scholarly journals In vitro and in vivo roles of glucocorticoid and vitamin D receptors in the control of cardiomyocyte proliferative potential

2019 ◽  
Author(s):  
Stephen Cutie ◽  
Dominic Lunn ◽  
Guo N. Huang
Keyword(s):  
Cell Cycle ◽  
Vitamin D ◽  
Cardiac Injury ◽  
Dominant Negative ◽  
Mutant Mice ◽  
Proliferative Potential ◽  
Vitamin D Receptors ◽  
Receptor Alpha

ABSTRACTCardiomyocyte (CM) proliferative potential varies considerably across species. While lower vertebrates and neonatal mammals retain robust capacities for CM proliferation, adult mammalian CMs lose proliferative potential due to cell-cycle withdrawal and polyploidization, failing to mount a proliferative response to regenerate lost CMs after cardiac injury. The decline of murine CM proliferative potential occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extrauterine life. We recently demonstrated that thyroid hormone (TH) signaling functions as a primary factor driving CM proliferative potential loss in vertebrates. Whether other hormonal pathways govern this process remains largely unexplored. Here we showed that agonists of glucocorticoid receptor (GR) and vitamin D receptor (VDR) suppressed neonatal CM proliferation in vitro. We next examined CM nucleation and proliferation in mutant mice lacking GR or VDR specifically in CMs, but we observed no difference between mutant and control littermates. Additionally, we generated compound mutant mice that lack GR or VDR and express dominant-negative TH receptor alpha in their CMs, and similarly observed no increase in CM proliferative potential compared to dominant-negative TH receptor alpha mice alone. Thus, although GR and VDR activation in cultured CMs is sufficient to inhibit CM proliferation, they seem to be dispensable for CM cell-cycle exit and binucleation in vivo. In addition, given the recent report that VDR activation in zebrafish promotes CM proliferation and tissue regeneration, our results suggest distinct roles of VDR in zebrafish and rodent CM cell-cycle regulation.

scholarly journals Epimers of Vitamin D: A Review

2020 ◽  
Vol 21 (2) ◽  
pp. 470 ◽  
Author(s):  
Bashar Al-Zohily ◽  
Asma Al-Menhali ◽  
Salah Gariballa ◽  
Afrozul Haq ◽  
Iltaf Shah
Keyword(s):  
Vitamin D ◽  
Biological Activity ◽  
Hydroxyl Group ◽  
Vitamin D Metabolites ◽  
Dihydroxyvitamin D3 ◽  
In Vivo Models ◽  
Vitamin D Receptors ◽  
Potential Factors

In this review, we discuss the sources, formation, metabolism, function, biological activity, and potency of C3-epimers (epimers of vitamin D). We also determine the role of epimerase in vitamin D-binding protein (DBP) and vitamin D receptors (VDR) according to different subcellular localizations. The importance of C3 epimerization and the metabolic pathway of vitamin D at the hydroxyl group have recently been recognized. Here, the hydroxyl group at the C3 position is orientated differently from the alpha to beta orientation in space. However, the details of this epimerization pathway are not yet clearly understood. Even the gene encoding for the enzyme involved in epimerization has not yet been identified. Many published research articles have illustrated the biological activity of C3 epimeric metabolites using an in vitro model, but the studies on in vivo models are substantially inadequate. The metabolic stability of 3-epi-1α,25(OH)2D3 has been demonstrated to be higher than its primary metabolites. 3-epi-1 alpha, 25 dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3) is thought to have fewer calcemic effects than non-epimeric forms of vitamin D. Some researchers have observed a larger proportion of total vitamin D as C3-epimers in infants than in adults. Insufficient levels of vitamin D were found in mothers and their newborns when the epimers were not included in the measurement of vitamin D. Oral supplementation of vitamin D has also been found to potentially cause increased production of epimers in mice but not humans. Moreover, routine vitamin D blood tests for healthy adults will not be significantly affected by epimeric interference using LC–MS/MS assays. Recent genetic models also show that the genetic determinants and the potential factors of C3-epimers differ from those of non-C3-epimers.Most commercial immunoassays techniques can lead to inaccurate vitamin D results due to epimeric interference, especially in infants and pregnant women. It is also known that the LC–MS/MS technique can chromatographically separate epimeric and isobaric interference and detect vitamin D metabolites sensitively and accurately. Unfortunately, many labs around the world do not take into account the interference caused by epimers. In this review, various methods and techniques for the analysis of C3-epimers are also discussed. The authors believe that C3-epimers may have an important role to play in clinical research, and further research is warranted.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Vitamin D receptors and anti-proliferative effects of vitamin D derivatives in human pancreatic carcinoma cells in vivo and in vitro

1997 ◽  
Vol 76 (8) ◽  
pp. 1017-1020 ◽  
Author(s):  
KW Colston ◽  
SY James ◽  
EA Ofori-Kuragu ◽  
L Binderup ◽  
AG Grant
Keyword(s):  
Vitamin D ◽  
Pancreatic Carcinoma ◽  
Carcinoma Cells ◽  
Vitamin D Receptors

scholarly journals The effects of purified recombinant murine interleukin-3 and/or purified natural murine CSF-1 in vivo on the proliferation of murine high- and low-proliferative potential colony-forming cells: demonstration of in vivo synergism

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 401-403
Author(s):  
DE Williams ◽  
G Hangoc ◽  
S Cooper ◽  
HS Boswell ◽  
RK Shadduck ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Active Cell ◽  
Proliferative Potential ◽  
Interleukin 3 ◽  
Low Concentrations ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Purified natural murine L cell (macrophage) colony-stimulating factor (nCSF-1) and purified recombinant murine interleukin-3 (rIL-3) were administered to normal or lactoferrin-pretreated mice 20 to 24 hours before sacrifice. rIL-3 and nCSF-1 administered separately increased the percentage of macrophage high-proliferative potential colony- forming cells (HPP-CFC) and low-proliferative potential colony-forming cells (LPP-CFC) in active cell cycle. Endotoxin was not detected in the samples of nCSF-1 or rIL-3 with the Limulus lysate test, and the in vitro and in vivo hematopoietic stimulatory effects of both molecules were abolished or markedly reduced by 30 minutes' treatment at 100 degrees C, which demonstrates that the effects noted in vivo were not due to endotoxin. Combinations of low concentrations of rIL-3 and nCSF- 1, which by themselves were inactive, increased the percentage of HPP- CFC and LPP-CFC in active cell cycle in a synergistic fashion. No significant change in the number of HPP-CFC or LPP-CFC per femur or femoral nucleated cellularity was observed. Thus, rIL-3 and nCSF-1 can synergize to effect the proliferation of the same cell populations in vivo.

Involvement of P-TEFb/CDK9 in Cooperative Transcriptional Activation of Megakaryocytic Genes by GATA-1 and RUNX1 and in Programming of Megakaryocytic Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1227-1227
Author(s):  
Kamaleldin E. Elagib ◽  
Ivailo S. Mihaylov ◽  
Lorrie L. Delehanty ◽  
Grant C. Bullock ◽  
Kevin D. Ouma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Program Analysis ◽  
Dominant Negative ◽  
In Vivo Model ◽  
Megakaryocytic Differentiation ◽  
Human Cd34 ◽  
Unique Cell

Abstract Programming of megakaryocytic differentiation requires precise coordination of multiple signal transduction and transcription pathways. Previous in vivo and in vitro studies have implicated RUNX1 and GATA-1 as transcription factors that collaborate in the execution of this program. Analysis of the mechanism for the synergy of these two factors revealed induction of RUNX1 hyperphosphorylation by GATA-1 coexpression. A pharmacologic screen identified roscovitine as an inhibitor of the transcriptional cooperation, implicating a cyclin-dependent kinase (Cdk). A screen employing a panel of dominant-negative Cdk mutants identified Cdk9 as a critical component of the GATA-1-RUNX1 cooperation. In addition, HEXIM1, an endogenous Cdk9 inhibitor, similarly blocked transcriptional synergy. Furthermore, two kinase inhibitory compounds, DRB and flavopiridol, also blocked GATA-1-RUNX1 cooperation at concentrations specific for Cdk9 inhibition. Regarding the mechanism for GATA-1 induction of RUNX1 phosphorylation, coimmunoprecipitation experiments showed GATA-1 binding to both Cdk9 and cyclinT1. To examine the role of P-TEFb in primary megakaryocytic differentiation, human CD34+ cells in megakaryocytic cultures underwent treatment with 50 nM flavopiridol, a dose selective for Cdk9 inhibition. This treatment blocked megakaryocytic polyploidization while having no effect on the cell cycle properties of the non-megakaryocytic cells in the cultures. The treatment also impaired upregulation of CD41. Extending these findings to an in vivo model system, mice underwent treatment with daily low dose flavopiridol (5–7 mg/kg/day), a regimen previously shown to have no toxicity. Wild type C57BL/6 (wt BL/6) mice were compared with the ΔneoΔHS strain (GATA-1Lo) which has diminished GATA-1 expression in megakaryocytes. After only 1 week of treatment, the GATA-1Lo mice developed worsening thrombocytopenia associated with new-onset anemia, with several dying after 2 weeks of treatment. Flow cytometry on marrow from the treated GATA-1Lo mice revealed a marked expansion of abnormal megakaryocytes showing coexpression of CD71 plus CD41 and loss of polyploidization. Marrow and spleen histology showed extensive replacement by immature-appearing megakaryocytes with hypolobulated nuclei, as well as frequent pyknotic megakaryocytes. The control mice, flavopiridol treated wt BL/6 and saline treated GATA-1Lo, displayed none of these abnormalities. Additional experiments determined the flavopiridol effect on the GATA-1Lo mice to be completely reversible, with normalization of all parameters 2 weeks after ending treatment. In aggregate, these data implicate P-TEFb recruitment by GATA-1 in mediating cooperative activation of megakaryocytic promoters with RUNX1. This pathway may depend in part on the direct phosphorylation of RUNX1 by Cdk9. In mice, a synthetic lethal relationship between megakaryocytic GATA-1 deficiency and Cdk9 inhibition exists, manifesting as a fulminant but reversible megakaryocytic proliferative disorder reminiscent of the Down syndrome-associated megakaryocyte proliferations. A model is proposed in which P-TEFb, as a component of GATA-1-RUNX1 transcriptional complexes, plays an integral role in the specific programming of megakaryocytic differentiation, with particular importance in the unique cell cycle changes associated with this lineage.

scholarly journals Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

1994 ◽  
Vol 14 (8) ◽  
pp. 5501-5509 ◽  
Author(s):  
D Finley ◽  
S Sadis ◽  
B P Monia ◽  
P Boucher ◽  
D J Ecker ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Turnover ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Sole Source ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Polyubiquitin Gene

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

scholarly journals Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL- 6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 30-37 ◽  
Author(s):  
SO Peters ◽  
EL Kittler ◽  
HS Ramshaw ◽  
PJ Quesenberry
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Factor ◽  
Proliferative Potential ◽  
Interleukin 3 ◽  
Marrow Cells

Abstract In vitro incubation of bone marrow cells with cytokines has been used as an approach to expand stem cells and to facilitate retroviral integration. Expansion of hematopoietic progenitor cells has been monitored by different in vitro assays and in a few instances by in vivo marrow renewal in myeloablated hosts. This is the first report of studies, using two competitive transplant models, in which cytokine-treated cells, obtained from nonpretreated donors (eg, 5-fluorouracil), were competed with normal cells. A basic assumption is that the expansion of progenitors assayed in vitro as high- and low-proliferative potential colony-forming cells (HPP- and LPP-CFCs) indicates an expansion of stem cells which will repopulate in vivo. This study shows that culture of marrow cells with four cytokines (stem cell factor, interleukin-3 [IL-3], IL-6, IL-11) induces significant expansion and proliferation of HPP-CFC and LPP-CFC. Cell-cycle analysis showed that these hematopoietic progenitors were induced to actively cell cycle by culture with these cytokines. In the first competitive transplant model, which uses Ly5.2/Ly5.1 congenic mice, cytokine-cultured Ly5.2 cells competed with noncultured Ly5.1 cells led to 5% +/- 1% engraftment at 12 weeks and to 4% +/- 2% engraftment at 22 weeks posttransplantation for the cytokine exposed cells. Noncultured Ly5.2 cells competed with cultured Ly5.1 cells led to 70% +/- 1% engraftment at 12 weeks and to 93% +/- 2% engraftment at 22 weeks posttransplantation. In the second model, which uses BALB/c marrow of opposite genders, cultured male cells lead to 13% +/- 9% engraftment at 10 weeks and 2% +/- 1% engraftment at 14 weeks posttransplantation; noncultured male cells lead to 70% +/- 2% and 95% +/- 2% engraftment at 10 and 14 weeks posttransplantation, respectively. Data presented here from two different competitive transplant studies show a defect of cytokine expanded marrow related to cell cycle activation which manifests as defective long-term repopulating capability in irradiated host mice. The engraftment defect is more profound at longer time intervals, suggesting that the most striking effect may be on long-term repopulating cells.

scholarly journals Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

1999 ◽  
Vol 10 (11) ◽  
pp. 3927-3941 ◽  
Author(s):  
Holger Bastians ◽  
Leana M. Topper ◽  
Gary L. Gorbsky ◽  
Joan V. Ruderman
Keyword(s):  
Cell Cycle ◽  
Metaphase Plate ◽  
S Phase ◽  
Dominant Negative ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

scholarly journals Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53

2000 ◽  
Vol 14 (3) ◽  
pp. 278-288 ◽  
Author(s):  
Nabil H. Chehab ◽  
Asra Malikzay ◽  
Michael Appel ◽  
Thanos D. Halazonetis
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Tumor Suppressor Protein

Chk2/hcds1, the human homolog of theSaccharomyces cerevisiae RAD53/SPK1 andSchizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G2 in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.

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