过表达RSK4m1对人乳腺癌细胞MDA-MB-231生长和侵袭的影响

目的探讨过表达p90核糖体S6蛋白激酶4变异体1(ribosomal protein S6 kinase 4 variant 1,RSK4m1)对人乳腺癌MDA-MB-231细胞生长和侵袭的影响。方法通过负载RSK4m1慢病毒在人乳腺癌MDA-MB-231细胞中稳定过表达RSK4m1。以MDA-MB-231亲本细胞为空白对照组(Con组)、转染空载病毒的MDA-MB-231细胞为阴性对照组(Mock组)、转染过表达RSK4m1的MDA-MB-231细胞为实验组(OE组)。采用qRT-PCR和Westen Blot法检测各组RSK4m1mRNA及蛋白的表达;CCK-8法检测细胞增殖情况;细胞划痕实验检测细胞的迁移能力;Transwell小室侵袭实验检测细胞的侵袭能力。结果 OE组中RSK4m1 mRNA及蛋白的表达量均明显高于Con组及Mock组(P<0.05)。CCK-8实验显示,24 h、48 h、72 h和96 h时OE组细胞增殖均低于Mock组和Con组(P<0.05);细胞划痕实验显示,细胞培养24 h后,OE组细胞迁移率显著低于Con组和Mock组[(14.53±0.64)%vs(25.67±2.44)%、(24.47±2.25)%,P<0.05]。Transwell小室侵袭实验显示,OE组细胞侵袭能力显著低于Con组和Mock组[(35.00±5.57)个vs(66.70±5.86)个、(58.67±4.16)个,P<0.05]。结论过表达RSK4m1可显著抑制人乳腺癌MDA-MB-231细胞增殖、迁移和侵袭能力,为RSK4m1可能成为乳腺癌的治疗新靶点提供了实验依据。

p85S6K sustains synaptic GluA1 to ameliorate cognitive deficits in Alzheimer’s disease

Background Ribosomal protein S6 kinase 1(S6K1)is a serine-threonine kinase that has two main isoforms:p70S6K(70-kDa isoform)and p85S6K(85-kDa isoform).p70S6K,with its upstream mammalian target of rapamycin(mTOR),has been shown to be involved in learning and memory and participate in the pathophysiology of Alzheimer’s dis-ease(AD).However,the function of p85S6K has long been neglected due to its high similarity to p70S6k.The role of p85S6K in learning and memory is still largely unknown.Methods We fractionated the postsynaptic densities to illustrate the differential distribution of p85S6K and p70S6K.Coimmunoprecipitation was performed to unveil interactions between p85S6K and the GluA1 subunit of AMPA receptor.The roles of p85S6K in synaptic targeting of GluA1 and learning and memory were evaluated by specific knockdown or overexpression of p85S6K followed by a broad range of methodologies including immunofluorescence,Western blot,in situ proximity ligation assay,morphological staining and behavioral examination.Further,the expression level of p85S6K was measured in brains from AD patients and AD model mice.Results p85S6K,but not p70S6K,was enriched in the postsynaptic densities.Moreover,knockdown of p85S6K resulted in defective spatial and recognition memory.In addition,p85S6K could interact with the GluA1 subunit of AMPA receptor through synapse-associated protein 97 and A-kinase anchoring protein 79/150.Mechanistic studies demonstrated that p85S6K could directly phosphorylate GluA1 at Ser845 and increase the amount of GluA1 in syn-apses,thus sustaining synaptic function and spine densities.Moreover,p85S6K was found to be specifically decreased in the synaptosomal compartment in the brains of AD patients and AD mice.Overexpression of p85S6K ameliorated the synaptic deficits and cognitive impairment in transgenic AD model mice.Conclusions These results strongly imply a significant role for p85S6K in maintaining synaptic and cognitive function by interacting with GluA1.The findings provide an insight into the rational targeting of p85S6K as a therapeutic potential for AD.

核糖体S6蛋白激酶4变异体基因表达对乳腺癌细胞增殖的影响

目的探讨核糖体S6蛋白激酶4变异体(RSK4m)对乳腺癌细胞株MDA-MB-231增殖的影响。方法将RSK4m的过表达载体稳定转染至乳腺癌M DA-M B-231细胞株中,分别设一个阴性对照组(mock组)及过表达RSK4m1组(OE1组)、过表达RSK4m2组(OE2组)、过表达RSK4m3组(OE3组),采用实时荧光定量PCR(qRTPCR)和Western blotting检测RSK4m的mRNA和蛋白的表达,CCK-8试剂盒检测转染后M DA-M B-231细胞的生长增殖,免疫共沉淀试验(CO-IP)验证热休克蛋白与RSK4变异体的相互作用。结果成功转染RSK4m慢病毒表达载体至乳腺癌MDA-MB-231细胞,其mRNA和蛋白水平均过表达(P均<0.05)。OE1组、OE2组、OE3组在24、48、72、96 h的细胞抑制率(%)与mock组相比,48、72、96 h的差异具有统计学意义(P<0.05),CO-IP结果证实热休克蛋白与RSK4m存在相互作用关系。结论 RSK4m可抑制乳腺癌细胞株MDA-MB-231的增殖,并可能与RSK4w的抑制增殖作用存在着不同的调控机制。

AMPK-associated signaling to bridge the gap between fuel metabolism and hepatocyte viability

The adenosine monophosphate-activated protein kinase (AMPK) and p70 ribosomal S6 kinase-1 pathway may serve as a key signaling flow that regulates energy metabolism; thus, this pathway becomes an attractive target for the treatment of liver diseases that result from metabolic derangements. In addition, AMPK emerges as a kinase that controls the redox-state and mitochondrial function, whose activity may be modulated by antioxidants. A close link exists between fuel metabolism and mitochondrial biogenesis. The relationship between fuel metabolism and cell survival strongly implies the existence of a shared signaling network, by which hepatocytes respond to challenges of external stimuli. The AMPK pathway may belong to this network. A series of drugs and therapeutic candidates enable hepatocytes to protect mitochondria from radical stress and increase cell viability, which may be associated with the activation of AMPK, liver kinase B1, and other molecules or components. Consequently, the components downstream of AMPK may contribute to stabilizing mitochondrial membrane potential for hepatocyte survival. In this review, we discuss the role of the AMPK pathway in hepatic energy metabolism and hepatocyte viability. This information may help identify ways to prevent and/or treat hepatic diseases caused by the metabolic syndrome. Moreover, clinical drugs and experimental therapeutic candidates that directly or indirectly modulate the AMPK pathway in distinct manners are discussed here with particular emphasis on their effects on fuel metabolism and mitochondrial function.

Hepatitis C virus infection and insulin resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.

Effect of Metformin-Induced Stimulation on the Expression of Insulin Receptor Substrate 1 through Negative Regulation of P70S6k

Objective:The aim is to study the effects of metformin on the expression of 70 kDa ribosomal protein S6 kinase(P70S6k),insulin receptor substrate 1(IRS-1),and IRS-1Ser307 phosphorylation in human luteinized granulosa cells.Methods:Granulosa cells in the experimental group were cultured in M199 medium containing 0.1 mmol/L metformin for 24 h and those in control group were cultured in M199 medium.The expression levels of P70S6k and IRS-1 mRNA were detected by reverse-transcriptiom polymerase chain reaction(RT-PCR)and real-time PCR.P70S6k,IRS-1,p-ser307-IRS-1,and p-thr389-P70S6k protein expression levels were detected by immunofluorescence and western blotting.Results:P70S6k mRNA level was higher and IRS-1 was significantly lower in the experimental group than those in the control group.IRS-1 and p-ser307-IRS-1 were expressed in cell plasma,and P70S6k and p-thr389-P70S6k were expressed in cell nucleus.The results of Western blot analysis indicated that the expression levels of P70S6k,p-thr389-P70S6k,IRS-1,and p-ser307-IRS-1 proteins had significant difference between the experimental group and the control group.Compared to the control group,the relative intensity illustrated that the expression levels of P70S6K and p-thr389-P70S6k significantly increased in the experimental group;however,those of IRS-1 and p-ser307-IRS-1 proteins significantly decreased.Conclusion:Metformin can inhibit the P70S6k mRNA and protein expression levels in the granulosa cells and improve insulin sensitivity by regulating IRS-1 expression through Akt/P70S6k/IRS-1-dependent pathway.

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator

The mammalian target of rapamycin （mTOR） pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I （10 pg）, induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase （p70 S6K） and eukaryotic initiation factor 4E-binding protein 1 （4E-BP1）, in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4E- BP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses （spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity）. Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

Oral everolimus inhibits intimal proliferation in injured carotid artery in rats

Background Everolimus, a derivative of sirolimus, is a potent immunosuppressant that has important anti-proliferative properties. In the present study, we demonstrated the inhibiting neointimal hyperplasia in injured carotid arteries in rats by using two different doses of everolimus administrated via the oral route for a long time. Methods A rat model of carotid artery injury was established by balloon inflation. Eighty rats were randomly divided into the sham-operated group （n=20）, injury group （n=20）, low dosage of everolimus group （n=20）, and high dosage of everolimus group （n=20）. The low close of everolimus （1.5 mg/kg） was given one day before injuring the carotid artery by balloon, followed by 0.75 mg/kg per day for 28 days via intragastric gavage. High dose everolimus （2.5 mg/kg） was given one day before injuring the carotid artery by balloon, followed by 1 mg/kg per day for 28 days. Expression of eukaryotic translation initiation factor 4E （elF-4E） and phosphorylation of ribosomal proteinS6 kinase 1 （P70S6K） were determined by reverse transcription-polymerase chain reaction and Western blotting analysis. Results In the injured carotid artery, neointimal hyperplasia was normally observed four weeks after injury. Everolimus inhibited neointimal hyperplasia after balloon injury in a dose dependent manner. At the same time, the study demonstrated that everolimus reduced the expression of P-P70S6K, elF-4E, transforming growth factor （TGF）-131 and of proliferating cell nuclear antigen （PCNA）. Conclusions Everolimus significantly inhibited neointimal hyperplasia of the injured carotid artery. The effect depended on dosaqe and was associated with the reduction of phosphorylation of P70S6K and the elF-4E expression level.