scholarly journals Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2

2011 ◽  
Vol 22 (16) ◽  
pp. 2946-2956 ◽  
Author(s):  
Vittoria Cenni ◽  
Alberto Bavelloni ◽  
Francesca Beretti ◽  
Francesca Tagliavini ◽  
Lucia Manzoli ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Muscle Cells ◽  
Protein Characterization ◽  
Stress Conditions ◽  
Kinase Assay ◽  
In Vivo Studies ◽  
Specific Substrate ◽  
Mutant Form ◽  
Response To Stress

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

scholarly journals Multifaceted effects of soluble human CD6 in experimental cancer models

2020 ◽  
Vol 8 (1) ◽  
pp. e000172 ◽  
Author(s):  
Inês T Simões ◽  
Fernando Aranda ◽  
Sergi Casadó-Llombart ◽  
María Velasco-de Andrés ◽  
Cristina Català ◽  
...  
Keyword(s):  
T Cell ◽  
Cancer Cells ◽  
Ex Vivo ◽  
Lymphoid Organ ◽  
Protein Characterization ◽  
In Vivo Studies ◽  
Soluble Form ◽  
Cell Contact

BackgroundCD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor.MethodsHigh sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEμTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins.ResultsThrough a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells.ConclusionsEvidence is provided for the disruption of CD6 receptor–ligand interactions as a feasible immunomodulatory approach in cancer.

scholarly journals Heterozygous Disruption of the TATA-Binding Protein Gene in DT40 Cells Causes Reduced cdc25B Phosphatase Expression and Delayed Mitosis

2001 ◽  
Vol 21 (7) ◽  
pp. 2435-2448 ◽  
Author(s):  
Moonkyoung Um ◽  
Jun Yamauchi ◽  
Shigeaki Kato ◽  
James L. Manley
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
In Vivo Studies ◽  
Mutant Form ◽  
Lower Growth ◽  
Protein Levels ◽  
Species Specific ◽  
Dt40 Cells

ABSTRACT TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases. Although it has been intensively analyzed in vitro and inSaccharomyces cerevisiae, in vivo studies of vertebrate TBP have been limited. We applied gene-targeting techniques using chicken DT40 cells to generate heterozygous cells with one copy of theTBP gene disrupted. Such TBP-heterozygous (TBP-Het) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G2/-M- than G1-phase cells, and a high proportion of sub-G1, presumably apoptotic, cells. Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs. To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G2/M transition, and unexpectedly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells. Providing an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly and specifically reduced. These properties were all due to decreased TBP levels, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant form lacking the species-specific N-terminal domain. Our results indicate that small changes in TBP concentration can have profound effects on cell growth in vertebrate cells.

scholarly journals Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response

2005 ◽  
Vol 25 (3) ◽  
pp. 921-932 ◽  
Author(s):  
Jingshi Shen ◽  
Erik L. Snapp ◽  
Jennifer Lippincott-Schwartz ◽  
Ron Prywes
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Competitive Binding ◽  
Competition Model ◽  
Misfolded Proteins ◽  
Endoplasmic Reticulum Stress Response ◽  
Specific Substrate ◽  
Mutant Form ◽  
Dynamic Binding

ABSTRACT Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

scholarly journals A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Binyam Mogessie ◽  
Daniel Roth ◽  
Zainab Rahil ◽  
Anne Straube
Keyword(s):  
Cell Differentiation ◽  
Cell Fusion ◽  
Muscle Cell ◽  
Cell Elongation ◽  
Myogenic Differentiation ◽  
Muscle Cells ◽  
Microtubule Array ◽  
Muscle Cell Differentiation ◽  
Microtubule Transport

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes.

scholarly journals MEK1/2 in Rhabdomyosarcoma

2019 ◽  
Author(s):  
Kenneth A. Crawford ◽  
Megan M. Cleary ◽  
Cora A. Ricker ◽  
Matthew N. Svalina ◽  
John F. Shern ◽  
...  
Keyword(s):  
Cell Growth ◽  
Myogenic Differentiation ◽  
Synergistic Effects ◽  
Mek Inhibitor ◽  
Differentiation Therapy ◽  
Muscle Stem Cells ◽  
Response To Stress ◽  
High Content Analysis ◽  
Growth Evaluation

AbstractAlveolar and embryonal rhabdomyosarcoma (RMS) are soft-tissue cancers that affect children, adolescents, and young adults. Sometimes referred to as muscle cancer, RMS is a cancer of muscle and non-muscle origin that phenocopies incompletely differentiated myoblasts or activated satellite (muscle stem) cells. Interestingly, embryonal RMS (ERMS) has been observed to undergo terminal myogenic differentiation in response to stress induced by chemotherapy and radiation therapy4, 9, 24. Given the propensity of rhabdomyosarcoma to differentiation, in this report we explore the use of differentiation therapy combining MEK inhibitor (MEKi) cobimetinib and chemotherapy as a strategy to halt RMS growth. We evaluated a representative panel of RMS cell lines with cobimetinib and chemotherapy in two dosing schedules that mimic clinical use followed by cell growth evaluation and high content analysis (differentiation) assays. We uncovered that cobimetinib does not have significant additive or synergistic effects on cell differentiation or cell growth with chemotherapy in RMS and can have unanticipated antagonistic effects; specifically, pre-exposure of cobimetinib to cells can decrease the effectiveness of chemotherapy-mediated cell growth inhibition in vitro. Although differentiation-therapy is still a potential viable strategy in RMS, our data do not support MEKi/chemotherapy co-treatment in this context.

scholarly journals STK33/ERK2 signal pathway contribute the tumorigenesis of colorectal cancer HCT15 cells

2019 ◽  
Vol 19 ◽  
Author(s):  
Shengjun Zhang ◽  
Minli Liu ◽  
Haoyu Wu ◽  
Kaiyu Wang
Keyword(s):  
Colorectal Cancer ◽  
Ex Vivo ◽  
Kinase Assay ◽  
In Vivo Studies ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Upstream Kinase ◽  
Colorectal Cancer Patients

: Serine/threonine kinase 33 (STK33) is a serine/threonine kinase, and participates in many apoptotic process. Herein, we found that the extracellular signal-regulated kinase 2 (ERK2) was a substrate of STK33. STK33 phosphorylated ERK2and increased the activity of ERK2 and promote the tumorigenesis of colorectal cancer HCT15 cells. Clinical simple showed that STK33 was highly expression in colorectal cells and tissues. Ex vivo and in vivo studies demonstrated that STK33 accelerate tumorigenic properties in JB6C141 cells and athymic nude rats. In vitro kinase assay results indicated that STK33 can phosphorylate ERK2. Ex vivo studies further showed that STK33 can bind with ERK2 and take part in the regulation of ERKs signaling pathway. In short, our results showed that STK33 is a novel upstream kinase of ERK2. It may provide a better prospect for STK33 based prevention and treatment for colorectal cancer patients.

scholarly journals AChR β-Subunit mRNAs Are Stabilized by HuR in a Mouse Model of Congenital Myasthenic Syndrome With Acetylcholinesterase Deficiency

2020 ◽  
Vol 13 ◽  
Author(s):  
Jennifer Karmouch ◽  
Perrine Delers ◽  
Fannie Semprez ◽  
Nouha Soyed ◽  
Julie Areias ◽  
...  
Keyword(s):  
Rna Binding ◽  
Myogenic Differentiation ◽  
Muscle Cells ◽  
Synaptic Cleft ◽  
Congenital Myasthenic Syndrome ◽  
Β Subunit ◽  
Mrna Stabilization ◽  
Myasthenic Syndrome

Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR β-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR β-subunit transcripts. This HuR/AChR transcript interaction induces AChR β-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR β-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.

scholarly journals STK33/ERK2 signal pathway contribute the tumorigenesis of colorectal cancer HCT15 cells

2019 ◽  
Vol 39 (3) ◽  
Author(s):  
Shengjun Zhang ◽  
Haoyu Wu ◽  
Kaiyu Wang ◽  
Minli Liu
Keyword(s):  
Colorectal Cancer ◽  
Ex Vivo ◽  
Kinase Assay ◽  
In Vivo Studies ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Upstream Kinase ◽  
Colorectal Cancer Patients

AbstractSerine/threonine kinase 33 (STK33) is a serine/threonine kinase and participates in many apoptotic process. Herein, we found that the extracellular signal-regulated kinase 2 (ERK2) was a substrate of STK33. STK33 phosphorylated ERK2 and increased the activity of ERK2 and promote the tumorigenesis of colorectal cancer HCT15 cells. Clinical simple showed that STK33 was highly expression in colorectal cells and tissues. Ex vivo and in vivo studies demonstrated that STK33 accelerate tumorigenic properties in NCM460 cells and athymic nude rats. In vitro kinase assay results indicated that STK33 can phosphorylate ERK2. Ex vivo studies further showed that STK33 can bind with ERK2 and take part in the regulation of ERKs signaling pathway. In short, our results showed that STK33 is a novel upstream kinase of ERK2. It may provide a better prospect for STK33 based prevention and treatment for colorectal cancer patients.

scholarly journals HigB1 Toxin in Mycobacterium tuberculosis Is Upregulated During Stress and Required to Establish Infection in Guinea Pigs

2021 ◽  
Vol 12 ◽  
Author(s):  
Arun Sharma ◽  
Kalpana Sagar ◽  
Neeraj Kumar Chauhan ◽  
Balaji Venkataraman ◽  
Nidhi Gupta ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pigs ◽  
Physiological Role ◽  
Stress Conditions ◽  
Expression Of Genes ◽  
Response To Stress ◽  
Cumulative Evidence ◽  
Host Tissues

The extraordinary expansion of Toxin Antitoxin (TA) modules in the genome of Mycobacterium tuberculosis has received significant attention over the last few decades. The cumulative evidence suggests that TA systems are activated in response to stress conditions and are essential for M. tuberculosis pathogenesis. In M. tuberculosis, Rv1955-Rv1956-Rv1957 constitutes the only tripartite TAC (Toxin Antitoxin Chaperone) module. In this locus, Rv1955 (HigB1) encodes for the toxin and Rv1956 (HigA1) encodes for antitoxin. Rv1957 encodes for a SecB-like chaperone that regulates HigBA1 toxin antitoxin system by preventing HigA1 degradation. Here, we have investigated the physiological role of HigB1 toxin in stress adaptation and pathogenesis of Mycobacterium tuberculosis. qPCR studies revealed that higBA1 is upregulated in nutrient limiting conditions and upon exposure to levofloxacin. We also show that the promoter activity of higBA1 locus in M. tuberculosis is (p)ppGpp dependent. We observed that HigB1 locus is non-essential for M. tuberculosis growth under different stress conditions in vitro. However, guinea pigs infected with higB1 deletion strain exhibited significantly reduced bacterial loads and pathological damage in comparison to the animals infected with the parental strain. Transcriptome analysis suggested that deletion of higB1 reduced the expression of genes involved in virulence, detoxification and adaptation. The present study describes the role of higB1 toxin in M. tuberculosis physiology and highlights the importance of higBA1 locus during infection in host tissues.

Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells

2019 ◽  
Vol 40 (12) ◽  
pp. 803-809 ◽  
Author(s):  
Eva K. Langendorf ◽  
Anja Klein ◽  
Pol M. Rommens ◽  
Philipp Drees ◽  
Ulrike Ritz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cell Viability ◽  
Human Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
In Vivo Studies ◽  
Human Skeletal Muscle Cells ◽  
Regulated Gene Expression

AbstractThe use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

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