Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

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Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase

Journal of Cell Science ◽

10.1242/jcs.109.2.289 ◽

1996 ◽

Vol 109 (2) ◽

pp. 289-300 ◽

Cited By ~ 3

Author(s):

T.R. Jackson ◽

I.J. Blader ◽

L.P. Hammonds-Odie ◽

C.R. Burga ◽

F. Cooke ◽

...

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Specific Inhibitor ◽

Morphological Differentiation ◽

Sympathetic Neuron ◽

Neuronal Morphology ◽

Regulatory Subunit ◽

Membrane Reorganization ◽

Phosphoinositide 3 Kinase

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.

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Wnt-3a and Dvl Induce Neurite Retraction by Activating Rho-Associated Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.24.10.4487-4501.2004 ◽

2004 ◽

Vol 24 (10) ◽

pp. 4487-4501 ◽

Cited By ~ 93

Author(s):

Shosei Kishida ◽

Hideki Yamamoto ◽

Akira Kikuchi

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Mammalian Cells ◽

Planar Cell Polarity ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Binding Domain ◽

Serum Starvation ◽

Neurite Retraction ◽

Rho Kinase Inhibitor

ABSTRACT Dvl is a key protein that transmits the Wnt signal to the canonical β-catenin pathway and the noncanonical planar cell polarity (PCP) pathway. We studied the roles of Rho-associated kinase (Rho-kinase), which is activated by Dvl in the PCP pathway of mammalian cells. The expression of Dvl-1, Wnt-1, or Wnt-3a activated Rho-kinase in COS cells, and this activation was inhibited by the Rho-binding domain of Rho-kinase. The expression of Dvl-1 in PC12 cells activated Rho and inhibited nerve growth factor (NGF)-induced neurite outgrowth. This inhibition was reversed by a Rho-kinase inhibitor but not by a c-Jun N-terminal kinase inhibitor. Dvl-1 also inhibited serum starvation-dependent neurite outgrowth of N1E-115 cells, and expression of the Rho-binding domain of Rho-kinase reversed this inhibitory activity of Dvl-1. Dvl-1 mutants that did not activate Rho-kinase did not inhibit the neurite outgrowth of N1E-115 cells. Furthermore, the purified Wnt-3a protein activated Rho-kinase and inhibited the NGF-dependent neurite outgrowth of PC12 cells. Wnt-3a-dependent neurite retraction was also prevented by a Rho-kinase inhibitor and a Dvl-1 mutant that suppresses Wnt-3a-dependent activation of Rho-kinase. These results suggest that Wnt-3a and Dvl regulate neurite formation through Rho-kinase and that PC12 and N1E-115 cells are useful for analyzing the PCP pathway.

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Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1016741109 ◽

2012 ◽

Vol 109 (5) ◽

pp. 1578-1583 ◽

Cited By ~ 7

Author(s):

J. Plavicki ◽

S. Mader ◽

E. Pueschel ◽

P. Peebles ◽

G. Boekhoff-Falk

Keyword(s):

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Olfactory System ◽

Homeobox Gene

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The p110 Isoform of the CDP/Cux Transcription Factor Accelerates Entry into S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2441-2455.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2441-2455 ◽

Cited By ~ 38

Author(s):

Laurent Sansregret ◽

Brigitte Goulet ◽

Ryoko Harada ◽

Brian Wilson ◽

Lam Leduy ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Cell Cycle Progression ◽

S Phase ◽

Proteolytic Processing ◽

Experimental Conditions ◽

Division Rate ◽

Cycle Progression ◽

Processing Event

ABSTRACT The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.

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Adapter Protein SH2-Bβ Undergoes Nucleocytoplasmic Shuttling: Implications for Nerve Growth Factor Induction of Neuronal Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3633-3647.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3633-3647 ◽

Cited By ~ 24

Author(s):

Linyi Chen ◽

Christin Carter-Su

Keyword(s):

Plasma Membrane ◽

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Nuclear Export ◽

Adapter Protein ◽

Wild Type ◽

Nerve Growth

ABSTRACT The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-Bβ. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-Bβ to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-Bβ to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-Bβ(L231A, L233A). These data provide strong evidence that SH2-Bβ shuttles constitutively between the nucleus and cytoplasm. However, SH2-Bβ needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-Bβ on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.

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The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.102.3.821 ◽

1986 ◽

Vol 102 (3) ◽

pp. 821-829 ◽

Cited By ~ 122

Author(s):

C Richter-Landsberg ◽

B Jastorff

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Morphological Differentiation ◽

Intracellular Camp ◽

Nerve Growth ◽

Process Formation ◽

Dependent Protein

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.

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Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1.

The EMBO Journal ◽

10.1002/j.1460-2075.1995.tb00317.x ◽

1995 ◽

Vol 14 (24) ◽

pp. 6268-6279 ◽

Cited By ~ 221

Author(s):

V. Gawantka ◽

H. Delius ◽

K. Hirschfeld ◽

C. Blumenstock ◽

C. Niehrs

Keyword(s):

Homeobox Gene ◽

Spemann Organizer

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Merkel Cell Polyomavirus T Antigens Induce Merkel Cell-Like Differentiation in GLI1-Expressing Epithelial Cells

Cancers ◽

10.3390/cancers12071989 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1989

Author(s):

Thibault Kervarrec ◽

Mahtab Samimi ◽

Sonja Hesbacher ◽

Patricia Berthon ◽

Marion Wobser ◽

...

Keyword(s):

Transcription Factor ◽

Epithelial Cells ◽

Human Keratinocytes ◽

Merkel Cell Polyomavirus ◽

Bhlh Transcription Factor ◽

Merkel Cell ◽

T Antigens ◽

Differentiation Potential ◽

Transcription Factor 1

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an epithelial neoplasia bearing Merkel cell (MC) differentiation potential. Accordingly, we hypothesized that MC progenitors may represent an origin of MCPyV-positive MCC. To sustain this hypothesis, phenotypic comparison of trichoblastomas and physiologic human MC progenitors was conducted revealing GLI family zinc finger 1 (GLI1), Keratin 17 (KRT 17), and SRY-box transcription factor 9 (SOX9) expressions in both subsets. Furthermore, GLI1 expression in keratinocytes induced transcription of the MC marker SOX2 supporting a role of GLI1 in human MC differentiation. To assess a possible contribution of the MCPyV T antigens (TA) to the development of an MC-like phenotype, human keratinocytes were transduced with TA. While this led only to induction of KRT8, an early MC marker, combined GLI1 and TA expression gave rise to a more advanced MC phenotype with SOX2, KRT8, and KRT20 expression. Finally, we demonstrated MCPyV-large T antigens’ capacity to inhibit the degradation of the MC master regulator Atonal bHLH transcription factor 1 (ATOH1). In conclusion, our report suggests that MCPyV TA contribute to the acquisition of an MC-like phenotype in epithelial cells.

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Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.276-281.1984 ◽

1984 ◽

Vol 4 (2) ◽

pp. 276-281

Author(s):

W E Mercer ◽

C Avignolo ◽

R Baserga

Keyword(s):

Cell Proliferation ◽

Monoclonal Antibodies ◽

Mammalian Cells ◽

P53 Protein ◽

S Phase ◽

Human Cells ◽

Antigenic Determinants ◽

3T3 Cells ◽

Species Specific

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.

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