SH3P2 is a negative regulator of cell motility whose function is inhibited by ribosomal S6 kinase-mediated phosphorylation

Although the extracellular signal-regulated kinase (ERK) pathway functions downstream of Ras in induction of the cell motility response, the detailed molecular mechanism by which this pathway regulates cell motility has remained elusive. The application of a functional expression cloning strategy to discover proteins that regulate cell motility has resulted in the identification of an SH3 domain-containing protein, SH3P2. Overexpression of SH3P2 in HeLa S3 cells inhibited cell motility, whereas RNA interference-mediated depletion of SH3P2 enhanced motility in various tumor cell lines, suggesting that SH3P2 functions as a negative regulator of cell motility. The expression level of SH3P2 alone did not correlate well with the motility of tumor cells, however. SH3P2 was phosphorylated on Ser(202) by ribosomal S6 kinase (RSK) in an ERK pathway-dependent manner, and such phosphorylation inhibited the ability of SH3P2 to suppress cell motility. The RSK inhibitor BI-D1870 suppressed SH3P2 phosphorylation and tumor cell motility as effectively as did the MEK inhibitor PD184352. Furthermore, expression of the unphosphorylatable SH3P2 mutant SH3P2(S202A) inhibited tumor cell motility, indicating that phosphorylation of SH3P2 at Ser(202) is a key determinant of such motility. These results suggest that SH3P2 is an essential molecule that functions downstream of the ERK pathway to modulate cell motility.     

PI3K/Akt/mTOR的信号传导通路在恶性肿瘤中的研究进展

真核生物对诸如生长因子受体、激素、细胞因子等刺激产生应答,激活细胞内不同信号传导通路,其中PI3K／Akt／mTOR是与细胞增殖和细胞凋亡关系最密切的信号传导通路之一。随着研究的不断深入,该通路在多种常见肿瘤如乳腺癌、结直肠癌、前列腺癌、卵巢癌、肝癌、肺癌及淋巴瘤等的发生发展中凸现出日益重要的作用,并为肿瘤的治疗提供了新的靶点。     

Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E

The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. We find that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E-Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size.     

Activation of the PI3K/Akt/mTOR/p70S6K Pathway is Involved in S100A4-induced Viability and Migration in Colorectal Cancer Cells

The S100 protein family member S100A4 regulates various cellular functions. Previous studies have shown that elevated expression of S100A4 is associated with progression and metastasis of colorectal cancer (CRC). However, little is known about whether and how S100A4 contributes to CRC development. In our present study, the elevated expression of S100A4 in CRC tissues compared to matched adjacent normal tissues was confirmed by immunohistochemistry, semi-quantitative RT-PCR and Western blot. Adenovirus-mediated S100A4 overexpression obviously enhanced viability and migration of CRC cells, which was detected by MTT assay and transwell assay, respectively. Additionally, S100A4 overexpression increased the phosphorylation levels of Akt, mTOR and p70S6K. These effects of S100A4 were abolished by treatment with either the specific PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or the specific mTOR/p70S6K inhibitor rapamycin. Furthermore, overexpression of S100A4 resulted in upregulation of VEGF and downregulation of E-cadherin, which were strongly reversed by either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or rapamycin. Altogether, our results demonstrate that activation of the PI3K/Akt/mTOR/p70S6K signaling pathway is involved in S100A4-induced viability, migration, upregulation of VEGF and downregulation of E-cadherin in CRC cells.     

鲍曼不动杆菌外膜蛋白A通过Akt/mTOR/p70S6K信号通路引起RAW264.7细胞自噬

目的:分析鲍曼不动杆菌标准株ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白A(outer membrane protein A,Omp A)对RAW264. 7细胞的自噬诱导作用。方法:建立Omp A刺激RAW264. 7细胞的模型,通过细胞免疫荧光染色、Western blot和透射电子显微镜检测Omp A对RAW264. 7细胞自噬的影响。结果:Omp A可以引起自噬蛋白LC3B-II表达升高,并抑制Akt/mTOR/p70S6K的磷酸化水平;雷帕霉素可以进一步降低mTOR和p70S6K磷酸化,并提高Omp A引起的LC3B-II表达升高。结论:鲍曼不动杆菌Omp A通过Akt/mTOR/p70S6K信号通路引起RAW264. 7细胞自噬。这为将来进一步研究鲍曼不动杆菌引起自噬的分子机制及找到对抗其感染的新方法提供理论依据。     

Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonie epithelial cells (HT-29/B6)

We investigated the inhibitory action of K⁺ channel blockers on carbachol-stimulated Ca²⁺ entry into human Cl^–-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K⁺ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 M) caused a clearly biphasic intracellular calcium (Ca_i response: Ca_i was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca²⁺ and represented a new steady state between Ca²⁺ entry and exit across the plasma membrane. A monophasic Ca²⁺ response was induced in the absence of external Ca²⁺ and after the initial peak Ca_i returned to baseline. The Ca_i plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 M) or the inorganic Ca²⁺ channel blocker lanthanum (effective concentration for 50% inhibition of Ca₁ plateau EC₅₀=68±18 nM), but it was unaffected by the organic Ca²⁺ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K⁺ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca²⁺ entry. The Ca_i plateau was calculated to be 50% inhibited by barium (96±2 M), lidocaine (74±3 M) and NPPB (27±10 M). The Ca_i plateau was transiently increased by 1 M and 10 M NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl^– channels (so that the membrane potential approached V _K). The resting Ca_i was transiently increased by 50 M and 300 M NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca²⁺ release from internal Ca stores. We conclude that carbachol-stimulated Ca²⁺ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca_i levels to K⁺ channel blockers indicates that there are feedback relationships among rates of Ca²⁺ entry, activity of Ca²⁺-activated K⁺ and Cl^– channels and membrane potential.     

Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response

Ribosome biogenesis has been associated with regulation of cell growth and cell division, but the molecular mechanisms that integrate the effect of ribosome biogenesis on these processes in mammalian cells remain unknown. To study the effect of impaired ribosome functions in vivo, we conditionally deleted one or two alleles of the 40S ribosomal protein S6 gene in T cells in the mouse. While complete deletion of S6 abrogated T-cell development, hemizygous expression did not have any effect on T-cell maturation in the thymus, but inhibited the accumulation of T cells in the spleen and lymph nodes, as a result of their decreased survival in the peripheral lymphoid organs. Additionally, TCR-mediated stimulation of S6-heterozygous T cells induced a normal increase in their size, but cell cycle progression was impaired. Genetic inactivation of p53 tumor suppressor rescued development of S6-homozygous null thymocytes and proliferative defect of S6-heterozygous T cells. These results demonstrate the existence of a p53-dependent checkpoint mechanism that senses changes in the fidelity of the translational machinery to prevent aberrant cell division or eliminate defective T cells in vivo. Failure to activate this checkpoint response could potentially lead to a development of pathological processes such as tumors and autoimmune diseases.     

CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent

OBJECTIVE: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE2 and PGI2 secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. MATERIALS AND METHODS: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE2, PGI2 and IL-6 synthesis. RESULTS: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154+ D1.1 cells but not CD154- B2.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE2 and PGI2 secretion triggered by CD154+ D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE2 triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. CONCLUSIONS: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.     

Oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling

Oleanolic acid has significant pharmacological activities, such as anti-tumor, regulating blood sugar level and liver protection, which are more effective compared with free aglyconeoleanolic acid. However, it is still unknown if oleanolic acid affects the proliferation of human bladder cancer. We utilized T24 cells to study the effect of oleanolic acid on the proliferation and apoptosis of human bladder cancer. In this study, we found that the anti-cancer effect of oleanolic acid significantly suppressed cell proliferation and increased apoptosis and caspase-3 activity of T24 cells. Furthermore, Akt, mTOR and S6K protein expression was greatly inhibited in T24 cells under oleanolic acid treatment. Meanwhile, ERK1/2 of phosphorylation protein expression was significantly promoted by oleanolic acid treatment. Taken together, we provided evidences that oleanolic acid was Akt/mTOR/S6K and ERK1/2 signaling-targeting anti-tumor agent. These findings represent new evidences that oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, and oleanolic acid may be used to prevent human bladder cancer.     

mTOR/S6K1在2型糖尿病Wistar大鼠肝脏和肌肉的表达

目的应用Wistar大鼠制备2型糖尿病的动物模型,观察肝脏和肌肉组织mTOR/Rps6kb1(S6K1)表达的变化。方法雄性Wistar成年大鼠高脂饲料喂养8周,腹腔注射链脲佐菌素(STZ)25mg/kg,注射后第4周检测空腹血糖、胰岛素水平,对结果进行统计分析;解剖大鼠,取肝脏和肌肉组织,应用RT-PCR和Western blot检测肝脏和肌肉组织mTOR及S6K1表达。结果与正常对照组比较,模型组大鼠血糖值升高(0.05),胰岛素水平下降(0.05),肝脏和肌肉中的mTOR/S6K1蛋白和mRNA表达均升高(0.05)。结论 mTOR及S6K1在2型糖尿病大鼠肝脏和肌肉过表达,提示其可能参与2型糖尿病发病过程。     

Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase,a key mediator of mTOR function

Current understanding of the mechanisms by which cell growth is regulated lags significantly behind our knowledge of the complex processes controlling cell cycle progression. Recent studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The key mTOR effectors of cell growth are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (S6K). Here we will review the current models for mTOR dependent regulation of ribosome function and biogenesis as well as its role in coordinating growth factor and nutrient signaling to facilitate homeostasis of cell growth and proliferation. We will place particular emphasis on the role of S6K1 signaling and will highlight the points of cross talk with other key growth control pathways. Finally, we will discuss the impact of S6K signaling and the consequent feedback regulation of the PI3K/Akt pathway on disease processes including cancer.     

Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function

Current understanding of the mechanisms by which cell growth is regulated lags significantly behind our knowledge of the complex processes controlling cell cycle progression. Recent studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The key mTOR effectors of cell growth are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (S6K). Here we will review the current models for mTOR dependent regulation of ribosome function and biogenesis as well as its role in coordinating growth factor and nutrient signaling to facilitate homeostasis of cell growth and proliferation. We will place particular emphasis on the role of S6K1 signaling and will highlight the points of cross talk with other key growth control pathways. Finally, we will discuss the impact of S6K signaling and the consequent feedback regulation of the PI3K/Akt pathway on disease processes including cancer.     

IL3-dependent cells die by apoptosis on removal of their growth factor

The IL3-dependent cell line FDCP-2 dies within 32 h of removal of IL3. Electron microscope studies indicate that 22 h after IL3 removal the nuclei are condensed, but the morphology of mitochondria and ribosomes is preserved. This pattern is characteristic of apoptosis. IL3 removal also results in the fragmentation of DNA into nucleosome-sized pieces, suggesting that an endonuclease is activated. The protein synthesis inhibitor, cycloheximide, enhances survival on IL3 removal, suggesting that death is an active process. The nuclease inhibitor, aurintricarboxylic acid, also enhances survival, suggesting a causal role for DNA fragmentation in apoptosis.     

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.     

通心络通过PI-3K/Akt/HIF信号通路改善血管内皮细胞缺氧损伤

目的: 观察通心络对缺氧血管内皮细胞中缺氧诱导因子(HIF)及其上游信号转导通路PI-3K/Akt的影响,探讨PI-3K/Akt/HIF信号通路在通心络改善血管内皮细胞抗缺氧损伤中的作用。方法: 将体外培养的人脐静脉血管内皮细胞(HUVECs)分为常氧对照组、通心络组、缺氧组和缺氧+通心络组。采用CCK-8试剂盒检测各组细胞的活性,Western blotting 检测HIF-1α、Bcl-2、Mcl-1、Bax表达变化及Akt的磷酸化情况。利用HIF-1α的显性负性突变体(DN-HIF)瞬时转染HUVECs,流式细胞仪分析细胞的凋亡情况。利用PI-3K和Akt的显性负性突变体瞬时转染HUVECs,探讨PI-3K/Akt信号通路在通心络改善血管内皮细胞抗缺氧损伤中的作用。结果: 与常氧条件下比较,通心络对缺氧内皮细胞虽有明显的促增殖作用,但程度明显减弱。通心络可显著上调HIF-1α、Bcl-2、Mcl-1蛋白表达水平及Akt磷酸化水平,且下调Bax的表达。利用DN-HIF抑制HIF-1α的活化后,通心络提高缺氧HUVECs活性的程度明显下降,但仍可一定程度上降低细胞凋亡百分率。进一步利用PI-3K显性负性突变体(Δp85)和Akt显性负性突变体(DN-Akt)阻断HIF-1α上游信号通路PI-3K/Akt后,通心络上调缺氧HUVECs HIF-1α蛋白表达的作用明显减弱,促进Akt磷酸化的作用基本消失。结论: 在缺氧条件下,通心络上调HUVECs HIF-1α蛋白表达水平,促进抗凋亡因子同时抑制促凋亡因子的表达,降低细胞凋亡率,从而提高缺氧细胞的增殖率,这些作用在一定程度上依赖于PI-3K/Akt/HIF通路的活性。     

ATP-mediated protein kinase B Akt/mammalian target of rapamycin mTOR/p70 ribosomal S6 protein p70S6 kinase signaling pathway activation promotes improvement of locomotor function after spinal cord injury in rats

The protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6K) signaling pathway, as a central controller of cell growth, proliferation, survival, and differentiation in response to extracellular signals, growth factors, nutrient availability, energy status of the cell, and stress, has recently gained attention in neuroscience. The effects of this signaling pathway on repair of spinal cord injury (SCI), however, have not been well elucidated. ATP is increasingly recognized as an important regulator of signal transduction pathways, and plays important roles in functional recovery after nervous system injury. In the present study, we examined the ATP-induced changes of the Akt/mTOR/p70S6K signaling pathway in injured spinal cord of adult rats and potential therapeutic effects of this pathway on SCI-induced locomotor dysfunction. SCI was produced by extradural weight-drop using modified Allen's stall with damage energy of 50 g-cm force. The rats were divided into four groups: SCI plus ATP, SCI plus saline, SCI plus ATP and rapamycin, and sham-operated. Using immunostaining studies, Western blot analyses and real-time qualitative RT-PCR analyses, we demonstrated that the Akt/mTOR/p70S6K signaling pathway is present in the injured spinal cord and the expression of its components at the protein and mRNA levels is significantly elevated by exogenous administration of ATP following SCI. We observed the effectiveness of the activated Akt/mTOR/p70S6K signaling pathway in improving locomotor recovery, significantly increasing the expression of nestin, neuronal nuclei (NeuN), neuron specific enolase (NSE), and neurofilament 200 (NF200), and relatively inhibiting excessive reactive astrogliosis after SCI in a rapamycin-sensitive manner. We concluded that ATP injection produced a significant activation of the Akt/mTOR/p70S6K signaling pathway in the injured spinal cord and that enhancement of rapamycin-sensitive signaling produces beneficial effects on SCI-induced motor function defects and repair potential. We suggest that modulation of this protein kinase signaling pathway activity should be considered as a potential therapeutic strategy for SCI.     

mTOR_P70S6K信号通路在小儿脑胶质瘤中的表达及其临床意义

目的:探讨mTOR_P70S6K信号通路在小儿脑胶质瘤组织中的表达及其临床意义。方法:随机选取脑胶质瘤患儿88例,采用SP免疫组化方法,检测脑胶质瘤组织和正常脑组织中mTOR蛋白及P70S6K蛋白的表达。并分析其与脑胶质瘤组织临床分级之间的关系。结果:mTOR蛋白及P70S6K蛋白在脑胶质瘤组织中的阳性表达率分别为81.58%(31/38)和78.95%(30/38),明显高于其在正常脑组织中的阳性表达率[分别为15%(3/20)和10%(2/20)](P<0.01)。mTOR蛋白和P70S6K蛋白在脑胶质瘤组织中的表达呈正相关(r=0.68,P<0.05),且两者在正常脑组织中的表达亦正相关性(r=0.89,P<0.01)mTOR蛋白及P70S6K蛋白阳性表达率在脑胶质瘤高级别组(Ⅲ~Ⅳ级)中的表达均明显高于低级别组(Ⅰ~Ⅱ级),说明随着肿瘤级别的升高二者的表达也增强。结论:mTOR_P70S6K信号通路在小儿脑胶质瘤中发生了特异性的激活。该通路可能与小儿脑胶质瘤的发生、发展密切相关。     

Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis

The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6(P-/-)). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6(P-/-) mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6(P+/+) MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6(P-/-) MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic beta-cells in adult rpS6(P-/-) mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.