MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal

MEK/ERK signaling plays a crucial role in a diverse set of cellular functions including cell proliferation, differentiation and survival, and recently has been reported to negatively regulate mouse embryonic stem cell (mESC) self-renewal by antagonizing STAT3 activity. However, its role in human ESCs (hESCs) remains unclear. Here we investigated the functions of MEK/ERK in controlling hESC activity. We demonstrated that MEK/ERK kinases were targets of fibroblast growth factor (FGF) pathway in hESCs. Surprisingly, we found that, in contrast to mESCs, high basal MEK/ERK activity was required for maintaining hESCs in an undifferentiated state. Inhibition of MEK/ERK activity by specific MEK inhibitors PD98059 and U0126, or by RNA interference, rapidly caused the loss of self-renewal capacity. We also showed that MEK/ERK signaling cooperated with phosphoinositide 3-kinase (PI3K)/AKT signaling in maintaining hESC pluripotency. However, MEK/ERK signaling had little or no effect on regulating hESC proliferation and survival, in contrast to PI3K/AKT signaling. Taken together, these findings reveal the unique and crucial role of MEK/ERK signaling in the determination of hESC cell fate and expand our understanding of the molecular mechanisms behind the FGF pathway maintenance of hESC pluripotency. Importantly, these data make evident the striking differences in the control of self-renewal between hESCs and mESCs.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

Cannabinoids inhibit ethanol-induced activation of liver toxicity in rats through JNK/ERK/MAPK signaling pathways

Cannabinoids (CBs) are psychoactive compounds, with reported anticancer, anti-inflammatory, and anti-neoplastic properties. The study was aimed at assessing the hepatoprotective effects of CB against ethanol (EtOH)-induced liver toxicity in rats. The animals were divided into seven groups: control (Group I) and Group II were treated with 50% ethanol (EtOH 5 mg/kg). Groups III, IV, and VI were treated with (EtOH + CB 10 mg/kg), (EtOH + CB 20 mg/kg), and (EtOH + CB 30 mg/kg), respectively. Groups V and VII consisted of animals treated with 20 and 30 mg/kg, of CB, respectively. Biochemical analysis revealed that Group IV (EtOH + CB 20 mg/kg) had reduced levels of ALT—alanine transferase, AST—aspartate aminotransferase, ALP—alanine peroxidase, MDA—malondialdehyde and increased levels of GSH-reduced glutathione. Histopathological analysis of liver and kidney tissues showed that EtOH + CB (20 and 30 mg/kg) treated animal groups exhibited normal tissue architecture similar to that of the control group. ELISA revealed that the inflammatory markers were reduced in the animal groups that were treated with EtOH + CB 20 mg/kg, in comparison to the animals treated only with EtOH. The mRNA expression levels of COX-2, CD-14, and MIP-2 showed a remarkable decrease in EtOH + CB treated animal groups to control groups. Western blot analysis revealed that CB downregulated p38/JNK/ERK thereby exhibiting its hepatoprotective property by inhibiting mitogen-activated protein kinase pathways. Thus, our findings suggest that CB is a potential candidate for the treatment of alcohol-induced hepatotoxicity.     

Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.     

Primordial germ cell development in zebrafish

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. These cells follow a characteristic developmental path that is manifested in specialized regulation of basic cell functions and behavior making them an attractive system for studying cell fate specification, differentiation and migration. This review summarizes studies aimed at understanding the development of this cell population in zebrafish and compares these results with those obtained in other model organisms.     

Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines

The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.     

Stromal cell derived factor-1 acutely promotes neural progenitor cell proliferation in vitro by a mechanism involving the ERK1/2 and PI-3K signal pathways

Stromal cell derived factor-1 (SDF-1), a member of the chemotactic cytokine family, has attracted attention in recent years. It participates in diverse processes such as the regulation of neuronal migration and activation of CD4+ T cells; it is also a co-receptor for human immunodeficiency virus-1 (HIV-1). Here, we show that the proliferation of neural progenitor cells dissociated from rat cortex and cultured in vitro with basic fibroblast growth factor (bFGF) is stimulated by SDF-1. PD98059 and wortmannin, which are, respectively, specific inhibitors of the extracellular regulated kinase1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI-3K) signal pathways, markedly attenuate this stimulation of proliferation. These findings indicate that SDF-1 acutely promotes the proliferation of NPCs in vitro involving the ERK1/2 and PI-3 kinase pathways, suggesting that it plays a basic role in the development of neural progenitors.     

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our fi nding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

Cooperative action of ADMP- and BMP-mediated pathways in regulating cell fates in the zebrafish gastrula.

It was shown in Xenopus and chick that Spemann's organizer activity is regulated through the negative action of Anti-Dorsalizing Morphogenetic Protein (ADMP). We report the characterization and functional properties of admp in zebrafish. admp expression profile is consistent with a role in the organizer, including the tail organizer. We studied admp function through overexpression experiments, with the use of a dominant-negative form (TR-ADMP) and of an antisense morpholino-modified oligonucleotide. Our results indicate that the ADMP pathway causes the restriction of anterior and axial fates and that ADMP, BMP2b, and BMP7 pathways have distinct actions but cooperate in establishing proper dorso-ventral regionalization. This is shown by partial rescue of the dorsalized mutant snailhouse and of the ventralized mutant chordino, upon admp and tr-admp RNA injection, respectively. Moreover, ADMP and BMP7 probably form heterodimers as shown by the ability of TR-ADMP and BMP7 to antagonize each other. We observed that a MYC-tagged ADMP was secreted and detected in the extracellular space, suggesting that admp could act at a distance. Simultaneous local inhibition of bmp function at the blastoderm margin and impairment of ADMP secretion led to the induction of secondary head structures, confirming that the two pathways cooperatively regulate organizer formation and activity.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

The chitin catabolic cascade in the marine bacterium Vibrio cholerae: characterization of a unique chitin oligosaccharide deacetylase

Chitin, one of the most abundant organic substances in nature, is consumed by marine bacteria, such as Vibrio cholerae, via a multitude of tightly regulated genes (Li and Roseman 2004, Proc Natl Acad Sci USA. 101:627-631). One such gene, cod, is reported here. It encodes a chitin oligosaccharide deacetylase (COD), when cells are induced by chitobiose, (GlcNH(2))(2), or crude crab shells. COD was molecularly cloned (COD-6His), overproduced, and purified to apparent homogeneity. COD is secreted at all stages of growth by induced V. cholerae. The gene sequence predicts a 26 N-terminal amino acid signal peptide not found in the isolated protein. COD is very active with chitin oligosaccharides, is virtually inactive with GlcNAc, and slightly active with colloidal ([(3)H]-N-acetyl)-chitin. The oligosaccharides are converted almost quantitatively to products lacking one acetyl group. The latter were characterized by mass spectrometry (ESI-MS), and treatment with nitrous acid. COD catalyzes the following reactions (n = 2-6): (GlcNAc)(n)--> GlcNAc-GlcNH(2)-(GlcNAc)(n-2) + Ac(-). That is, COD hydrolyzes the N-acetyl groups attached to the penultimate GlcNAc residue. The gene bank sequence data show that cod is highly conserved in Vibrios and Photobacteria. One such gene encodes a deacetylase isolated from V. alginolytics (Ohishi et al. 1997, Biosci Biotech Biochem. 61:1113-1117; Ohishi et al. 2000, J Biosci Bioeng. 90:561-563), that is specific for (GlcNAc)(2), but inactive with higher oligosaccharides. The COD enzymatic products, GlcNAc-GlcNH(2)-(GlcNAc)(n), closely resemble those obtained by hydrolysis of the chitooligosaccharides with Nod B: GlcNH(2)-(GlcNAc)(3-4). The latter are key intermediates in the biosynthesis of Nod factors, critically important in communications between the symbiotic nitrogen fixing bacteria and plants. Conceivably, the COD products play equally important roles in cellular communications that remain to be defined.     

Autophagy and mTOR pathways in mouse embryonic stem cell,lung cancer and somatic fibroblast cell lines

Embryonic developmental stages and regulations have always been one of the most intriguing aspects of science. Since the cancer stem cell discovery, striking for cancer development and recurrence, embryonic stem cells and control mechanisms, as well as cancer cells and cancer stem cell control mechanisms become important research materials. It is necessary to reveal the similarities and differences between somatic and cancer cells which are formed of embryonic stem cells divisions and determinations. For this purpose, mouse embryonic stem cells (mESCs), mouse skin fibroblast cells (MSFs) and mouse lung squamous cancer cells (SqLCCs) were grown in vitro and the differences between these three cell lines signalling regulations of mechanistic target of rapamycin (mTOR) and autophagic pathways were demonstrated by immunofluorescence and real-time polymerase chain reaction. Expressional differences were clearly shown between embryonic, cancer and somatic cells that mESCs displayed higher expressional level of Atg10, Hdac1 and Cln3 which are related with autophagic regulation and Hsp4, Prkca, Rhoa and ribosomal S6 genes related with mTOR activity. LC3 and mTOR protein levels were lower in mESCs than MSFs. Thus, the mechanisms of embryonic stem cell regulation results in the formation of somatic tissues whereas that these cells may be the causative agents of cancer in any deterioration.     

Down-regulation of ERK1 and ERK2 activity during differentiation of the intestinal cell line HT-29

The role and regulation of signal transduction pathways in proliferation and differentiation of intestinal epithelial cells are still poorly understood. However, growing evidences have been recently accumulated demostrating that mitogen-activated protein kinases (MAPKs) play a pivotal function in the normal development of intestine. We have investigated, in the intestinal cell line HT-29, the regulation (namely activity and phosphorylation degree) of MAP kinases ERK 1 (p44) and ERK 2 (p42) during differentiation. Addition of fetal calf serum to HT-29 undifferentiated resting cells caused a rapid phosphorylation of both ERKs and an increase of their specific kinase activity. Moreover, nuclear translocation of ERK 1 and ERK 2 occurred concurrently to their activation, leading to the conclusion that ERK 1 and ERK 2 are classically regulated when quiescent HT-29 cells are induced to proliferate. Butyrate addition to the intestinal cell line resulted in terminal differentiation and in a selective down-regulation of ERK 2 activity (and phosphorylation degree) without any effect on ERK 1. Conversely, when HT-29 cells were differentiated by repeated passages in a glucose-free medium, we observed a progressive dephosphorylation and inactivation of p42 and p44 kinases along with the failure of serum to activate both the enzymes. Our findings suggest that, during the differentiation of intestinal cells, remarkable changes occur in ERK 1 and ERK 2 control mechanisms leading to an unresponsivness of MAP kinase pathway.     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.