OBJECTIVE: To investigate the effect of β-arrestin1 gene on senescence of T-ALL cells and its possible mechanism. METHODS: The bone marrow specimens of T-ALL patients and controls were collected, the expression of β-arrestin1 and β-arrestin1 in the T-ALL patients was detected by RT-PCR and Western blot, respectively, and the relation of β-arrestin1 expression with the clinical pathologic characteristics and the prognosis of T-ALL patients was analyzed statistically. The stable Jurkat cell line with knocked down or overexpressed β-arrestin1 was constructed, the CCK method was used to detect the Jurkat cell number, the β-gal staining was used to analyze the effect of β-arrestin1 on senescence of Jurkat cells, the cross analysis of RNA-Seg data and KEGG data was performed for screening the possible signaling pathway, and Western blot was performed for varifying the key sites of signaling pathway. RESULTS: The β-arrestin1 expression in specimens of T-ALL patients decreased (P＜0.01), moreover the β-arrestin1 expression negatively related with peripheral blood cell number (r=-0.601), the blasts in peripheral blood (r=-0.516) and extramedullary infiltration (r=-0.359), while positively related with the response to chemotherapy (r=0.393). The detection of stable Jurkat cell line with knocked-down and overexpressed β-arrestin1 found that the β-arrestin 1 could decrease the Jurkat cell number and accelarate the senescence of Jurkat cells (P＜0.05). The cross analysis of RNA-Seg data and KEGG data showed that the senescence of T-ALL cells may be regulated via RAS-P16-PRb-E2F1 by β-arrestin 1. Western bolt confirmed that β-arrestin1 promoted the expression of Ras and p16, and decreased the expression of pRB and E2F1 (P＜0.05). CONCLUSIONS β-arrestin1 accelerates the senescence of Jurkat cells via Ras-p16-pRb-E2F1, and delays the progression in T-ALL, which may provide a new hypothesis for the pathogenesis of T-ALL.
Cellular Senescence ; Humans ; Jurkat Cells ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Prognosis ; beta-Arrestin 1 ; genetics

OBJECTIVE: To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft. METHODS: Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen. RESULTS: We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts. CONCLUSIONS β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.
Animals ; Disease Progression ; Heterografts ; Humans ; Mice ; T-Lymphocytes ; Transplantation, Heterologous ; beta-Arrestin 1

OBJECTIVE: To investigate the effect of β-arrestin1 on the concentration of reactive oxygen species (ROS) in the mitochondria of acute T-lymphocytic leukemia (T-ALL) cells and its possible mechanisms. METHODS: The stable T-ALL cell line with knocked down β-arrestin1 (Jurkat Siβ1) was constructed. Flow cytometry and probe assays were used to detect ROS content in cell and mitochondrial, respectively. The relationship between β-arrestin1 and microRNA was detected, analyzed and Q-PCR confirmed by microRNA microarray. The target genes of microRNA were predicated by miRbase software, identified by Western blot, and validated by Dual luciferase reporter gene. RESULTS: Jurkat Siβ1 stable cell line was successfully constructed and it was found that ROS content was slightly reduced in Jurkat Siβ1 at the whole cell level, and the ROS content was also significantly reduced in mitochondria. MicroRNA microarray analysis revealed that multiple T-ALL related microRNAs showed differentially expressed, in which the expression of miR-652-5p was significantly increased in Jurkat Siβ1 (P<0.05 fold>2.0), and Q-PCR showed that miR-652-5p was nearly 5-fold up-regulated in Jurkat Siβ1. miRbase predicted that the P62 gene was the target gene of miR-652-5p which could regulates mitochondrial function. P62 protein showed highly expressed in stably knocked down miR-652-5p in Jurkat cells. Dual luciferase reporter gene assay confirms that P62 was the target gene of miR-652-5p. CONCLUSION β-arrestin1 can decreases the expression of miR-652-5p and deregulates the translational inhibition of P62 mRNA, thus to increase ROS content in mitochondria of T-ALL cells.
Humans ; MicroRNAs/genetics* ; Mitochondria ; Oxygen ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; RNA, Messenger ; beta-Arrestin 1

A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.
Humans ; beta-Arrestin 2/metabolism* ; HEK293 Cells ; Adrenergic beta-Agonists/pharmacology* ; Isoproterenol/pharmacology* ; Receptors, Adrenergic, beta-2/metabolism* ; Norepinephrine/pharmacology*

Animals ; Arrestins ; metabolism ; Hypoxia ; metabolism ; Male ; Prefrontal Cortex ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; metabolism ; beta-Arrestin 1 ; beta-Arrestins

Besides its role in desensitization and internalization of receptors, β-arrestin2 facilitates G protein-independent signaling through its ability to scaffold various signaling molecules. β-arrestin2 is widely distributed in the central nervous system, and mediates signal transduction of brain circuit. The aim of the present study was to investigate the role of β-arrestin2 in reward behaviors induced by cocaine. We assessed the conditioned place preference (CPP) induced by low (10 mg/kg), moderate (20 mg/kg) and high (30 mg/kg) doses of cocaine in Arrb2(-/-) mice and Arrb2(+/+) controls. In the Arrb2(-/-) mice, moderate and high, but not low, dose of cocaine induced pronounced increases of CPP scores, which were higher than those in the Arrb2(+/+) mice. Moreover, cocaine-induced locomotor activity was significantly lower in Arrb2(-/-) mice than that of Arrb2(+/+) littermate controls. Taken together, our results suggest a potential role of β-arrestin2 in the cocaine-induced rewarding behaviors.
Animals ; Arrestins ; physiology ; Behavior, Animal ; drug effects ; Cocaine ; pharmacology ; Mice ; Mice, Knockout ; Motor Activity ; drug effects ; Reward ; beta-Arrestin 2 ; beta-Arrestins

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.
Arrestin ; Arrestins ; Computational Biology ; Cytosol ; Dopamine ; Family Characteristics ; GTP-Binding Proteins ; Membranes ; Phosphorylation ; Receptors, Dopamine D1 ; Receptors, Dopamine D2 ; Receptors, Dopamine

Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.
Arrestin ; genetics ; metabolism ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Endothelial Cells ; cytology ; drug effects ; Humans ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism ; pharmacology

AIMTo evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats.

METHODSThe study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined.

RESULTSThe fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control.

CONCLUSIONThe difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.

Animals ; Arrestin ; biosynthesis ; Culture Media, Conditioned ; Cycloheximide ; pharmacology ; Epididymis ; cytology ; drug effects ; growth & development ; Male ; Protein Synthesis Inhibitors ; pharmacology ; Rats ; Seminiferous Epithelium ; cytology ; drug effects ; physiology ; Seminiferous Tubules ; metabolism ; Sperm Count ; Testis ; drug effects ; growth & development

PURPOSE: Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)2-Fe2+, as a spin trap. METHODS: Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer. RESULTS: The ESR three-line spectrum (g=2.04; a(N)=12.5 G) obtained was characteristic of the adduct [(MGD)2-Fe2+-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite. CONCLUSIONS: The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.
Uveitis/immunology/*metabolism ; Thiocarbamates ; Spin Trapping ; Spin Labels ; Sorbitol/analogs & derivatives ; Retina/metabolism ; Reactive Nitrogen Species/*metabolism ; Rats, Inbred Lew ; Rats ; Peptide Fragments/immunology ; Humans ; Electron Spin Resonance Spectroscopy ; Choroid/metabolism ; Autoimmune Diseases/immunology/*metabolism ; Arrestin/immunology ; Animals