scholarly journals Unexpected maturation of PI3K and MAPK-ERK signaling in fetal ovine cardiomyocytes

2014 ◽  
Vol 307 (8) ◽  
pp. H1216-H1225 ◽  
Author(s):  
N. N. Chattergoon ◽  
S. Louey ◽  
P. J. Stork ◽  
G. D. Giraud ◽  
K. L. Thornburg
Keyword(s):  
Cell Cycle ◽  
Intracellular Signaling ◽  
Cardiac Development ◽  
Hormonal Control ◽  
Binucleated Cells ◽  
Age Related ◽  
Fetal Cardiomyocytes ◽  
Near Term ◽  
In Cells

In the first two-thirds of gestation, ovine fetal cardiomyocytes undergo mitosis to increase cardiac mass and accommodate fetal growth. Thereafter, some myocytes continue to proliferate while others mature and terminally differentiate into binucleated cells. At term (145 days gestational age; dGA) about 60% of cardiomyocytes become binucleated and exit the cell cycle under hormonal control. Rising thyroid hormone (T3) levels near term (135 dGA) inhibit proliferation and stimulate maturation. However, the degree to which intracellular signaling patterns change with age in response to T3 is unknown. We hypothesized that in vitro activation of ERK, Akt, and p70S6K by two regulators of cardiomyocyte cell cycle activity, T3 and insulin like growth factor-1 (IGF-1), would be similar in cardiomyocytes at gestational ages 100 and 135 dGA. IGF-1 and T3 each independently stimulated phosphorylation of ERK, Akt, and p70S6K in cells at both ages. In the younger mononucleated myocytes, the phosphorylation of ERK and Akt was reduced in the presence of IGF-1 and T3. However, the same hormone combination led to a dramatic twofold increase in the phosphorylation of these signaling proteins in the 135 dGA cardiomyocytes—even in cells that were not proliferating. In the older cells, both mono- and binucleated cells were affected. In conclusion, fetal ovine cardiomyocytes undergo profound maturation-related changes in signaling in response to T3 and IGF-1, but not to either factor alone. Differences in age-related response are likely to be related to milestones in fetal cardiac development as the myocardium prepares for ex utero life.

Apoptosis Susceptibility and Cell-Cycle Distribution in Cells from Myelodysplastic Syndrome Patients: Modulatory In-Vitro Effects of G-CSF and Interferon-α

2004 ◽  
Vol 45 (7) ◽  
pp. 1437-1443 ◽  
Author(s):  
Maria R Ricciardi ◽  
Maria T Petrucci ◽  
Chiara Gregorj ◽  
Vincenza Martini ◽  
Anna Levi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myelodysplastic Syndrome ◽  
Interferon Α ◽  
Cell Cycle Distribution ◽  
In Vitro Effects ◽  
In Cells

scholarly journals IQGAP1 binds AMPK and is required for maximum AMPK activation

2020 ◽  
pp. jbc.RA120.016193
Author(s):  
Andrew C. Hedman ◽  
Zhigang Li ◽  
Laëtitia Gorisse ◽  
Swetha Parvathaneni ◽  
Chase J. Morgan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Intracellular Signaling ◽  
Energy Homeostasis ◽  
Molecular Mechanisms ◽  
Direct Interaction ◽  
Mitogen Activated Protein Kinase ◽  
Null Cell ◽  
Ampk Signaling ◽  
In Cells

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals SCFFbxw5 targets MCAK in G2/M to facilitate ciliogenesis in the following cell cycle

2021 ◽  
Author(s):  
Jörg Schweiggert ◽  
Gregor Habeck ◽  
Sandra Hess ◽  
Felix Mikus ◽  
Klaus Meese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Primary Cilia ◽  
Protein Microarrays ◽  
Mitotic Progression ◽  
Cellular Processes ◽  
Ubiquitin E3 Ligase ◽  
M Phase ◽  
Cancer Types ◽  
In Cells

AbstractThe microtubule depolymerase Kif2C/MCAK plays important roles in various cellular processes and is frequently overexpressed in different cancer types. Despite the importance of its correct abundance, remarkably little is known about how MCAK levels are regulated in cells.Using comprehensive screening on protein microarrays, we identified 161 candidate substrates of the multi-subunit ubiquitin E3 ligase SCFFbxw5, including MCAK. In vitro reconstitution assays demonstrate that MCAK and its closely related orthologs Kif2A and Kif2B become efficiently polyubiquitylated by neddylated SCFFbxw5 and Cdc34, without requiring preceding modifications. In cells, SCFFbxw5 targets MCAK for proteasomal degradation specifically during G2/M. While this seems largely dispensable for mitotic progression, loss of Fbxw5 leads to increased MCAK levels at basal bodies, which impair formation of primary cilia in the following G1. We have thus identified a novel regulatory event of ciliogenesis that occurs already within the G2/M phase of the preceding cell cycle.

scholarly journals Thyroid hormone signaling and consequences for cardiac development

2019 ◽  
Vol 242 (1) ◽  
pp. T145-T160 ◽  
Author(s):  
Natasha N Chattergoon
Keyword(s):  
Thyroid Hormone ◽  
Cardiovascular Health ◽  
Neuronal Development ◽  
Cardiac Development ◽  
Perinatal Period ◽  
Organ Systems ◽  
Fetal Cardiomyocytes ◽  
Maturation Stages ◽  
Fetal Size ◽  
Near Term

The fetal heart undergoes its own growth and maturation stages all while supplying blood and nutrients to the growing fetus and its organs. Immature contractile cardiomyocytes proliferate to rapidly increase and establish cardiomyocyte endowment in the perinatal period. Maturational changes in cellular maturation, size and biochemical capabilities occur, and require, a changing hormonal environment as the fetus prepares itself for the transition to extrauterine life. Thyroid hormone has long been known to be important for neuronal development, but also for fetal size and survival. Fetal circulating 3,5,3′-triiodothyronine (T3) levels surge near term in mammals and are responsible for maturation of several organ systems, including the heart. Growth factors like insulin-like growth factor-1 stimulate proliferation of fetal cardiomyocytes, while thyroid hormone has been shown to inhibit proliferation and drive maturation of the cells. Several cell signaling pathways appear to be involved in this complicated and coordinated process. The aim of this review was to discuss the foundational studies of thyroid hormone physiology and the mechanisms responsible for its actions as we speculate on potential fetal programming effects for cardiovascular health.

scholarly journals Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

1995 ◽  
Vol 15 (11) ◽  
pp. 6430-6442 ◽  
Author(s):  
C A Pritchard ◽  
M L Samuels ◽  
E Bosch ◽  
M McMahon
Keyword(s):  
Protein Kinases ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Prolonged Exposure ◽  
Feedback Regulation ◽  
Biochemical Properties ◽  
3T3 Cells ◽  
In Cells

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.

scholarly journals Dysregulation of the Polo-Like Kinase Pathway in CD4+ T Cells Is Characteristic of Pathogenic Simian Immunodeficiency Virus Infection

2004 ◽  
Vol 78 (3) ◽  
pp. 1464-1472 ◽  
Author(s):  
Pavel Bostik ◽  
Geraldine L. Dodd ◽  
Francois Villinger ◽  
Ann E. Mayne ◽  
Aftab A. Ansari
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Simian Immunodeficiency Virus ◽  
Intracellular Signaling ◽  
Immunodeficiency Virus ◽  
M Phase ◽  
Siv Infection

ABSTRACT CD4+ T-cell dysfunction highlighted by defects within the intracellular signaling cascade and cell cycle has long been characterized as a direct and/or indirect consequence of human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM). Dysregulation of the M phase of the cell cycle is a well-documented effect of HIV or SIV infection both in vivo and in vitro. In this study the effect of SIV infection on the modulation of two important regulators of the M phase—polo-like kinases Plk3 and Plk1—was investigated. We have previously shown that Plk3 is markedly downregulated in CD4+ T cells from SIV-infected disease-susceptible RM but not SIV-infected disease-resistant sooty mangabeys (SM), denoting an association of downregulation with disease progression. Here we show that, in addition to the downregulation, Plk3 exhibits aberrant activation patterns in the CD4+ T cells from SIV-infected RM following T-cell receptor stimulation. Interestingly, in vitro SIV infection of CD4+ T cells leads to the upregulation, rather than downregulation, of Plk3, suggesting that different mechanisms operate in vitro and in vivo. In addition, CD4+ T cells from RM with high viral loads exhibited consistent and significant upregulation of Plk1, concurrent with an aberrant activation-induced Plk1 response, suggesting complex mechanisms of SIV-induced M-phase abnormalities in vivo. Altogether this study presents a novel mechanism underlying M-phase defects observed in CD4+ T cells from HIV or SIV-infected disease-susceptible humans and RM which may contribute to aberrant T-cell responses and disease pathogenesis.

scholarly journals Plant-Derived Molecules α-Boswellic Acid Acetate, Praeruptorin-A, and Salvianolic Acid-B Have Age-Related Differential Effects in Young and Senescent Human Fibroblasts In Vitro

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 141
Author(s):  
Anna Lewinska ◽  
Lakshman Sodagam ◽  
Dominika Bloniarz ◽  
Karsten Siems ◽  
Maciej Wnuk ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Metabolic Activity ◽  
Salvianolic Acid B ◽  
Low Doses ◽  
Boswellic Acid ◽  
Replicative Lifespan ◽  
Differential Effects ◽  
Salvianolic Acid ◽  
Age Related

Testing and screening of plant-derived molecules on normal human cells in vitro is a widely used approach for discovering their eventual health beneficial effects for human ageing and longevity. As little is known about age-associated differential effects of such molecules, here we report that young (<25% replicative lifespan completed) and near-senescent (>90% replicative lifespan completed) human skin fibroblasts exposed for 1–15 days to a wide range of concentrations (0.1–100 μM) of the three selected phytochemicals, namely α-boswellic acid acetate (ABC), praeruptorin-A (PTA), and salvianolic acid-B (SAB) had age-related differential effects. The parameters studied were the metabolic activity (MTT assay), cellular morphological phenotype, one-step growth characteristics, expression of genes involved in the cell cycle regulation and cytokine network genes, protein levels of p53, cytosolic superoxide dismutase (SOD1) and microtubule-associated protein 1A/1B-light chain 3 (LC3), and the extent of protein carbonylation and protein aggregation as a sign of oxidative stress. All three compounds showed biphasic hormetic dose response by stimulating cell growth, survival and metabolic activity at low doses (up to 1 μM), while showing inhibitory effects at high doses (>10 μM). Furthermore, the response of early passage young cells was different from that of the late passage near-senescent cells, especially with respect to the expression of cell cycle-related and inflammation-related genes. Such studies have importance with respect to the use of low doses of such molecules as health-promoting and/or ageing-interventions through the phenomenon of hormesis.

scholarly journals Platelet-Derived Growth Factor Receptor Association with Na+/H+ Exchanger Regulatory Factor Potentiates Receptor Activity

2000 ◽  
Vol 20 (22) ◽  
pp. 8352-8363 ◽  
Author(s):  
Stuart Maudsley ◽  
A. Musa Zamah ◽  
Nadeem Rahman ◽  
Jeremy T. Blitzer ◽  
Louis M. Luttrell ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Intracellular Signaling ◽  
Pdz Domain ◽  
Growth Factor Receptor ◽  
Cell Types ◽  
Platelet Derived Growth Factor ◽  
Regulatory Factor ◽  
In Cells

ABSTRACT Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na+/H+ exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the β2-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.

Sign in / Sign up

Export Citation Format

Share Document