Abstract P448: Role For Btg1 And Btg2 In Postnatal Cardiomyocyte Cell Cycle Arrest And Maturation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Nivedhitha Velayutham ◽  
Frank N van Leeuwen ◽  
Blanca Scheijen ◽  
Katherine E Yutzey
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Mitotic Activity ◽  
Mitotic Arrest ◽  
Neonatal Rat ◽  
Heart Weight ◽  
Cell Cycle Regulators ◽  
Double Knockout ◽  
Cross Sectional ◽  
Cycle Arrest

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

scholarly journals Hypoxia-Inducible Factor 1α Is Essential for Cell Cycle Arrest during Hypoxia

2003 ◽  
Vol 23 (1) ◽  
pp. 359-369 ◽  
Author(s):  
Nobuhito Goda ◽  
Heather E. Ryan ◽  
Bahram Khadivi ◽  
Wayne McNulty ◽  
Robert C. Rickert ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Cycle Arrest ◽  
Cellular Response ◽  
Hypoxia Inducible Factor ◽  
Growth Arrest ◽  
Cell Types ◽  
Low Oxygen ◽  
Double Knockout ◽  
Cycle Arrest

ABSTRACT A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1α gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1α influenced p53, we also created double-knockout (HIF-1α null, p53 null) strains and cells. In both cell types, loss of HIF-1α abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1α genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1α causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1α-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1α dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.

Platelet Factor 4 (PF4) Causes Cell Cycle Arrest in Megakaryocytes (Megs) by Inactivating CDC2 (CDK1) and CDK2

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1238-1238
Author(s):  
Liqing Xiao ◽  
Mortimer Poncz ◽  
Michele Lambert
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Platelet Count ◽  
Steady State ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Cell Surface Receptor ◽  
Brdu Incorporation ◽  
Cell Cycle Regulators ◽  
Cycle Arrest

Abstract Abstract 1238 PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A microRNA program controls the transition of cardiomyocyte hyperplasia to hypertrophy and stimulates mammalian cardiac regeneration

2021 ◽  
Author(s):  
Andrea Raso ◽  
Ellen Dirkx ◽  
Vasco Sampaio-Pinto ◽  
Hamid el Azzouzi ◽  
Ryan J. Cubero ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gene Delivery ◽  
Cell Cycle Arrest ◽  
Regulatory Function ◽  
Viral Gene ◽  
Postnatal Life ◽  
Mouse Heart ◽  
Cell Cycle Regulators ◽  
Induce Cell Cycle Arrest ◽  
Cycle Arrest

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b∼25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b∼25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b∼25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b∼25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b∼25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

HSP27 regulates p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac H9c2 cells: p21 upregulation and G2/M phase cell cycle arrest

2008 ◽  
Vol 294 (4) ◽  
pp. H1736-H1744 ◽  
Author(s):  
C. D. Venkatakrishnan ◽  
Kathy Dunsmore ◽  
Hector Wong ◽  
Sashwathi Roy ◽  
Chandan K. Sen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Heat Shock ◽  
Cell Cycle Arrest ◽  
Transcriptional Activity ◽  
Neonatal Rat ◽  
Phase Cell ◽  
H9c2 Cells ◽  
Cycle Arrest ◽  
M Phase

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-1+/+) and HSF-1-knockout (HSF-1−/−) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-α (PFT-α) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-1+/+ cells, PFT-α attenuated Dox-induced toxicity. However, in HSF-1−/− cells (which express a very low level of HSP27 compared with HSF-1+/+ cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1+/+ and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G2/M phase cell cycle arrest in Dox-treated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death.

Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovaryThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 621-628 ◽  
Author(s):  
Xu-Xia Sui ◽  
Li-Li Luo ◽  
Jin-Jie Xu ◽  
Yu-Cai Fu
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Immunofluorescent Staining ◽  
Oocyte Nucleus ◽  
Caspase 8 ◽  
Double Labeling ◽  
Cycle Arrest ◽  
Old Rats

Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a.

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