scholarly journals P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Elena Alcalde-Estévez ◽  
Ana Asenjo-Bueno ◽  
Patricia Sosa ◽  
Patricia Plaza ◽  
Diego Rodríguez-Puyol ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Muscle Regeneration ◽  
Uremic Toxins ◽  
C2c12 Cells ◽  
Cyclin B ◽  
Skeletal Muscle Regeneration ◽  
Regeneration Process ◽  
Cycle Arrest ◽  
Advanced Stages ◽  
And Function

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

scholarly journals Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Michael E. O’Brien ◽  
James Londino ◽  
Marcus McGinnis ◽  
Nathaniel Weathington ◽  
Jessica Adair ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Cell Cycle Progression ◽  
Myogenic Differentiation ◽  
Core Promoter ◽  
Skeletal Muscle Regeneration ◽  
Skeletal Myogenesis ◽  
Necrosis Factor Alpha ◽  
Tnf Α

ABSTRACT FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp −160 to +42 within the proximal 5′ flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine. IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

scholarly journals Distinct Phosphoisoforms of the XenopusMcm4 Protein Regulate the Function of the Mcm Complex

2000 ◽  
Vol 20 (10) ◽  
pp. 3667-3676 ◽  
Author(s):  
Inna Pereverzeva ◽  
Elizabeth Whitmire ◽  
Bettina Khan ◽  
Martine Coué
Keyword(s):  
Cell Cycle ◽  
Cyclin B ◽  
Detailed Characterization ◽  
Cyclin Dependent Kinases ◽  
Origins Of Replication ◽  
Mcm Complex ◽  
Initiation Of Dna Replication ◽  
And Function ◽  
Mitotic Phosphorylation

ABSTRACT Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.

scholarly journals IL-4 and SDF-1 Increase Adipose Tissue-Derived Stromal Cell Ability to Improve Rat Skeletal Muscle Regeneration

2020 ◽  
Vol 21 (9) ◽  
pp. 3302
Author(s):  
Małgorzata Zimowska ◽  
Karolina Archacka ◽  
Edyta Brzoska ◽  
Joanna Bem ◽  
Areta M. Czerwinska ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Muscle Regeneration ◽  
Structure And Function ◽  
Skeletal Muscle Regeneration ◽  
Stem Cell Markers ◽  
Myogenic Factors ◽  
And Function

Skeletal muscle regeneration depends on the satellite cells, which, in response to injury, activate, proliferate, and reconstruct damaged tissue. However, under certain conditions, such as large injuries or myopathies, these cells might not sufficiently support repair. Thus, other cell populations, among them adipose tissue-derived stromal cells (ADSCs), are tested as a tool to improve regeneration. Importantly, the pro-regenerative action of such cells could be improved by various factors. In the current study, we tested whether IL-4 and SDF-1 could improve the ability of ADSCs to support the regeneration of rat skeletal muscles. We compared their effect at properly regenerating fast-twitch EDL and poorly regenerating slow-twitch soleus. To this end, ADSCs subjected to IL-4 and SDF-1 were analyzed in vitro and also in vivo after their transplantation into injured muscles. We tested their proliferation rate, migration, expression of stem cell markers and myogenic factors, their ability to fuse with myoblasts, as well as their impact on the mass, structure and function of regenerating muscles. As a result, we showed that cytokine-pretreated ADSCs had a beneficial effect in the regeneration process. Their presence resulted in improved muscle structure and function, as well as decreased fibrosis development and a modulated immune response.

scholarly journals Modulation of Caspase Activity Regulates Skeletal Muscle Regeneration and Function in Response to Vasopressin and Tumor Necrosis Factor

PLoS ONE ◽  
2009 ◽  
Vol 4 (5) ◽  
pp. e5570 ◽  
Author(s):  
Viviana Moresi ◽  
Gisela Garcia-Alvarez ◽  
Alessandro Pristerà ◽  
Emanuele Rizzuto ◽  
Maria C. Albertini ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tumor Necrosis Factor ◽  
Muscle Regeneration ◽  
Tumor Necrosis ◽  
Skeletal Muscle Regeneration ◽  
Caspase Activity ◽  
And Function ◽  
Necrosis Factor

Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1317-1317
Author(s):  
Jonathan K. Alder ◽  
Robert W. Georgantas ◽  
Richard L. Hildreth ◽  
Xiaobing Yu ◽  
Curt I. Civin
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fetal Liver ◽  
Family Members ◽  
Hematopoietic Cells ◽  
Normal Numbers ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Klf4 Expression ◽  
And Function

Abstract Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.

scholarly journals Interferon Regulatory Factor 6 Promotes Cell Cycle Arrest and Is Regulated by the Proteasome in a Cell Cycle-Dependent Manner

2008 ◽  
Vol 28 (7) ◽  
pp. 2235-2243 ◽  
Author(s):  
Caleb M. Bailey ◽  
Daniel E. Abbott ◽  
Naira V. Margaryan ◽  
Zhila Khalkhali-Ellis ◽  
Mary J. C. Hendrix
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Cell Cycle Arrest ◽  
Interferon Regulatory Factor ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Regulatory Factor ◽  
Cycle Arrest ◽  
Interferon Regulatory Factor 6 ◽  
And Function

ABSTRACT Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G0 phase of the cell cycle.

scholarly journals Foxk1 recruits the Sds3 complex and represses gene expression in myogenic progenitors

2012 ◽  
Vol 446 (3) ◽  
pp. 349-357 ◽  
Author(s):  
Xiaozhong Shi ◽  
David C. Seldin ◽  
Daniel J. Garry
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Muscle Regeneration ◽  
Cell Cycle Progression ◽  
Adaptor Protein ◽  
Skeletal Muscle Regeneration ◽  
Cycle Progression ◽  
Repression Complex

Previous studies have established that Foxk1 (forkhead box k1) plays an important role in skeletal muscle regeneration. Foxk1 regulates the cell-cycle progression of myogenic progenitors by repressing the cell-cycle inhibitor gene p21. However, the underlying mechanism is not well understood. In the present study, we report the identification of Sds3 (suppressor of defective silencing 3) as an adaptor protein that recruits the Sin3 [SWI (switch)-independent 3]–HDAC (histone deacetylase) repression complex and binds Foxk1. Using GST (glutathione transferase) pull-down assays, we defined the interaction between the Foxk1 FHA (forkhead-associated domain) domain and phospho-Thr49 in Sds3. We demonstrated that the transcriptional repression of Foxk1 is dependent on the Sin3–Sds3 repression complex, and knockdown of Sds3 results in cell-cycle arrest. We further identified the protein kinase CK2 as the protein kinase for Sds3 Thr49 and demonstrated that the protein kinase activity of CK2 is required for proper cell-cycle progression. Analysis of CK2 mutant mice reveals perturbation of skeletal muscle regeneration due to the dysregulation of cell-cycle kinetics. Overall, these studies define a CK2–Sds3–Foxk1 cascade that modulates gene expression and regulates skeletal muscle regeneration.

scholarly journals ARHGEF3 regulates skeletal muscle regeneration and strength through autophagy

2020 ◽  
Author(s):  
Jae-Sung You ◽  
Nilmani Singh ◽  
Adriana Reyes-Ordonez ◽  
Nidhi Khanna ◽  
Zehua Bao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Mammalian Target Of Rapamycin ◽  
Akt Signaling ◽  
Skeletal Muscle Regeneration ◽  
Myoblast Differentiation ◽  
Independent Manner ◽  
And Function ◽  
Old Mice

SummarySkeletal muscle regeneration is essential for restoring muscle function upon injury and for the maintenance of muscle health with aging. ARHGEF3, a Rho-specific GEF, negatively regulates myoblast differentiation via mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling in a GEF-independent manner in vitro. Here, we investigated ARHGEF3’s role in skeletal muscle regeneration by creating ARHGEF3 KO mice. These mice exhibited no discernible phenotype under normal conditions. Upon injury, however, ARHGEF3 deficiency enhanced the mass, fiber size and function of regenerating muscles in both young and aged mice. Surprisingly, these effects were not mediated by mTORC2-Akt signaling, but by the GEF activity of ARHGEF3. Furthermore, ARHGEF3 KO promoted muscle regeneration through activation of autophagy, a process that is also critical for maintaining muscle strength. Accordingly, in old mice, ARHGEF3 depletion prevented muscle weakness by restoring autophagy flux. Collectively, our findings identify an unexpected link between ARHGEF3 and autophagy-related muscle pathophysiology.

scholarly journals Cell Cycle Dysregulation by Human Cytomegalovirus: Influence of the Cell Cycle Phase at the Time of Infection and Effects on Cyclin Transcription

1998 ◽  
Vol 72 (5) ◽  
pp. 3729-3741 ◽  
Author(s):  
Bryan S. Salvant ◽  
Elizabeth A. Fortunato ◽  
Deborah H. Spector
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Hcmv Infection ◽  
Cyclin A ◽  
S Phase ◽  
Cyclin B ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Time Of Infection

ABSTRACT Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G0, G1, and S). In all cases, infection led to cell cycle arrest. Cells infected in G0 or G1phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G0, G1, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.

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