Induced neural progenitor cell-derived extracellular vesicles promote neural progenitor cell survival via extracellular signal-regulated kinase pathway.
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ABSTRACT:
SUBMITTER: Ma Y
PROVIDER: S-EPMC8611769 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neural stem/progenitor cells (NPCs) are known to have potent therapeutic effects in neurological disorders through the secretion of extracellular vesicles (EVs). Despite the therapeutic potentials, the numbers of NPCs are limited in the brain, curbing the further use of EVs in the disease treatment. To overcome the limitation of NPC numbers, we used a three transcription factor (Brn2, Sox2, and Foxg1) somatic reprogramming approach to generate induced NPCs (iNPCs) from mouse fibroblasts and astrocytes. The resulting iNPCs released significantly higher numbers of EVs compared with wild-type NPCs (WT-NPCs). Furthermore, iNPCs-derived EVs (iNPC-EVs) promoted NPC function by increasing the proliferative potentials of WT-NPCs. Characterizations of EV contents through proteomics analysis revealed that iNPC-EVs contained higher levels of growth factor-associated proteins that were predicted to activate the down-stream extracellular signal-regulated kinase (ERK) pathways. As expected, the proliferative effects of iNPC-derived EVs on WT-NPCs can be blocked by an ERK pathway inhibitor. Our data suggest potent therapeutic effects of iNPC-derived EVs through the promotion of NPC proliferation, release of growth factors, and activation of ERK pathways. These studies will help develop highly efficient cell-free therapeutic strategies for the treatment of neurological diseases.
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Project description:The extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase that phosphorylates and regulates various transcription factors in response to growth factors and extracellular stresses. To address its biological function during the development of the peripheral nervous system (PNS), we have engineered a novel model of sympathetic neurons in which the erk5 gene can be deleted in vitro. Our data provide for the first time genetic evidence that ERK5 is required to mediate the survival response of neurons to nerve growth factor. Increased cell death associated with the loss of ERK5 is caused by elevated expression of the BH3-only members of the Bcl-2 family, Bad and Bim. Further investigation indicated that ERK5 suppresses the transcription of the bad and the bim genes by Ca(2+)/cAMP response element-binding protein and Forkhead box O3a, respectively. Consistently, we found that the phosphorylation of both p90 ribosomal S6 kinase and protein kinase B is impaired in neurons lacking ERK5. Together these findings reveal a novel signaling mechanism that promotes neuronal survival during the development of the PNS.
| S-EPMC2670276 | biostudies-literature
Project description:Activation of the extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in mediating a diverse array of cellular functions including cell differentiation, proliferation, and inflammatory responses. In this review, we will discuss approaches to identify inhibitors of ERK proteins through targeting ATP-dependent and ATP-independent mechanisms. Given the diversity of ERK substrates and the importance of ERK signaling in normal cell functions, emphasis will be placed on the methods for identifying small molecular weight compounds that are substrate selective through ATP-independent interactions and potentially relevant to inflammatory processes. The approach for selective targeting of ERK substrates takes advantage of the basic understanding of unique ERK docking domains that are thought to interact with specific amino acid sequences on substrate proteins. Computer aided drug design (CADD) can facilitate the high throughput screening of millions of compounds with the potential for selective interactions with ERK docking domains and disruption of substrate interactions. As such, the CADD approach significantly reduces the number of compounds that will be evaluated in subsequent biological assays and greatly increases the hit rate of biologically active compounds. The potentially active compounds are evaluated for ERK protein binding using spectroscopic and structural biology methods. Compounds that show ERK interactions are then tested for their ability to inhibit substrate interactions and phosphorylation as well as ERK-dependent functions in whole organism or cell-based assays. Finally, the relevance of substrate-selective ERK inhibitors in the context of inflammatory disease will be discussed.
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Project description:Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.
| S-EPMC3268412 | biostudies-literature
Project description:In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.
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Project description:BackgroundGlioblastomas are lethal brain tumors under the current combinatorial therapeutic strategy that includes surgery, chemo- and radio-therapies. Extensive changes in the tumor microenvironment is a key reason for resistance to chemo- or radio-therapy and frequent tumor recurrences. Understanding the tumor-nontumor cell interaction in TME is critical for developing new therapy. Glioblastomas are known to recruit normal cells in their environs to sustain growth and encroachment into other regions. Neural progenitor cells (NPCs) have been noted to migrate towards the site of glioblastomas, however, the detailed mechanisms underlying glioblastoma-mediated NPCs' alteration remain unkown.MethodsWe collected EVs in the culture medium of three classic glioblastoma cell lines, U87 and A172 (male cell lines), and LN229 (female cell line). U87, A172, and LN229 were co-cultured with their corresponding EVs, respectively. Mouse NPCs (mNPCs) were co-cultured with glioblastoma-derived EVs. The proliferation and migration of tumor cells and mNPCs after EVs treatment were examined. Proteomic analysis and western blotting were utilized to identify the underlying mechanisms of glioblastoma-derived EVs-induced alterations in mNPCs.ResultsWe first show that glioblastoma cell lines U87-, A172-, and LN229-derived EVs were essential for glioblastoma cell prolifeartion and migration. We then demonstrated that glioblastoma-derived EVs dramatically promoted NPC proliferation and migration. Mechanistic studies identify that glioblastoma-derived EVs achieve their functions via activating PI3K-Akt-mTOR pathway in mNPCs. Inhibiting PI3K-Akt pathway reversed the elevated prolfieration and migration of glioblastoma-derived EVs-treated mNPCs.ConclusionOur findings demonstrate that EVs play a key role in intercellular communication in tumor microenvironment. Inhibition of the tumorgenic EVs-mediated PI3K-Akt-mTOR pathway activation might be a novel strategy to shed light on glioblastoma therapy. Video Abstract.
| S-EPMC8756733 | biostudies-literature
Project description:Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-?1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-?1.
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Project description:The ERK1/2 pathway is one of the most commonly dysregulated pathways in human cancers and controls many vital cellular processes. Although many ERK1/2 kinase substrates have been identified, the diversity of ERK1/2 mediated processes suggests the existence of additional targets. Here, we identified Deoxyhypusine synthase (DHPS), an essential hypusination enzyme regulating protein translation, as a major and direct-binding protein of ERK1/2. Further experiments showed that ERK1/2 phosphorylate DHPS at Ser-233 site. The Ser-233 phosphorylation of DHPS by ERK1/2 is important for its function in cell proliferation. Moreover, we found that higher DHPS expression correlated with poor prognosis in lung adenocarcinoma and increased resistance to inhibitors of the ERK1/2 pathway. In summary, our results suggest that ERK1/2-mediated DHPS phosphorylation is an important mechanism that underlies protein translation and that DHPS expression is a potent biomarker of response to therapies targeting ERK1/2-pathway.
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Project description:The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.
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