Serum Response Factor Is Required for Immediate-Early Gene Activation yet Is Dispensable for Proliferation of Embryonic Stem Cells

ABSTRACT Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf −/− mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf −/− ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf−/− ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf −/− ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

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Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.200106008 ◽

2002 ◽

Vol 156 (4) ◽

pp. 737-750 ◽

Cited By ~ 120

Author(s):

Gerhard Schratt ◽

Ulrike Philippar ◽

Jürgen Berger ◽

Heinz Schwarz ◽

Olaf Heidenreich ◽

...

Keyword(s):

Focal Adhesion ◽

Serum Response Factor ◽

Es Cells ◽

Embryonic Stem ◽

Cell Spreading ◽

Stress Fibers ◽

Response Factor ◽

Es Cell ◽

And Migration ◽

Serum Response

The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf(−/−) embryonic stem (ES) cells, we demonstrate that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), β1-integrin, talin, zyxin, and vinculin were downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf(−/−) ES cells were accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescued F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf(−/−) ES cells, on the other hand, strongly induced expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, we demonstrate for the first time the importance of SRF for the formation of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. Our findings suggest an involvement of SRF in cell migratory processes in multicellular organisms.

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Oct-4 controls cell-cycle progression of embryonic stem cells

Biochemical Journal ◽

10.1042/bj20091439 ◽

2010 ◽

Vol 426 (2) ◽

pp. 171-181 ◽

Cited By ~ 69

Author(s):

Jungwoon Lee ◽

Yeorim Go ◽

Inyoung Kang ◽

Yong-Mahn Han ◽

Jungho Kim

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Cycle Progression ◽

Es Cells ◽

Embryonic Stem ◽

Functional Domains ◽

Es Cell ◽

Down Regulation ◽

Cycle Progression

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation.

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Dp1 Is Largely Dispensable for Embryonic Development

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7197-7205.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7197-7205 ◽

Cited By ~ 20

Author(s):

Matthew J. Kohn ◽

Sandra W. Leung ◽

Vittoria Criniti ◽

Monica Agromayor ◽

Lili Yamasaki

Keyword(s):

Cell Cycle ◽

Embryonic Development ◽

Cell Cycle Progression ◽

Target Genes ◽

Es Cells ◽

Embryonic Stem ◽

Wild Type ◽

Cycle Progression ◽

Cell Cycle Genes

ABSTRACT E2F/DP complexes activate or repress the transcription of E2F target genes, depending on the association of a pRB family member, thereby regulating cell cycle progression. Whereas the E2F family consists of seven members, the DP family contains only two (Dp1 and Dp2), Dp1 being the more highly expressed member. In contrast to the inactivation of individual E2F family members, we have recently demonstrated that loss of Dp1 results in embryonic lethality by embryonic day 12.5 (E12.5) due to the failure of extraembryonic lineages to develop and replicate DNA properly. To bypass this placental requirement and search for roles of Dp1 in the embryo proper, we generated Dp1-deficient embryonic stem (ES) cells that carry the ROSA26-LacZ marker and injected them into wild-type blastocysts to construct Dp1-deficient chimeras. Surprisingly, we recovered mid- to late gestational embryos (E12.5 to E17.5), in which the Dp1-deficient ES cells contributed strongly to most chimeric tissues as judged by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining and Western blotting. Importantly, the abundance of DP2 protein does not increase and the expression of an array of cell cycle genes is virtually unchanged in Dp1-deficient ES cells or chimeric E15.5 tissues with the absence of Dp1. Thus, Dp1 is largely dispensable for embryonic development, despite the absolute extraembryonic requirement for Dp1, which is highly reminiscent of the restricted roles for Rb and cyclins E1/E2 in vivo.

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Coordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00740.2019 ◽

2020 ◽

Vol 318 (6) ◽

pp. H1538-H1558

Author(s):

Allen Sam Titus ◽

Harikrishnan V ◽

Shivakumar Kailasam

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Serum Response Factor ◽

Cardiac Fibroblasts ◽

Apoptosis Resistance ◽

Cycle Progression ◽

Injured Myocardium ◽

Coordinated Regulation ◽

And Function ◽

Serum Response

Relative resistance to apoptosis and the ability to proliferate and produce a collagen-rich scar enable cardiac fibroblasts to play a central role in myocardial response to injury. This study reports novel findings that mitogen-stimulated cardiac fibroblasts exploit a common regulatory mechanism involving collagen receptor (DDR2)-dependent activation of ERK1/2 MAPK and serum response factor to achieve coordinated regulation of apoptosis resistance and cell cycle progression, which could facilitate their survival and function in the injured myocardium.

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Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

AJP Cell Physiology ◽

10.1152/ajpcell.00181.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1510-C1518 ◽

Cited By ~ 77

Author(s):

Nidhi Kapur ◽

Gregory A. Mignery ◽

Kathrin Banach

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Es Cells ◽

Embryonic Stem ◽

S Phase ◽

Calcium Oscillations ◽

Confocal Imaging ◽

Cycle Progression ◽

Intracellular Ca ◽

Mes Cells

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+signals during the G0phase, the transition from G1to S, and from G2to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+release and the refilling of intracellular stores by a store-operated Ca2+influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+oscillations were confined to the G1/S phase (∼70% oscillating cells vs. G2/M with ∼15% oscillating cells) of the cell cycle. ATP induced Ca2+oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+stores were not depleted, and all three IP3receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+release is necessary for cell cycle progression through G1/S. Because the IP3receptor sensitizer thimerosal induced Ca2+oscillations only in G1/S, we propose that changes in IP3receptor sensitivity or basal levels of IP3could be the basis for the G1/S-confined Ca2+oscillations.

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Human Papillomavirus Type 16 E7 Maintains Elevated Levels of the cdc25A Tyrosine Phosphatase during Deregulation of Cell Cycle Arrest

Journal of Virology ◽

10.1128/jvi.76.2.619-632.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 619-632 ◽

Cited By ~ 57

Author(s):

Don X. Nguyen ◽

Thomas F. Westbrook ◽

Dennis J. McCance

Keyword(s):

Cell Cycle ◽

Human Papillomavirus ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Tyrosine Phosphatase ◽

Early Gene ◽

Cycle Progression ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Cycle Arrest

ABSTRACT Essential to the oncogenic properties of human papillomavirus type 16 (HPV-16) are the activities encoded by the early gene product E7. HPV-16 E7 (E7.16) binds to cellular factors involved in cell cycle regulation and differentiation. These include the retinoblastoma tumor suppressor protein (Rb) and histone deacetylase (HDAC) complexes. While the biological significance of these interactions remains unclear, E7 is believed to help maintain cells in a proliferative state, thus establishing an environment that is conducive to viral replication. Most pathways that govern cell growth converge on downstream effectors. Among these is the cdc25A tyrosine phosphatase. cdc25A is required for G1/S transition, and its deregulation is associated with carcinogenesis. Considering the importance of cdc25A in cell cycle progression, it represents a relevant target for viral oncoproteins. Accordingly, the present study focuses on the putative deregulation of cdc25A by E7.16. Our results indicate that E7.16 can impede growth arrest induced during serum starvation and keratinocyte differentiation. Importantly, these E7-specific phenotypes correlate with elevated cdc25A steady-state levels. Reporter assays performed with NIH 3T3 cell lines and human keratinocytes indicate that E7 can transactivate the cdc25A promoter. In addition, transcriptional activation by E7.16 requires the distal E2F site within the cdc25A promoter. We further demonstrate that the ability of E7 to abrogate cell cycle arrest, activate cdc25A transcription, and increase cdc25A protein levels requires intact Rb and HDAC-1 binding domains. Finally, by using the cdk inhibitor roscovitine, we reveal that E7 activates the cdc25A promoter independently of cell cycle progression and cdk activity. Consequently, we propose that E7.16 can directly target cdc25A transcription and maintains cdc25A gene expression by disrupting Rb/E2F/HDAC-1 repressor complexes.

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Babam2 Regulates Cell Cycle Progression and Pluripotency in Mouse Embryonic Stem Cells as Revealed by Induced DNA Damage

Biomedicines ◽

10.3390/biomedicines8100397 ◽

2020 ◽

Vol 8 (10) ◽

pp. 397

Author(s):

Cheuk Yiu Tenny Chung ◽

Paulisally Hau Yi Lo ◽

Kenneth Ka Ho Lee

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Dna Damage ◽

Gamma Irradiation ◽

Cellular Senescence ◽

Cell Cycle Regulation ◽

Cell Cycle Progression ◽

Embryonic Stem ◽

Cycle Progression ◽

Cycle Regulation

BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2−/−) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2−/− mESCs following gamma irradiation. In addition, Babam2−/− mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2−/− mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.

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Caveolin-1 plays important role in EGF-induced migration and proliferation of mouse embryonic stem cells: involvement of PI3K/Akt and ERK

AJP Cell Physiology ◽

10.1152/ajpcell.00121.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. C935-C944 ◽

Cited By ~ 75

Author(s):

Jae Hong Park ◽

Ho Jae Han

Keyword(s):

Cell Cycle ◽

Cell Migration ◽

Kinase Inhibitor ◽

Thymidine Incorporation ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Akt Inhibitor ◽

Expression Levels ◽

Caveolin 1

The involvement of caveolin-1 in the regulation of embryonic stem (ES) cell growth by epidermal growth factor (EGF) is by no means clear cut. Thus we examined the relationship between EGF and caveolin-1 in mouse ES cell migration and proliferation. The results revealed that EGF increased Src, caveolin-1, focal adhesion kinase (FAK), Akt, and extracellular signal-regulated kinase-1/2 (ERK) phosphorylation levels. Especially, phosphorylation of caveolin-1 is attenuated by AG1478, herbimycin A (tyrosine kinase inhibitors), and pyrazolopyrimidine 2 (PP2, Src inhibitor) and EGF-induced ERK activation was blocked by PP2, methyl-β-cyclodextrin (MβCD), caveolin-1 small interfering RNA (siRNA), LY-294002 [phosphoinositol-3 kinase inhibitor (PI3K)], and Akt inhibitor. In addition, EGF promoted the cell migration, which was attenuated by PP2, caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. EGF also increased matrix metalloproteinase (MMP-2) expression levels and EGF-induced MMP2 expression was inhibited by caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. Furthermore, EGF-induced increase of cell cycle proteins expression level and [3H]thymidine incorporation was blocked by MMP inhibitor. EGF also significantly increases [3H]thymidine incorporation and cell number, which were significantly blocked by AG 1478, PP2, MβCD, caveolin-1 siRNA, FAK siRNA, LY-294002, and PD-98059 (ERK inhibitor). EGF-induced increase of protooncogenes (c- fos, c- myc, and c- Jun) and cell cycle regulatory proteins (cyclin D1, CDK4, cyclin E, and CDK2) expression levels were also attenuated by caveolin-1 siRNA and FAK siRNA. In conclusion, these results demonstrated that EGF-induced DNA synthesis and cell migration are mediated by caveolin-1, which is activated by Src, FAK, PI3K/Akt, ERK, and MMP-2 signals in mouse ES cells.

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T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

AJP Cell Physiology ◽

10.1152/ajpcell.00267.2011 ◽

2012 ◽

Vol 302 (3) ◽

pp. C494-C504 ◽

Cited By ~ 27

Author(s):

José A. Rodríguez-Gómez ◽

Konstantín L. Levitsky ◽

José López-Barneo

Keyword(s):

Cell Cycle ◽

Alkaline Phosphatase ◽

Es Cells ◽

Embryonic Stem ◽

Cell Types ◽

Mrna Levels ◽

Es Cell ◽

External Application ◽

Self Renewal ◽

Mouse Es Cells

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na+ currents as well as transient Ca2+ currents abolished by the external application of Ni2+. Biophysical and pharmacological data indicated that the Ca2+ current is predominantly mediated by T-type (Cav3.2) channels. The number of cells expressing T-type channels and Cav3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca2+ currents with Ni2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3.2) Ca2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca2+ entry mediated by Cav3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

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A Critical Role for Cyclin C in Promotion of the Hematopoietic Cell Cycle by Cooperation with c-Myc

Molecular and Cellular Biology ◽

10.1128/mcb.18.6.3445 ◽

1998 ◽

Vol 18 (6) ◽

pp. 3445-3454 ◽

Cited By ~ 27

Author(s):

Zhao-Jun Liu ◽

Takahiro Ueda ◽

Tadaaki Miyazaki ◽

Nobuyuki Tanaka ◽

Shinichiro Mine ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Line ◽

Cell Cycle Progression ◽

Critical Role ◽

S Phase ◽

Early Gene ◽

Cycle Progression ◽

Cyclin C ◽

Growth Properties

ABSTRACT Cyclin C, a putative G1 cyclin, was originally isolated through its ability to complement a Saccharomyces cerevisiae strain lacking the G1 cyclin geneCLN1-3. Unlike cyclins D1 and E, the other two G1 cyclins obtained by the same approach and subsequently shown to play important roles during the G1/S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression. In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G1/S phase upon IL-3 stimulation and reached a maximal level in the S phase. Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties. In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation. We found that cyclin C is able to cooperate functionally with c-Myc, but not c-Fos, to induce both BAF-B03 cell proliferation in a cytokine-independent fashion and the formation of cell clusters. Furthermore, cyclin C was primarily responsible for the induction of cdc2 gene expression. Our data define a novel role for cyclin C in the regulation of both the G1/S and G2/M phases of the cell cycle, and this effect appears to be independent of the activity of CDK8 in the control of transcription.

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