells may exert detrimental effect on microenvironment through the secretion of soluble factors and the release of extracellular vesicles, such as microparticles, key actors in ageing and cardiovascular diseases. We previously reported that sirtuin-1 (SIRT1) deficiency drives accelerated senescence and dysfunction of endothelial colony-forming cells (ECFC) in PT neonates. Because preterm birth (PT) increases the risk for cardiovascular diseases during neonatal period as well as at adulthood, we hypothesized that SIRT1 deficiency could control the biogenesis of microparticles as part of a senescence-associated secretory phenotype (SASP) of PT-ECFC and investigated the related molecular mechanisms. Compared to control ECFC, PT-ECFC displayed a SASP associated with increased release of endothelial microparticles (EMP), mediating a paracrine induction of senescence in naïve endothelial cells. SIRT1 level inversely correlated with EMP release and drives PT-ECFC vesiculation. Global transcriptomic analysis revealed changes in stress response pathways, specifically the MAPK pathway. We delineate a new epigenetic mechanism by which SIRT1 deficiency regulates MKK6/p38 MAPK /Hsp27 pathway to promote EMP biogenesis in senescent ECFC. These findings deepen our understanding of the role of ECFC senescence in the disruption of endothelial homeostasis and provide potential new targets towards the control of cardiovascular risk in individuals born preterm.

Background: Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Recent studies have revealed an association between autophagy and drug resistance., Methods: We previously synthesized a MAPK kinase-recombinant fusion protein, MAP2K6-FP, that contains 3 domains: a protein transduction domain TAT, a human ovarian cancer HO8910 cell-specific binding peptide, and a potential antitumor effector domain MKK6(E). In this study, we investigated the effect of MAP2K6-FP on HO8910 cells treated with paclitaxel., Results: The IC50 (concentration by which 50% cell growth was inhibited) was 20 μM for paclitaxel alone, 1.5 μg/mL for MAP2K6-FP alone, and 0.3 μg/mL for MAP2K6-FP and 15 μM for paclitaxel if combined, respectively. In addition, immunohistochemistry assay demonstrated that tumor tissues from ovarian cancer patients showed higher expression of LC-3, the autophagy-related protein, compared with normal ovarian tissues. MAP2K6-FP (0, 2.5, 5, 10, 20, and 40 μg/mL) dose-dependently increased the LC-3 expression in HO8910 cells. Immunofluorescence assay showed that paclitaxel alone increased the expression of LC-3 in HO8910 cells, which was further enhanced by the combination with MAP2K6-FP. Downregulation of LC-3 expression using LC-3 small interfering RNA inhibited the cytotoxicity effect of MAP2K6-FP. Furthermore, either MAP2K6-FP alone or in combination with paclitaxel increased the ratio of expressions of Beclin-1/Bcl-2, another autophagy-related markers, compared with paclitaxel alone., Conclusions: MAP2K6-FP enhanced the sensitiveness of paclitaxel for ovarian cancer via inducing autophagy.

Osteosarcoma (OS) is the most frequent primary bone tumor that affect children and adolescents. This tumor is highly aggressive with high risk of metastasis and the implementation of new drugs has not been successful. The search for biomarkers or new therapeutic targets is urgently needed and can help in advances of OS treatment. MAPKs are major signaling transduction molecules that play an important role in regulating a variety of cellular responses. DUSP1 is a phosphatase that dephosphorylates the MAPKs. Both MAPKs and DUSPs have been implicated as major modulators of critical signaling pathways that are dysregulated in various diseases. In a previous study, we found an increase in MAPK7 gene expression contributed for worst overall survival and treatment response. We analyzed gene expression of MAPK pathways that participate in MAPK7 regulation, and DUSP1 gene using paired 28 pre/post-chemotherapy and 12 metastasis OS samples. To understand the DUSP1 role in the pathogenesis of OS, we assessed the function of DUSP1 in four OS cell lines through a series of cellular assays combined with gene silencing technique. Our findings showed increased MAP2K6, MAP4K3, and DUSP1 gene expression in post-chemotherapy OS samples presenting poor prognosis. We also found that the suppression of DUSP1 gene expression resulted in decreased proliferation, migration, and invasion in OS cells. These results suggest that members of MAPK family may be possible prognostic markers in OS and DUSP1 has a relevant role in the OS pathogenesis and can be an attractive therapeutic target in new strategies of OS treatment., (© 2017 Wiley Periodicals, Inc.)

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses., (Copyright © 2017, American Association for the Advancement of Science.)

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra , which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E₂, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1β at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent., Competing Interests: The authors declare no conflict of interest.

Plants are continually exposed to numerous environmental stresses. To decrease damage caused by these potentially detrimental factors, various stress-related signaling cascades are activated in plants. One such stress-responsive signaling pathway, the mitogen-activated protein kinase (MAPK) module, plays a critical role in diverse plant stress responses. Here, we functionally characterized biotic and abiotic stress-responsive MAPK genes, IbMPK3 and IbMPK6, from sweetpotato. IbMPK3/6 contain totally 11 MAPK conserved subdomains and the phosphorylating motif TEY. Bacterially expressed IbMPK3/6 could be autophosphorylated in vitro, and these proteins phosphorylated universal kinase substrate, such as myelin basic protein. IbMPK3/6 transcripts were expressed in leaf, stem, and root of sweetpotato cultivars with storage roots of various colors. IbMPK3 and IbMPK6 were induced by various biotic/abiotic stress treatments. Furthermore, the kinase activity of IbMPK3/6 was induced during early NaCl, SA, H 2 O 2 , and ABA treatment. IbMPK3/6 were predominantly localized to the nucleus. To determine the biological functions of IbMPK3/6, we transiently expressed the IbMPK genes in tobacco (Nicotiana benthamiana) leaves, which resulted in enhanced tolerance to bacterial pathogen and increased expression of pathogenesis-related (PR) genes. These data demonstrate that IbMPK3 and IbMPK6 play significant roles in plant responses to environmental stress., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Oxaliplatin resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Herein, we show that miR-625-3p functionally induces oxaliplatin resistance in CRC cells, and identify the signalling networks affected by miR-625-3p. We show that the p38 MAPK activator MAP2K6 is a direct target of miR-625-3p, and, accordingly, is downregulated in non-responder patients of oxaliplatin therapy. miR-625-3p-mediated resistance is reversed by anti-miR-625-3p treatment and ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, reduction of p38 signalling by using siRNAs, chemical inhibitors or expression of a dominant-negative MAP2K6 protein induces resistance to oxaliplatin. Transcriptome, proteome and phosphoproteome profiles confirm inactivation of MAP2K6-p38 signalling as one likely mechanism of oxaliplatin resistance. Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38-regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

Cisplatin is a well-studied and widely used chemotherapeutic agent and is effective in the treatment of the advanced human non-small cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa and has been proved to have antioxidant and antitumor properties. XRCC1 is an important scaffold protein involved in base excision repair and plays an important role in the development of lung cancer. In this study, we characterize the role of curcumin in the cytotoxicity, p38 MAPK activation, and XRCC1 expression affected by cisplatin in NSCLC cells. We show that curcumin enhanced the cytotoxicity induced by cisplatin in two NSCLC cells, A549 and H1703. Treatment with cisplatin alone increased XRCC1 mRNA and protein expression through p38 MAPK activation. Moreover, SB2023580 (p38 inhibitor) decreased the XRCC1 mRNA and protein stability upon cisplatin treatment. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of p38 MAPK resulted in enhancing the cytotoxicity and cell growth inhibition induced by cisplatin. Curcumin inhibited the expression of XRCC1 in cisplatin-exposed NSCLC cells. Furthermore, transfection with constitutive active MKK6 or HA-p38 MAPK vectors rescued the XRCC1 protein level and also the cell survival suppressed by cisplatin and curcumin combination in A549 and H1703 cells. These findings suggested that the downregulation of XRCC1 expression by curcumin can enhance the chemosensitivity of cisplatin in NSCLC cells.

Since the 1940s, a proliferative gingival disease called hereditary hyperplastic gingivitis (HHG) has been described in the farmed silver fox, Vulpes vulpes (Dyrendahl and Henricson 1960). HHG displays an autosomal recessive transmission and has a pleiotropic relationship with superior fur quality in terms of length and thickness of guard hairs. An analogous human disease, hereditary gingival fibromatosis (HGF), is characterized by a predominantly autosomal dominant transmission and a complex etiology, occurring either as an isolated condition or as a part of a syndrome. Similar to HHG, the symptom most commonly associated with syndromic HGF is hypertrichosis. Here we explore potential mechanisms involved in HHG by comparison to known genetic information about hypertrichosis co-occurring with HGF, using an Affymetrix canine genome microarray platform, quantitative PCR, and candidate gene sequencing. We conclude that the mitogen-activated protein kinase pathway is involved in HHG, however despite involvement of the mitogen-activated protein kinase kinase 6 gene in congenital hypertrichosis with gingival fibromatosis in humans, this gene did not contain any fixed mutations in exons or exon-intron boundaries in HHG-affected foxes, suggesting that it is not causative of HHG in the farmed silver fox population. Differential up-regulation of MAP2K6 gene in HHG-affected foxes does implicate this gene in the HHG phenotype.

Testis determination in mammals is initiated by expression of SRY in somatic cells of the embryonic gonad. Genetic analyses in the mouse have revealed a requirement for mitogen-activated protein kinase (MAPK) signaling in testis determination: targeted loss of the kinases MAP3K4 and p38 MAPK causes complete XY embryonic gonadal sex reversal. These kinases occupy positions at the top and bottom level, respectively, in the canonical three-tier MAPK-signaling cascade: MAP3K, MAP2K, MAPK. To date, no role in sex determination has been attributed to a MAP2K, although such a function is predicted to exist. Here, we report roles for the kinases MAP2K3 and MAP2K6 in testis determination. C57BL/6J (B6) embryos lacking MAP2K3 exhibited no significant abnormalities of testis development, whilst those lacking MAP2K6 exhibited a minor delay in testis determination. Compound mutants lacking three out of four functional alleles at the two loci also exhibited delayed testis determination and transient ovotestis formation as a consequence, suggestive of partially redundant roles for these kinases in testis determination. Early lethality of double-knockout embryos precludes analysis of sexual development. To reveal their roles in testis determination more clearly, we generated Map2k mutant B6 embryos using a weaker Sry allele (Sry(AKR)). Loss of Map2k3 on this highly sensitized background exacerbates ovotestis development, whilst loss of Map2k6 results in complete XY gonadal sex reversal associated with reduction of Sry expression at 11.25 days postcoitum. Our data suggest that MAP2K6 functions in mouse testis determination, via positive effects on Sry, and also indicate a minor role for MAP2K3., (© 2016 by the Society for the Study of Reproduction, Inc.)

p38 mitogen-actived protein kinases (MAPKs) broadly exist from yeast to mammals and participate in diverse cellular responses to various stimuli, whose activation can be induced by the MAPK kinase 6 (MKK6). In this study, a novel MKK6 homolog from Litopenaeus vannamei (LvMKK6) was cloned and characterized. The transcript of LvMKK6 was 1465bp long with an open reading frame (ORF) of 987bp that encoded a polypeptide of 328 amino acids. LvMKK6 was a both cytoplasmic- and nuclear-localized protein and its expression was up-regulated with the treatment of different stimuli including LPS, Vibrio parahaemolyticus, Staphylococcus aureus, Poly (I:C) and white spot syndrome virus (WSSV). Overexpression of LvMKK6 could lead to activate the promoter activities of several antimicrobial peptides (AMPs) such as PEN4. The further investigation demonstrated that LvMKK6 could interact with and phosphorylate Lvp38, suggesting LvMKK6 was an activator of Lvp38. Knockdown of LvMKK6 caused attenuate expression of several AMPs and resulted in the higher mortality of shrimp under V. parahaemolyticus infection, suggesting LvMKK6 could play vital roles in defense against bacterial infection. Interestingly, silencing of LvMKK6 led to the lower virus loads and suppressed viral gene (VP28) expression during WSSV challenge. In addition, overexpression of LvMKK6 promoted the promoter activities of 19 WSSV immediate-early genes such as wsv069, wsv249, wsv108 and wsv403. These results suggested that LvMKK6 could be used by WSSV. Above all, these data provided experimental evidences that participation of LvMKK6 in regulating AMPs and host defense against bacteria, as well as the immune response to WSSV infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Biologically active peptides and proteins are novel agents that show promise in the development of anticancer drugs. Their relatively low cell permeability and poor tumor selectivity, however, impede their widespread applicability. In this study, we evaluated the tumor selectivity, cellular internalization, and biological activity of a cell-permeable ovarian cancer cell-specific therapeutic protein consisting of TAT-OSBP and constitutively active MKK6(E), an upstream kinase of the p38 signaling pathway that mediates cellular apoptosis. OSBP, a 7-amino-acid peptide with high affinity for human ovarian cancer HO8910 cells, was conjugated to the cell-penetrating peptide (TAT) to form a tumor-selective peptide (TAT-OSBP), which was further conjugated with EGFP or MKK6(E). Flow cytometry and fluorescent microscopy were performed to evaluate the tumor-targeted penetration of TAT-OSBP-EGFP. The inhibitory effects of TAT-OSBP-MKK6(E) were determined by cell proliferation and apoptosis assays. The internalization efficiency of TAT-OSBP-EGFP was significantly higher than that of TAT-EGFP. TAT-OSBP-EGFP selectively penetrated HO8910 cells. TAT-OSBP-MKK6(E) fusion protein inhibited cancer cell growth to varying degrees, with the highest level of inhibition in HO8910 cells. Moreover, TAT-OSBP-MKK6(E) significantly induced apoptosis of HO8910 cells. However, there was no significant difference in apoptosis in the normal ovarian epithelial cells treated with either TAT-OSBP-MKK6(E) or TAT-MKK6(E). Our results demonstrate that TAT-OSBP-MKK6(E) is a novel artificially designed molecule, which induces apoptosis and selectively targets human ovarian carcinoma HO8910 cells. Our study provides novel insights that may aid in the development of a new generation of anticancer drugs.

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Defects of long-range regulatory elements of dosage-sensitive genes represent an under-recognized mechanism underlying genetic diseases. Haploinsufficiency of SOX9, the gene essential for development of testes and differentiation of chondrocytes, results in campomelic dysplasia, a skeletal malformation syndrome often associated with sex reversal. Chromosomal rearrangements with breakpoints mapping up to 1.6 Mb up- and downstream to SOX9, and disrupting its distant cis-regulatory elements, have been described in patients with milder forms of campomelic dysplasia, Pierre Robin sequence, and sex reversal. We present an ∼1.58 Mb deletion mapping ∼1.28 Mb upstream to SOX9 that encompasses its putative long-range cis-regulatory element(s) and MAP2K6 in a patient with Pierre Robin sequence and osteopenia with multiple fractures. Low bone mass panel testing using massively parallel sequencing of 23 nuclear genes, including COL1A1 and COL1A2 was negative. Based on the previous mouse model of Map2k6, suggesting that Sox9 is likely a downstream target of the p38 MAPK pathway, and our previous chromosome conformation capture-on-chip (4C) data showing potential interactions between SOX9 promoter and MAP2K6, we hypothesize that deletion of MAP2K6 might have affected SOX9 expression and contributed to our patient's phenotype., (© 2015 Wiley Periodicals, Inc.)

Objective: To observe the influence of silent information regulator of transcription 1 (SIRT1) level by regulation of MKK6 over-expression in esophageal cancer cell Eca109, and then to explore the relationship between p38-MAPK-SIRT1 axis and progressing of esophageal cancer., Methods: MKK6 over-expression vector was successfully constructed firstly. Then the divided Eca109 cells were treated according to four groups: empty vector group, MKK6 over-xpression group (MKK6 group), MKK6-SIRT1 ShRNA group, MKK6-RES group. The expression of MKK6 and endogenous SIRT1 were tested by Western blot; cell proliferation capability was detected by MTT method; cell invasion force was observed by transwell method; and cell apoptosis was detected with flow cytometry., Results: pcDNA3.1 (+)/myc-His A-MKK6 over-expression vector was constructed successfully and proved by sequencing. MKK6's over-expressing could reduce the expression of endogenous SIRT1. The viability of Eca109 cells was decreased. The increasing of invasion and apoptosis was observed., Conclusion: There might be the p38-MAPK-SIRT1 regulation axis in Eca109 cells and affecting on a series of physiological characteristics of Eca109 cells.

To improve the selectivity of TAT-fusion proteins for targeted cancer therapy, we developed a novel TAT-based target-specific fusion protein, TAT-OSBP-1-MKK6(E), and evaluated its selectivity and anti-tumor activity in vitro and in vivo. The fusion protein containing TAT-OSBP-1-MKK6(E) has three functional domains: (1) the protein transduction domain of TAT, (2) the human ovarian cancer HO8910 cell-specific binding peptide (OSBP-1) and (3) the potential anti-tumor effector domain of MKK6(E). The transduction efficiency, selectivity, cytotoxicity and apoptotic effect of TAT-OSBP-1-MKK6(E) were examined using immunofluorescence, CCK8 assay and flow cytometry. The in vivo anti-tumor efficacy and target specificity of the fusion protein were evaluated using a nude mouse model with subcutaneous xenografts of human ovarian cancer HO8910 cells. Tumor-bearing mice were divided into three treatment groups that received tail vein injections of TAT-OSBP-1-MKK6(E), TAT-OSBP-1 or normal saline. Tumor growth inhibition was determined by tumor volume, weight and morphology. The distribution and apoptotic effect of TAT-OSBP-1-MKK6(E) were assessed by immunohistochemical staining and TUNEL assays. TAT-OSBP-1-MKK6(E) can be selectively internalized into human ovarian cancer HO8910 cells, rather than normal ovarian OSE cells. In vivo, the fusion protein was mainly expressed in the tumor xenograft, but not in ovary or liver tissues. As a result, TAT-OSBP-1-MKK6(E) significantly induced growth inhibition and apoptosis of tumor cells in vitro and in vivo, with limited effects in normal cells and tissues. TAT-OSBP-1-MKK6(E) treatment can selectively target HO8910 cells in vitro and in vivo, leading to growth inhibition and apoptosis of tumor cells. As such, TAT-OSBP-1-MKK6(E) may be a potential approach for ovarian cancer target therapy.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)