Identification of mitogen-activated protein kinase 7 inhibitors from natural products: Combined virtual screening and dynamic simulation studies.

Mitogen-activated protein kinase 7 (MAPK7) is a serine/threonine protein kinase that belongs to the MAPK family and plays a vital role in various cellular processes such as cell proliferation, differentiation, gene transcription, apoptosis, metabolism, and cell survival. The elevated expression of MAPK7 has been associated with the onset and progression of multiple aggressive tumors in humans, underscoring the potential of targeting MAPK7 pathways in therapeutic research. This pursuit holds promise for the advancement of anticancer drug development by developing potential MAPK7 inhibitors. To look for potential MAPK7 inhibitors, we exploited structure-based virtual screening of natural products from the ZINC database. First, the Lipinski rule of five criteria was used to filter a large library of ~90,000 natural compounds, followed by ADMET and pan-assay interference compounds (PAINS) filters. Then, top hits were chosen based on their strong binding affinity as determined by molecular docking. Further, interaction analysis was performed to find effective and specific compounds that can precisely bind to the binding pocket of MAPK7. Consequently, two compounds, ZINC12296700 and ZINC02123081, exhibited significant binding affinity and demonstrated excellent drug-like properties. All-atom molecular dynamics simulations for 200 ns confirmed the stability of MAPK7-ZINC12296700 and MAPK7-ZINC02123081 docked complexes. According to the molecular mechanics Poisson-Boltzmann surface area investigation, the binding affinities of both complexes were considerable. Overall, the result suggests that ZINC12296700 and ZINC02123081 might be used as promising leads to develop novel MAPK7 inhibitors. Since these compounds would interfere with the kinase activity of MAPK7, therefore, may be implemented to control cell growth and proliferation in cancer after required validations.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Pathophysiological Impact of the MEK5/ERK5 Pathway in Oxidative Stress.

Oxidative stress regulates many physiological and pathological processes. Indeed, a low increase in the basal level of reactive oxygen species (ROS) is essential for various cellular functions, including signal transduction, gene expression, cell survival or death, as well as antioxidant capacity. However, if the amount of generated ROS overcomes the antioxidant capacity, excessive ROS results in cellular dysfunctions as a consequence of damage to cellular components, including DNA, lipids and proteins, and may eventually lead to cell death or carcinogenesis. Both in vitro and in vivo investigations have shown that activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway is frequently involved in oxidative stress-elicited effects. In particular, accumulating evidence identified a prominent role of this pathway in the anti-oxidative response. In this respect, activation of krüppel-like factor 2/4 and nuclear factor erythroid 2-related factor 2 emerged among the most frequent events in ERK5-mediated response to oxidative stress. This review summarizes what is known about the role of the MEK5/ERK5 pathway in the response to oxidative stress in pathophysiological contexts within the cardiovascular, respiratory, lymphohematopoietic, urinary and central nervous systems. The possible beneficial or detrimental effects exerted by the MEK5/ERK5 pathway in the above systems are also discussed.

Extracellular signal-Regulated Kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity.

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFß-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

Acute pharmacological degradation of ERK5 does not inhibit cellular immune response or proliferation.

Recent interest in the role that extracellular signal-regulated kinase 5 (ERK5) plays in various diseases, particularly cancer and inflammation, has grown. Phenotypes observed from genetic knockdown or deletion of ERK5 suggested that targeting ERK5 could have therapeutic potential in various disease settings, motivating the development ATP-competitive ERK5 inhibitors. However, these inhibitors were unable to recapitulate the effects of genetic loss of ERK5, suggesting that ERK5 may have key kinase-independent roles. To investigate potential non-catalytic functions of ERK5, we report the development of INY-06-061, a potent and selective heterobifunctional degrader of ERK5. In contrast to results reported through genetic knockdown of ERK5, INY-06-061-induced ERK5 degradation did not induce anti-proliferative effects in multiple cancer cell lines or suppress inflammatory responses in primary endothelial cells. Thus, we developed and characterized a chemical tool useful for validating phenotypes reported to be associated with genetic ERK5 ablation and for guiding future ERK5-directed drug discovery efforts.

Overexpression of TRAF4 promotes lung cancer growth and EGFR-dependent phosphorylation of ERK5.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is overexpressed in a variety of carcinomas of different origins, but its role in tumorigenesis remains incompletely understood. Previous studies suggest that TRAF4 promotes epidermal growth factor receptor (EGFR) activation in non-small cell lung cancer (NSCLC). However, the downstream signaling pathway of TRAF4-mediated EGFR activation, as well as its effects on tumor cells, have not been fully elucidated. Here we report that TRAF4 overexpression is associated with increased activity of extracellular signal-regulated kinase 5 (ERK5) in NSCLC tissues. Activation of ERK5 was dependent on TRAF4-mediated EGFR activation, since inhibition of either TRAF4 or EGFR dramatically abolished phosphorylation of ERK5. Mechanistically, EGFR recruited mitogen-activated protein kinase kinase kinase 3 (MEKK3), an upstream kinase of ERK5, in a TRAF4-dependent manner. Thus, our data suggest that an EGFR-TRAF4-MEKK3-ERK5 axis promotes the proliferation of tumor cells, and this may be a potential target for therapeutic intervention of NSCLC.

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.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

The Hedgehog-GLI Pathway Regulates MEK5-ERK5 Expression and Activation in Melanoma Cells.

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.

[Phosphorylation of Smurf2 at Thr249 by Erk5 regulates TGF-ß signaling].

Vertebral bone and limb bone are formed by endochondral ossification, which is replaced with bone tissue by osteoblasts after cartilage formation. Bone growth is regulated by the balance between epiphyseal chondrocyte proliferation and ossification. We attempted to elucidate the mechanism of chondrocyte differentiation and maturation regulated by the Extracellular-signal-regulated kinase 5 (Erk5) signal. Erk5 is a serine/threonine kinase belonging to the mitogen-activated protein kinase (MAPK) family, which includes Erk1/2, JNK, and p38. Mesenchymal stem cell-specific Erk5-deficient mice exhibited the phenotype of deformities of the metatarsal bones, enlargement of the long bones in limbs, and overgrowth of cartilage tissue. Based on this result, we searched for factors that directly phosphorylate Erk5, and We demonstrated that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249 to activate its function and promotes ubiquitination-mediated degradation. The TGF-ß-Smad signal suppresses the proliferation of many cells and regulates the production of extracellular matrix. Our findings may lead to the development of novel drugs targeting TGF-ß associated diseases. In this paper, we investigated the function of Smurf2Thr249 phosphorylation and the possibility as new therapeutic target for various diseases.

circBTBD7 Promotes Immature Porcine Sertoli Cell Growth through Modulating miR-24-3p/MAPK7 Axis to Inactivate p38 MAPK Signaling Pathway.

Sertoli cells are the crucial coordinators to guarantee normal spermatogenesis and male fertility. Although circular RNAs (circRNAs) exhibit developmental-stage-specific expression in porcine testicular tissues and have been thought of as potential regulatory molecules in spermatogenesis, their functions and mechanisms of action remain largely unknown, especially in domestic animals. A novel circBTBD7 was identified from immature porcine Sertoli cells using reverse transcription PCR, Sanger sequencing, and fluorescence in situ hybridization assays. Functional assays illustrated that circBTBD7 overexpression promoted cell cycle progression and cell proliferation, as well as inhibited cell apoptosis in immature porcine Sertoli cells. Mechanistically, circBTBD7 acted as a sponge for the miR-24-3p and further facilitated its target mitogen-activated protein kinase 7 (MAPK7) gene. Overexpression of miR-24-3p impeded cell proliferation and induced cell apoptosis, which further attenuated the effects of circBTBD7 overexpression. siRNA-induced MAPK7 deficiency resulted in a similar effect to miR-24-3p overexpression, and further offset the effects of miR-24-3p inhibition. Both miR-24-3p overexpression and MAPK7 knockdown upregulated the p38 phosphorylation activity. The SB202190 induced the inhibition of p38 MAPK pathway and caused an opposite effect to that of miR-24-3p overexpression and MAPK7 knockdown. Collectively, circBTBD7 promotes immature porcine Sertoli cell growth through modulating the miR-24-3p/MAPK7 axis to inactivate the p38 MAPK signaling pathway. This study expanded our knowledge of noncoding RNAs in porcine normal spermatogenesis through deciding the fate of Sertoli cells.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.

The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion.

There is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior.

Extracellular signal-regulated kinases 2 (Erk2) and Erk5 in the central nervous system differentially contribute to central sensitization in male mice.

Nervous systems are designed to become extra sensitive to afferent nociceptive stimuli under certain circumstances such as inflammation and nerve injury. How pain hypersensitivity comes about is key issue in the field since it ultimately results in chronic pain. Central sensitization represents enhanced pain sensitivity due to increased neural signaling within the central nervous system (CNS). Particularly, much evidence indicates that underlying mechanism of central sensitization is associated with the change of spinal neurons. Extracellular signal-regulated kinases have received attention as key molecules in central sensitization. Previously, we revealed the isoform-specific function of extracellular signal-regulated kinase 2 (Erk2) in spinal neurons for central sensitization using mice with Cre-loxP-mediated deletion of Erk2 in the CNS. Still, how extracellular signal-regulated kinase 5 (Erk5) in spinal neurons contributes to central sensitization has not been directly tested, nor is the functional relevance of Erk5 and Erk2 known. Here, we show that Erk5 and Erk2 in the CNS play redundant and/or distinct roles in central sensitization, depending on the plasticity context (cell types, pain types, time, etc.). We used male mice with Erk5 deletion specifically in the CNS and found that Erk5 plays important roles in central sensitization in a formalin-induced inflammatory pain model. Deletion of both Erk2 and Erk5 leads to greater attenuation of central sensitization in this model, compared to deletion of either isoform alone. Conversely, Erk2 but not Erk5 plays important roles in central sensitization in neuropathic pain, a type of chronic pain caused by nerve damage. Our results suggest the elaborate mechanisms of Erk signaling in central sensitization.

Isoflurane post-conditioning contributes to anti-apoptotic effect after cerebral ischaemia in rats through the ERK5/MEF2D signaling pathway.

The mechanisms of brain protection during ischaemic reperfusion injury induced by isoflurane (ISO) post-conditioning are unclear. Myocyte enhancement factor 2 (MEF2D) has been shown to promote neural survival in a variety of models, in which multiple survival and death signals converge on MEF2D and modulate its activity. Here, we investigated the effect of MEF2D on the neuroprotective effects of ISO post-conditioning on rats after cerebral ischaemia/reperfusion (I/R) injury. Rats underwent middle cerebral artery occlusion (MCAO) surgery with ischaemia for 90 minutes and reperfusion for 24-48 hours. After MCAO, neurological status was assessed at 12, 24 and 48 hours by the Modified Neurological Severity Score (mNSS) test. The passive avoidance test (PAT) was used to assess cognition function. Histological and neuropathological evaluations were performed with HE staining and Nissl's staining, respectively. We measured the expression of MEF2D, ERK5, GFAP and caspase-3 by immunofluorescent staining and Western blotting, and TUNEL staining to assess the severity of apoptosis in hippocampal CA1 area. We found that MEF2D was involved in nerve protection after I/R injury, and post-treatment of ISO significantly promoted the phosphorylation of ERK5, increased MEF2D transcriptional activity, inhibited the expression of caspase-3 and played a role of brain protection.

Chimeric RNA ASTN2-PAPPAas aggravates tumor progression and metastasis in human esophageal cancer.

Transcription-induced chimeric RNAs are an emerging area of research into molecular signatures for disease biomarker and therapeutic target development. Despite their importance, little is known for chimeric RNAs-relevant roles and the underlying mechanisms for cancer pathogenesis and progression. Here we describe a unique ASTN2-PAPPAantisense chimeric RNA (A-PaschiRNA) that could be the first reported chimeric RNA derived from the splicing of exons and intron antisense of two neighboring genes, respectively. Aberrant A-PaschiRNA level in ESCC tissues was associated with tumor progression and patients' outcome. In vitro and in vivo studies demonstrated that A-PaschiRNA aggravated ESCC metastasis and enhanced stemness through modulating OCT4. Mechanistic studies demonstrated that ERK5-mediated non-canonical PAF1 activity was required for A-PaschiRNA-induced cancer malignancy. The study defined an undocumented function of chimeric RNAs in aggravating cancer stemness and metastasis.

ERK5 negatively regulates Kruppel-like factor 4 and promotes osteogenic lineage cell proliferation in response to MEK5 overexpression or fluid shear stress.

Aim of the study: Fluid shear stress (FSS) plays a critical role in osteoblast proliferation via extracellular signal-regulated kinase 5 (ERK5). Kruppel-like factor 4 (KLF4) knockout robustly enhances bone formation due to increased osteoblast differentiation and mineralization. However, the effect of KLF4 on osteoblast proliferation is unresolved. Therefore, the aim of our study was to investigate the effect of KLF4 on osteogenic lineage cell proliferation and the relationship between KLF4 and ERK5. Materials and methods: MC3T3-E1 cells were treated with FSS and/or KLF4 siRNA, cell viability was accessed by Edu labeling and CCK-8 assay, and proliferative gene expression were assessed by PCR array. Bone marrow stromal cells (BMSCs) were infected with adenovirus expressing KLF4 and/or constitutively active MEK5, cell viability was evaluated using crystal violet staining, colony formation assay, and cell WST1 assay. The levels of KLF4 and ERK5 phosphorylation were identified through qRT-PCR and western blot, respectively. Results: KLF4 expression was significantly down-regulated by FSS exposure, however, this was reversed by ERK5 siRNA. KLF4 overexpression inhibited colony formation efficiency and cell viability in BMSCs. Adenoviruses expressing constitutively active MEK5 increased ERK5 phosphorylation, which inhibited KLF4 expression, and promoted BMSC proliferation. FSS-induced osteoblast proliferation also involved elevation of Cyclin B2 and Cdc14b as well as repressed expression of P27. Conclusions: KLF4 negatively regulates osteogenic lineage cell proliferation, and ERK5 negatively regulates KLF4 expression and promotes osteogenic lineage cell proliferation.

MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance.

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

Inducible and conditional activation of ERK5 MAP kinase rescues mice from cadmium-induced olfactory memory deficits.

Cadmium (Cd) is a heavy metal that is one of the most toxic environmental pollutants throughout the world. We previously reported that Cd exposure impairs olfactory memory in mice. However, the underlying mechanisms for its neurotoxicity for olfactory function are not well understood. Since adult Subventricular zone (SVZ) and Olfactory Bulb (OB) neurogenesis contributes to olfaction, olfactory memory defects caused by Cd may be due to inhibition of neurogenesis. In this study, using bromodeoxyuridine (BrdU) labeling and immunohistochemistry, we found that 0.6 mg/L Cd exposure through drinking water impaired adult SVZ/OB neurogenesis in C57BL/6 mice. To determine if the inhibition of olfactory memory by Cd can be reversed by stimulating adult neurogenesis, we utilized the transgenic caMEK5 mouse strain to conditional stimulate of adult neurogenesis by activating the endogenous ERK5 MAP kinase signaling pathway. This was accomplished by conditionally induced expression of active MEK5 (caMEK5) in adult neural stem/progenitor cells. The caMEK5 mice were exposed to 0.6 mg/L Cd for 38 weeks, and tamoxifen was administered to induce caMEK5 expression and stimulate adult SVZ/OB neurogenesis during Cd exposure. Short-term olfactory memory test and sand-digging based, odor-cued olfactory learning and memory test were conducted after Cd and tamoxifen treatments to examine their effects on olfaction. Here we report that Cd exposure impaired short-term olfactory memory and odor-cued associative learning and memory in mice. Furthermore, the Cd-impaired olfactory memory deficits were rescued by the tamoxifen-induction of caMEK5 expression. This suggests that Cd exposure impairs olfactory function by affecting adult SVZ/OB neurogenesis in mice.