Study of the clinical significance of ETAR mRNA expression in high-grade serous ovarian cancer and the inhibitory effect of ETAR derived fusion polypeptide on cancer progression / 中华妇产科杂志

Objective: To investigate the clinical significance of endothelin A receptor (ETAR) expression in high-grade serous ovarian carcinoma (HGSOC). To design ETAR carboxyl terminal (ETAR-C) amino acids derived polypeptide and to study the inhibitory effect on ovarian epithelial carcinoma cells in vitro. Methods: (1) A total of 126 patients who received surgical treatment and were diagnosed with HGSOC by postoperative pathological examination in Central Hospital of Xuzhou from January 1, 2007 to December 31, 2017 were selected. All patients had completed clinicopathological data and follow-up data. Cancer tissue samples were collected and ETAR mRNA expression in HGSOC tissues was detected by reverse transcript-PCR. The clinical significance was analyzed. (2) ETAR-C fusion polypeptide was designed based on the sequence of carboxyl terminal amino acids of ETAR, expressed and purified in vitro. The effects of ETAR-C fusion polypeptide on migration and invasion ability of ovarian cancer SKOV3 and CAOV3 cells were detected by scratch test and invasion test, respectively. The effect of ETAR-C fusion polypeptide on chemosensitivity of cisplatin-resistant ovarian cancer SKOV3/cDDP and CAOV3/cDDP cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The effect of ETAR-C fusion polypeptide on β-arrestin-1 expression in ovarian cancer SKOV3 and CAOV3 cells was detected by western blot. Results: (1) The relative expression level of ETAR mRNA in HGSOC tissues was 18.6±5.1. Patients with HGSOC were divided into high ETAR mRNA expression (n=76) and low ETAR mRNA expression (n=50) with 61.7% as cut-off value analyzed by X-Tile software. High expression of ETAR mRNA was significantly correlated with abdominal water volume, platinum drug resistance, and cancer antigen 125 (CA125) value in HGSOC patients (all P<0.05), but was not related to the age of patients with HGSOC and the size of postoperative residual lesions (all P>0.05). The 5-year progression free survival rates were 18.4% and 28.0%, and the 5-year overall survival rates were 38.2% and 52.0% in HGSOC patients with high and low ETAR mRNA expression respectively, there were statistically significant differences (P=0.046, P=0.034). (2) The results of scratch test and invasion test showed that the scratch healing rate and cell invasion rate of SKOV3 or CAOV3 cells treated with endothelin-1 (ET-1) and ET-1+ETAR-C were respectively compared, and the differences were statistically significant (all P<0.05). MTT assay showed that the inhibition rates of ETAR-C fusion polypeptide treated in SKOV3/cDDP and CAOV3/cDDP cells were significantly higher than those of control cells after the addition of 4, 6, 8, 10, 12, and 24 μg/ml cisplatin (all P<0.05). Western blot analysis showed that the relative expression levels of β-arrestin-1 in SKOV3 or CAOV3 cells treated with ET-1 and ET-1+ETAR-C were 1.85±0.09 and 1.13±0.09 (SKOV3 cells), 2.14±0.15 and 1.66±0.12 (CAOV3 cells), respectively. The differences were statistically significant (all P<0.05). Conclusions: The prognosis of HGSOC patients with high expression of ETAR mRNA is significantly worse than those with low expression of ETAR mRNA. ETAR might be a new target for HGSOC treatment. The ETAR-C fusion polypeptide that interferes with the interaction of ETAR and β-arrestin-1 has good inhibitory effect on ovarian cancer cells in vitro, and might have clinical application potential.

ß-ARRESTIN/CONNEXIN 43 (Cx43) complex anchors ERKS outside the nucleus: a prerequisite for bisphosphonate antiapoptotic effect mediated by Cx43/ERK in osteocytes / El complejo ß-ARRESTINA/CONEXINA 43 (Cx43) retiene ERKS fuera del núcleo: un paso necesario para el efecto anti-apoptótico de los bisfosfonatos mediado por Cx34/ERK en osteocitos

Bisphosphonates (BPs) anti-fracture efficacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of connexin (Cx) 43 hemichannels and phosphorylation of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. Instead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaffolding protein ß-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red fluorescent protein (ERK2-RFP) co-localizes with Cx43 fused to green fluorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear accumulation in cells transfected with wild type ß-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant negative ß-arrestin mutant (dnARR) consisting of the ß-arrestin-clathrin binding domain that competes with endogenous ß-arrestin for binding to clathrin. Alendronate activates ERKs in dnARRtransfected cells as effectively as in cells transfected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is ineffective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, ß-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs. (AU)

La efectividad de los bisfosfonatos (BPs) en la prevención de fracturas puede deberse en parte a la inhibición de la apoptosis de osteocitos. Este efecto depende de la apertura de hemicanales de conexina (Cx) 43 y la fosforilación de quinasas reguladas por señales extracelulares (ERKs). Sin embargo, a diferencia de la activación de ERKs debida a otros estímulos, la vía de señalización Cx43/ERK activada por BPs no conlleva la acumulación de ERKs en el núcleo. El efecto anti-apoptótico de los BPs depende de la fosforilación de blancos citoplasmáticos de ERKs y es inhibido cuando las quinasas son retenidas en el núcleo. En este estudio hemos demostrado que ERKs y la proteína "scaffolding" ß-arrestina co-inmunoprecipitan con Cx43 en células osteocíticas MLO-Y4 y que alendronato aumenta esta asociación. Más aún, ERK2 fusionada a la proteína roja fluorescente (ERK2-RFP) co-localiza con Cx43 fusionada con la proteína verde fluorescente fuera del núcleo en células tratadas con vehículo o alendronato. Alendronato no indujo la acumulación nuclear de ERK en células transfectadas con ß-arrestina nativa (wtARR) o con un vector control, pero si lo hizo en células que expresan una forma dominante negativa de ß-arrestina (dnARR), consistente en el dominio de interacción entre ß-arrestina y clatrina, y que compite con ß-arrestina endógena por la unión a clatrina. Alendronato activa ERKs con la misma eficiencia en células transfectadas con dnARR o wtARR, demostrando que dnARR sólo interfiere con la localización subcelular de ERKs, pero no con su activación inducida por los BPs. Más aún, mientras alendronato inhibe apoptosis en células que expresan wtARR o vector control, es inefectivo en células que expresan dnARR. En conclusión, los BPs inducen la formación de un complejo que incluye Cx43, ß-arrestina y clatrina, el cual retiene ERKs fuera del núcleo y es indispensable para la sobrevida de los osteocitos inducida por estas drogas. (AU)

β-arrestin1 promotes chronic myeloid leukemia cell proliferation by activating JNK signaling pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the signaling pathways involved in β-arrestin1-induced proliferation of K562 cells.</p><p><b>METHODS</b>We established stable cell lines K562-siβ1 and K562-β1 by lentivirus-mediated β-arrestin1 knock-down or overexpression in K562 cells, with cells transfected with non-specific siRNA as the control (K562-Ctrl). The proliferation of these cells were evaluated by cell counting and CCK-8 assays. Western blotting was used to detect the expression of JNK and p-JNK in the cells, and co-immunoprecipitation (Co-IP) assay was employed to investigate the interaction between β-arrestin1 and Src.</p><p><b>RESULTS</b>K562-β1 cells showed significantly greater but K562-siβ1 cells had significantly lower proliferation ability and cell survival rate than K562-Ctrl cells. Western blotting showed that β-arrestin1 specifically enhanced the expression of p-JNK, and the JNK inhibitor SP600125 obviously suppressed p-JNK and cell proliferation of K562 cells. Co-IP assay revealed the binding of β-arrestin1 to Src.</p><p><b>CONCLUSIONS</b>In K562 cells, β-arrestin1 activates JNK signaling pathway by binding to Src to promote the cell proliferation.</p>

Role of DOR-β-arrestin1-Bcl2 signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis / 华中科技大学学报（医学）（英德文版）

This study was aimed to investigate the role of the delta-opioid receptor (DOR)-β-arrestin1-Bcl-2 signal transduction pathway in the pathogenesis of ulcerative colitis (UC) and the intervention effects of oxymatrine on UC. Forty Sprague-Dawley rats were divided into normal group, model group, oxymatrine-treated group and mesalazine-treated group (n=10 each) at random. The rat UC model was established by intra-colonic injection of trinitrobenzene sulfonic acid in the model group and two treatment groups. The rats in oxymatrine-treated group were subjected to intramuscular injection of oxymatrine [63 mg/(kg·day)] for 15 days, and those in mesalazine-treated group given mesalazine solution [0.5 g/(kg·day)] by gastric lavage for the same days. Animals in normal group and model group were administered 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the rats were sacrificed for the removal of colon tissues. The expression levels of DOR, β-arrestin1 and Bcl-2 were determined in colon tissues by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR), respectively. It was found that the expression levels of DOR, β-arrestin1 and Bcl-2 protein and mRNA were significantly increased in the model group as compared with the other groups (P<0.05). They were conspicuously decreased in both mesalazine-treated and oxymatrine-treated groups in contrast to the model group (P<0.05). No statistically significant difference was noted in these indices between mesalazine- and oxymatrinetreated groups (P>0.05). This study indicated that the DOR-β-arrestin1-Bcl-2 signal transduction pathway may participate in the pathogenesis of UC. Moreover, oxymatrine can attenuate the development of UC by regulating the DOR-β-arrestin1-Bcl-2 signal transduction pathway.

β-arrestin2 plays a critical role in reward behaviors induced by cocaine / 生理学报

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.

Effects of Chinese Fructus Mume formula and its separated prescription extract on insulin resistance in type 2 diabetic rats / 华中科技大学学报（医学）（英德文版）

The effect of Fructus Mume formula and its separated prescription extract on insulin resistance in type 2 diabetic rats was investigated. The rat model of type 2 diabetes was established by feeding on a high-fat diet for 8 weeks and by subsequently intravenous injection of small doses of streptozotocin. Rats in treatment groups, including the Fructus Mume formula treatment group (FM), the cold property herbs of Fructus Mume formula treatment group (CFM), the warm property herbs of Fructus Mume formula treatment group (WFM), were administrated with Fructus Mume formula and its separated prescription extract by gavage, while the rats in diabetic model group (DM) and metformin group (MET) were given by gavage with normal saline and metformin correspondingly. The body weight before and after treatment was measured, and the oral glucose tolerance test (OGTT) and the insulin release test (IRT) were performed. The homeostasis model assessment-insulin resistance index (HOMA-IR) was calculated. The protein and mRNA expression levels of Insr, β-arrestin-2, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues were detected by using Western blotting and RT-PCR respectively. The results demonstrated that, as compared with DM group, OGTT, IRT (0 h, 1 h) levels and HOMR-IR in treatment groups were all reduced, meanwhile their protein and mRNA expression levels of Insr, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues were obviously increased, and their protein and mRNA expression levels of β-arrestin-2 in the liver and skeletal muscle tissues were also markedly increased. It was suggested that the Fructus Mume formula and its separated prescription extracts could effectively improve insulin resistance in type 2 diabetic rats, which might be related to the up-regulated expression of Insr, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues, and β-arrestin-2 in the liver and skeletal muscle tissues.

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the β2-adrenoceptor (β2AR)-β-arrestin2-nuclear factor-κB (NF-κB) signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis.</p><p><b>METHODS</b>Forty SD rats were randomly divided into four groups, which included the normal control group, the model group, the mesalazine group and the oxymatrine treatment group, with 10 rats per group. Experimental colitis induced with trinitrobenzene sulfonic acid (TNBS) was established in each group except the normal control group. The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed. Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected. The expression of β2AR, β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h. Six rats died of lavage with 2 each in the normal control, the model group and the mesalazine group; and were not included in the analysis.</p><p><b>RESULTS</b>The rats in the model group suffered from looser stool and bloody purulent stool after modeling. But in the oxymatrine and mesalazine groups, looser stool and bloody purulent stool reduced after treatment. And the colonic wall in the model group was thickened and the colon length shortened. The colon mucosa was congested in multiple areas with edema, erosion, superficial or linear ulcer and scar formation, while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups (P<0.01). In colonic mucosa and in spleen lymphocytes, compared with the normal control group, the expression of NF-κBp65 were significantly increased (P<0.01) in the model group while the expressions of β 2AR and β-arrestin2 were significantly decreased (P<0.01). Compared with the model group, the expression of NF-κ Bp65 was significantly decreased in the mesalazine group (P<0.01) and oxymatrine treatment group (P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased (P<0.01). There were no statistically significant differences in the expression of β2AR, β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group (P>0.05).</p><p><b>CONCLUSIONS</b>The β2AR-β-arrestin2-NF-κB signal transduction pathway participated in the pathologic course of ulcerative colitis. Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.</p>

Parkinson disease drug screening based on the interaction between D(2) dopamine receptor and beta-arrestin 2 detected by capillary zone electrophoresis

Parkinson's disease is the second most common neurodegenerative disease in the world. Beta-arrestin-2 has been reported to be an important protein involved in D(2) dopamine receptor desensitization, which is essential to Parkinson's disease. Moreover, the potential value of pharmacological inactivation of G protein-coupled receptor kinase or arrestin in the treatment of patients with Parkinson's disease has recently been shown. We studied the interaction between D(2) dopamine receptor and beta-arrestin-2 and the pharmacological regulation of chemical compounds on such interaction using capillary zone electrophoresis. The results from screening more than 40 compounds revealed three compounds that remarkably inhibit the beta-arrestin-2/D(2) dopamine receptor interaction among them. These compounds are promising therapies for Parkinson's disease, and the method used in this study has great potential for application in large-scale drug screening and evaluation.

Pathogenetic mechanism of beta-arrestin1 in experimental colitis of rats and intervention effects of oxymatrine / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the pathogenetic mechanism of beta-arrestin1 in the rat's experimental colitis, whether the delta opioid receptor-beta-arrestin1 -Bcl-2 signal transduction pathway involves the pathological process of experimental colitis in rats, and whether oxymatrine could attenuate colitis through this pathway.</p><p><b>METHODS</b>Twenty-six SD rats were randomly divided into four groups, the normal control group, the model group, the mesalazine treated group and the oxymatrine treated group (8 rats in the last group and 6 each in the others). The colitis model was established with trinitrobenzene sulfonic acid (TNBS), and rats in the latter two groups were treated by oxymatrine (intramuscular injection) and mesalazine (3 mL solution gavaged) for 15 days, respectively, while rats in the former two groups were fed with equal volume of distilled water. Symptoms of diarrhea and bloody stool as well as colonic patho-histologic changes were observed, and changes in expressions of delta opioid receptor, beta-arrestin1 and Bcl-2 in rat's colon tissue and spleen T lymphocytes were detected with immuno-histochemistry and Western immune-blotting techniques, respectively.</p><p><b>RESULTS</b>In contrast to the normal control group, expressions of delta opioid receptor, beta-arrestin1 and Bcl-2 were significantly higher in the model group (P < 0.01); compared with the model group, they were significantly lower in the two treated groups (P < 0.01).</p><p><b>CONCLUSIONS</b>Delta opioid receptor-beta-arrestin1 -Bcl-2 signal transduction pathway participates in the pathogenesis of TNBS-induced experimental colitis in rats. Oxymatrine can intervene the signal transduction, which may be one of the mechanisms of oxymatrine in attenuating colitis in rats.</p>

Beta-arrestin and NF-kappaB, AP-1 activity in peripheral blood mononuclear cells of guinea pigs sensitized by trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the regulatory mechanism of immune response of guinea pigs sensitized by trichloroethylene (TCE), and the expression level of 3-arrestin, and the activity of NF-kappaB and AP-1 in peripheral blood mononuclear cells (PBMC) of guinea pigs sensitized by TCE.</p><p><b>METHODS</b>Guinea pigs were treated with TCE based on the guinea pig maximum response test (GPMT); Blank control group and DNCB positive control group were established. Scores of skin reaction were evaluated and used to determine whether or not allergy in guinea pig. Then TCE treated group was divided into sensitized group or un-sensitized group. The expression levels of beta-arrestin protein, activity of NF-kappaB and AP-1 in PBMC were detected by Western Blotting and EMSA, respectively. TNF-alpha level in serum was detected by ELISA kits.</p><p><b>RESULTS</b>No erythema or edema was found in the control group; part of guinea pigs treated with TCE developed erythema and edema, while obvious erythema and edema could be found in DNCB group. The sensitization rates were 71.4% and 100% in TCE and DNCB group, respectively. Compared with TCE un-sensitized group, expression of beta-arrestin and AP-1 activity were not significantly different in TCE sensitized group (P > 0.05). While the NF-kappaB activity was elevated obviously (P < 0.05). Compared with blank control groups [(32.118 +/- 12.550) pg/ml], serum TNF-alpha levels in TCE sensitized groups [(55.485 +/- 8.732) pg/ml] significantly elevated (P < 0.05);</p><p><b>CONCLUSION</b>In guinea pigs, beta-arrestin and AP-1 may not be activated, while the NF-kappaB activation is significant, and plays a immune regulatory role in the immune reaction of allergy induced by TCE.</p>