scholarly journals Ubiquitin-dependent degradation of HDAC4, a new regulator of random cell motility

2011 ◽  
Vol 22 (2) ◽  
pp. 278-289 ◽  
Author(s):  
Nadia Cernotta ◽  
Andrea Clocchiatti ◽  
Cristina Florean ◽  
Claudio Brancolini
Keyword(s):  
Growth Factors ◽  
Cell Motility ◽  
Histone Deacetylases ◽  
Glycogen Synthase ◽  
Consensus Sequence ◽  
Ubiquitin Proteasome System ◽  
Serum Starvation ◽  
Cellular Functions ◽  
Histone Deacetylase 4

HDAC4 (histone deacetylase 4) belongs to class IIa of histone deacetylases, which groups important regulators of gene expression, controlling pleiotropic cellular functions. Here we show that, in addition to the well-defined nuclear/cytoplasmic shuttling, HDAC4 activity is modulated by the ubiquitin–proteasome system. Serum starvation elicits the poly-ubiquitination and degradation of HDAC4 in nontransformed cells. Phosphorylation of serine 298 within the PEST1 sequence plays an important role in the control of HDAC4 stability. Serine 298 lies within a glycogen synthase kinase 3β consensus sequence, and removal of growth factors fails to trigger HDAC4 degradation in cells deficient in this kinase. GSK3β can phosphorylate HDAC4 in vitro, and phosphorylation of serine 302 seems to play the role of priming phosphate. We have also found that HDAC4 modulates random cell motility possibly through the regulation of KLF2 transcription. Apoptosis, autophagy, cell proliferation, and growth arrest were unaffected by HDAC4. Our data suggest a link between regulation of HDAC4 degradation and the control of cell motility as operated by growth factors.

scholarly journals Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

2006 ◽  
Vol 17 (2) ◽  
pp. 585-597 ◽  
Author(s):  
Fang Liu ◽  
Nabendu Pore ◽  
Mijin Kim ◽  
K. Ranh Voong ◽  
Melissa Dowling ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
Histone Deacetylases ◽  
Targeted Mutagenesis ◽  
Cellular Functions ◽  
Specific Expression ◽  
Protein Levels ◽  
Histone Deacetylase 4 ◽  
First Time

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

Polyamine-based analogues as biochemical probes and potential therapeutics

2007 ◽  
Vol 35 (2) ◽  
pp. 356-363 ◽  
Author(s):  
T. Boncher ◽  
X. Bi ◽  
S. Varghese ◽  
R.A. Casero ◽  
P.M. Woster
Keyword(s):  
Histone Deacetylases ◽  
Biologically Active ◽  
Cellular Functions ◽  
Antiparasitic Activity ◽  
Polyamine Analogues ◽  
Naturally Occurring ◽  
Polyamine Derivatives ◽  
Structural Classes ◽  
Potential Therapeutics

The polyamines putrescine, spermidine and spermine are ubiquitous polycationic compounds that are found in nearly every cell type, and are required to support a wide variety of cellular functions. The existence of multiple cellular effector sites for naturally occurring polyamines implies that there are numerous targets for polyamine-based therapeutic agents. Through a programme aimed at the synthesis and evaluation of biologically active polyamine analogues, our laboratory has identified three distinct structural classes of polyamine derivatives that exhibit promising biological activity in vitro. We have synthesized more than 200 symmetrically and unsymmetrically substituted alkylpolyamines that possess potent antitumour or antiparasitic activity, depending on their backbone architecture and terminal alkyl substituents. Along similar lines, we have developed novel polyamino(bis)guanidines and polyaminobiguanides that are promising antitrypanosomal agents and that interfere with biofilm formation in the pathogenic bacterium Yersinia pestis. Finally, we recently reported a series of PAHAs (polyaminohydroxamic acids) and PABAs (polyaminobenzamides) that inhibit HDACs (histone deacetylases), and in some cases are selective for individual HDAC isoforms. These studies support the hypothesis that polyamine-based small molecules can be developed for use as biochemical probes and as potential therapies for multiple diseases.

119 FGF2 AND FGF10 STIMULATES BOVINE AND OVINE TROPHOBLAST CELL MIGRATION

2011 ◽  
Vol 23 (1) ◽  
pp. 164
Author(s):  
Q. E. Yang ◽  
K. Zhang ◽  
M. I. Giassetti ◽  
M. Ozawa ◽  
S. E. Johnson ◽  
...  
Keyword(s):  
Cell Migration ◽  
Growth Factors ◽  
P38 Mapk ◽  
Trophoblast Cell ◽  
Mitogen Activated Protein Kinase ◽  
Epifluorescence Microscopy ◽  
Migration And Invasion ◽  
Serum Starvation ◽  
Migration Assay

Following hatching, bovine and ovine conceptuses undergo a phase of massive development and remodelling that causes elongation and filamentation. Proper trophoblast cell development and interaction with the uterus is critical for the establishment and maintenance of pregnancy. Various growth factors, including several fibroblast growth factors (FGFs), are produced by the uterus, and at least two of these, FGF2 and FGF10, are released into uterus lumen during early pregnancy. Microarray analysis found that gene products associated with migration and invasion were altered in bovine blastocysts exposed to FGF2 or 10. The objective of this work was to determine if FGF2 and FGF10 impact bovine and ovine trophoblast cell migration. The ability of FGF2 and FGF10 to influence migratory ability of trophoblast cells was examined by using an in vitro transwell migration assay. The bovine trophoblast line, CT1, was used in the first study. After serum starvation, CT1 cells were seeded on the top of each transwell membrane (50 000/transwell) in the presence of vehicle, 0.5, 5, or 50 ng mL–1 bovine recombinant FGF2 or human recombinant FGF10. After 12 h, the transwell was fixed and stained with Hoechst 33342 (0.5 μg mL–1). Migrated cells were counted on five non-overlapping areas of each filter using epifluorescence microscopy. Supplementation with 0.5 ng mL–1 FGF2 increased the number of migrated CT1 cells when compared with controls (268.3 ± 58.3 v. 167.3 ± 47.7; P < 0.01). Supplementation with 5 or 50 ng mL–1 FGF2 further increased the number of migrated CT1 cells (297.0 ± 51.4 and 429.4 ± 98.3, respectively; P < 0.001). Adding 0.5 ng mL–1 FGF10 did not affect CT1 migration but providing 5 or 50 ng mL–1 FGF10 increased CT1 migration (399.8 ± 29.7 and 392.7 ± 58.6 v. 194.2 ± 40.3 for controls; P < 0.005). A subsequent study utilised the ovine trophoblast line, oTR1 in the migration assay (30 000 cells/transwell; 8 h migration assay). Adding 0.5 ng mL–1 FGF2 or FGF10 did not affect oTR1 migration number but exposure to holdout 5 or 50 ng mL–1 FGF2 or FGF10 increased oTR1 migrated cell numbers v. controls (P < 0.05). In a subsequent study, p38 mitogen-activated protein kinase (MAPK), ERK1/2 and JNK signalling cascades utilised by FGF2 and FGF10 in oTR1 cells were investigated. Western blot analysis indicated that both FGF2 and FGF10 induced ERK1/2 and p38 MAPK phosphorylation status. Interestingly FGF10 activated JNK but not p38 MAPK. Taken together, FGF2 and FGF10 stimulate trophoblast cell migration. This response could be mediated by an ERK1/2- or p38 MAPK-dependent system. This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.

scholarly journals Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Daichao Xu ◽  
Bing Shan ◽  
Byung-Hoon Lee ◽  
Kezhou Zhu ◽  
Tao Zhang ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Signaling ◽  
Ubiquitin Proteasome System ◽  
Specific Protein ◽  
Negative Control ◽  
Common Mechanism ◽  
Wide Range ◽  
Specific Protease ◽  
Ubiquitin Proteasome

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.

scholarly journals Inhibition of ABCC6 Transporter Modifies Cytoskeleton and Reduces Motility of HepG2 Cells via Purinergic Pathway

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1410
Author(s):  
Angela Ostuni ◽  
Monica Carmosino ◽  
Rocchina Miglionico ◽  
Vittorio Abruzzese ◽  
Fabio Martinelli ◽  
...  
Keyword(s):  
Cell Motility ◽  
Hepg2 Cells ◽  
Purinergic Signaling ◽  
Atp Release ◽  
Cellular Functions ◽  
Migration Assay ◽  
Reverse Transcription Pcr ◽  
Cell Immunity ◽  
Gene And Protein Expression

ABCC6, belonging to sub-family C of ATP-binding cassette transporter, is an ATP-dependent transporter mainly present in the basolateral plasma membrane of hepatic and kidney cells. Although the substrates transported are still uncertain, ABCC6 has been shown to promote ATP release. The extracellular ATP and its derivatives di- and mono-nucleotides and adenosine by acting on specific receptors activate the so-called purinergic pathway, which in turn controls relevant cellular functions such as cell immunity, inflammation, and cancer. Here, we analyzed the effect of Abcc6 knockdown and probenecid-induced ABCC6 inhibition on cell cycle, cytoskeleton, and motility of HepG2 cells. Gene and protein expression were evaluated by quantitative Reverse Transcription PCR (RT-qPCR) and western blot, respectively. Cellular cycle analysis was evaluated by flow cytometry. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by in vitro wound-healing migration assay. Cell migration is reduced both in Abcc6 knockdown HepG2 cells and in probenecid treated HepG2 cells by interfering with the extracellular reserve of ATP. Therefore, ABCC6 could contribute to cytoskeleton rearrangements and cell motility through purinergic signaling. Altogether, our findings shed light on a new role of the ABCC6 transporter in HepG2 cells and suggest that its inhibitor/s could be considered potential anti-metastatic drugs.

scholarly journals NAA80 is actin’s N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

2018 ◽  
Vol 115 (17) ◽  
pp. 4399-4404 ◽  
Author(s):  
Adrian Drazic ◽  
Henriette Aksnes ◽  
Michaël Marie ◽  
Malgorzata Boczkowska ◽  
Sylvia Varland ◽  
...  
Keyword(s):  
Cell Motility ◽  
Posttranslational Modifications ◽  
Actin Binding ◽  
Signaling Proteins ◽  
Cellular Functions ◽  
Cytoskeletal Organization ◽  
Organelle Trafficking ◽  
Lamellipodia Formation ◽  
In Cells

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin’s cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin’s N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80’s role as actin’s NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Inhibitors of Neuroblastoma Cell Colony Growth That Increase Lysosome Accumulation

2020 ◽  
Author(s):  
Daniel Herp ◽  
Johannes Ridinger ◽  
Dina Robaa ◽  
Stephen A. Shinsky ◽  
Karin Schmidtkunz ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Neuroblastoma Cell ◽  
Hdac Inhibitors ◽  
Anticancer Therapy ◽  
Colony Growth ◽  
Autophagic Flux ◽  
Enzymatic Conversion ◽  
Lysine Deacetylase

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, esp. cancer. First HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement e.g. in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of the other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like spermine or spermidine. Hence, it also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin labelled acetyl spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10 mediated spermidine deacetylation in-vitro. Among those are potent inhibitors of neuroblastoma colony growth in culture that show accumulation of lysosomes, implicating disturbance of autophagic flux.

MALIGNANT TUMORS ASSOCIATED WITH METABOLIC SYNDROME: THE EXPRESSION PROFILE OF THE TRANSCRIPTIONAL AND GROWTH FACTORS

2017 ◽  
Vol 63 (6) ◽  
pp. 817-823
Author(s):  
Natalya Yunusova ◽  
Irina Kondakova ◽  
Sergey Afanasev ◽  
Larisa Kolomiets ◽  
Alena Chernyshova
Keyword(s):  
Quality Of Life ◽  
Metabolic Syndrome ◽  
Growth Factors ◽  
Cell Motility ◽  
Expression Profile ◽  
Malignant Tumors ◽  
Molecular Targets ◽  
Factors Associated ◽  
High Incidence

The study of the pathogenetic features of malignant tumors associated with metabolic syndrome (MS) is relevant because of high incidence of these tumors. Investigations of the mechanisms of involvement of MS in the pathogenesis of cancer reasonably supplemented by the study of transcription and growth factors associated with energy imbalance of the cell and involved in proliferation, apoptosis, angiogenesis, cell motility and inflammation. More research is needed to identify the most promising molecular targets for therapy of malignant tumors associated with MS with a view to increasing the survival and quality of life of these patients.

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