A signaling loop of REST, TSC2 and β-catenin governs proliferation and function of PC12 neural cells

Tomasoni, Romana; Negrini, Sara; Fiordaliso, Stefania; Klajn, Andrijana; Tkatch, Tatiana; Mondino, Anna; Meldolesi, Jacopo; D’Alessandro, Rosalba

doi:10.1242/jcs.087551

Cited by 26 publications

(32 citation statements)

References 63 publications

(78 reference statements)

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“…For transient Rac1 downregulation, the cells, seeded on slides coated with polylysine, were exposed for 48 h to the shRNA (Tomasoni et al, 2001), scrambled or specific. The latter, chosen out of four based on efficacy, was as follows: CGAGGACTCAAGACAGTGTTTGATGAAGC.…”

Section: Methodsmentioning

confidence: 99%

Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes

Racchetti¹,

D’Alessandro

Meldolesi³

2011

Glia

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Cultured astrocytes exhibit a flat/epitelioid phenotype much different from the star-like phenotype of tissue astrocytes. Upon exposure to treatments that affect the small GTPase Rho and/or its effector ROCK, however, flat astrocytes undergo stellation, with restructuring of cytoskeleton and outgrowth of processes with lamellipodia, assuming a phenotype closer to that exhibited in situ. The mechanisms of this change are known only in part. Using the ROCK blocker drug Y27632, which induces rapid (tens of min), dose-dependent and reversible stellations, we focused on two specific aspects of the process: its dependence on small GTPases and the large surface expansion of the cells. Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug. In contrast cdc42, the other G-protein often involved in phenotype changes, appeared not involved. The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes. Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool. A whole-cell process like stellation of cultured astrocytes might be irrelevant in the brain tissue. However, local restructuring of the cytoskeleton coordinate with surface expansion, occurring at critical cell sites and sustained by mechanisms analogous to those of stellation, might be of importance in both astrocyte physiology and pathology. © 2011 Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes

Racchetti¹,

D’Alessandro

Meldolesi³

2011

Glia

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“…Our previous studies had shown mTORC1 to be moderately more active, and mTORC2 much less active in the high REST PC12-27 cells compared to the wtPC12 cells (Tomasoni et al, 2011). In order to investigate the possible involvement in these differences of the NGF receptor signaling and of its mTORC1-dependent feed-back inhibition, we analyzed the direct read-outs of mTORC1 and mTORC2, P-S6(S235/236) and P-Akt(S473), respectively.…”

Section: Resultsmentioning

confidence: 97%

“…On the one hand, many neural cell-specific proteins encoded by REST target genes, such as those of regulated neurosecretion, are lacking (D'Alessandro et al, 2008); on the other hand, PC12-27 cells exhibit accelerated cell proliferation, controlled by a signaling loop in which REST operates together with the GAP protein TSC2 and the co-transcription factor β-catenin (Tomasoni et al, 2011). The activities of the two mTOR complexes were found to be dissociated: that of mTORC1 was slightly higher, that of mTORC2 was very low (Tomasoni et al, 2011), much lower than those of wtPC12, of other neural cells and of various types of neurons (Carson et al, 2013; Mazei-Robison et al, 2011; Urbanska et al, 2012). Whether and to what extent these properties of PC12-27 cells depend on their defective NGF signaling had never been established.…”

Section: Introductionmentioning

confidence: 99%

NGF signaling in PC12 cells: the cooperation of p75NTR with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade

2013

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SummaryPC12-27, a PC12 clone characterized by high levels of the transcription repressor REST and by very low mTORC2 activity, had been shown to be unresponsive to NGF, possibly because of its lack of the specific TrkA receptor. The neurotrophin receptor repressed by high REST in PC12-27 cells, however, is shown now to be not TrkA, which is normal, but p75NTR, whose expression is inhibited at the transcriptional level. When treated with NGF, the PC12-27 cells lacking p75NTR exhibited a defective TrkA autophosphorylation restricted, however, to the TrkA(Y490) site, and an impairment of the PI3K signaling cascade. This defect was sustained in part by a mTORC1-dependent feed-back inhibition that in wtPC12 cells appeared marginal. Transfection of p75NTR to a level and surface distribution analogous to wtPC12 did not modify various high REST-dependent properties of PC12-27 cells such as high β-catenin, low TSC2 and high proliferation rate. In contrast, the defective PI3K signaling cascade and its associated mTORC2 activity were largely rescued together with the NGF-induced neurite outgrowth response. These changes were not due to p75NTR alone but required its cooperation with TrkA. Our results demonstrate that, in PC12, high REST induces alterations of NGF signaling which, however, are indirect, dependent on the repression of p75NTR; and that the well-known potentiation by p75NTR of the TrkA signaling does not concern all the effects induced by NGF but primarily the PI3K cascade and its associated mTORC2, a complex known to play an important role in neural cell differentiation.

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“…Additionally, there is crosstalk between the PI3K/Akt, ERK, and Wnt/β-catenin signaling pathway participating in maintenance of mES cells self-renewal [33,35]. Furthermore, using genetic complementation and knockdown approaches, Tomasoni R et al found that a signaling loop of REST, GTPase-activating protein tuberin (TSC2) and the transcriptional co-factor β-catenin determines proliferation and function of PC12 neural cells [36]. Whether purarin-induced activation of PI3K signaling affects TSC2 and Wnt/β-catenin signaling pathway is still unknown.…”

Section: Discussionmentioning

confidence: 99%

Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway

Yin

Cui

et al. 2015

Cell Physiol Biochem

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Background/Aims: Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms. Methods: RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal. Results: Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal. Conclusion: Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

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A signaling loop of REST, TSC2 and β-catenin governs proliferation and function of PC12 neural cells

Cited by 26 publications

References 63 publications

Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes

Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes

NGF signaling in PC12 cells: the cooperation of p75NTR with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade

Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway

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