Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.     

ESAT6通过mTOR抑制自噬并促进BCG增殖

目的 研究mTOR在结核杆菌毒力因子ESAT6诱导的自噬抑制以及促进BCG增殖中的作用。方法 PCMV-HA-ESAT6质粒转染Raw264.7细胞,用蛋白免疫印迹检测LC3、P62、P-mTOR和P-70S6K表达水平;用mTOR阻断剂Torin1联合ESAT6转染以及分别作用于Raw264.7细胞后,免疫印迹检测P62和P-mTOR表达水平,LysoTracker Red染色观察溶酶体变化,BCG增殖实验计数各组菌落数。结果 ESAT6转染细胞后,细胞P62、P-mTOR和P-70S6K表达水平显著增高,LC3I完成向LC3II的转化;联合Torin1的ESAT6转染组和Torin1处理组的P-mTOR和P62无显著变化,溶酶体无变化,BCG菌落数减少。结论 ESAT6诱导的自噬抑制和BCG的增殖依赖于mTOR的活化。     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.     

Synthesis and structure–activity relationships of PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway by PI3K/mTOR dual inhibitors provides a promising new approach to the treatment of cancers. In this Letter, we identified structurally novel and potent PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives. Their synthesis and structure–activity relationships are reported.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

Redd1 inhibits the invasiveness of non-small cell lung cancer cells

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.     

SP600125 negatively regulates the mammalian target of rapamycin via ATF4-induced Redd1 expression

SP600125 (SAPK Inhibitor II) is reported to function as a reversible ATP competitive inhibitor of c-Jun N-terminal kinase (JNK). In the present study, we show that SP600125 induces a dose-dependent decrease in mTOR activity, as assessed by reduced phosphorylation of the downstream targets S6K1 and S6, and a significant increase in the expression of Redd1. Knockdown of Redd1 expression by siRNA resulted in a recovery of decreased S6 phosphorylation by SP600125. Overexpression of ATF4 upregulated the expression of Redd1, while suppression of ATF4 expression by siRNA enhanced the level of S6 phosphorylation by downregulating the SP600125-induced increase in Redd1 expression. Together, these results indicate that SP600125 inhibits mTOR activity via an ATF4-induced increase in Redd1 expression.     

Arecoline and the 30–100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified ≥10 kDa fraction (ANE ≥ 10 K) and subsequently to the 30–100 kDa fraction (ANE 30–100 K). ANE and ANE ≥10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE ≥ 10 K, and ANE 30–100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30–100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser²⁴⁴⁸. In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser²⁴⁴⁸ phosphorylation are capable of triggering apoptosis and autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Shyun-Yeu Liu, Mei-Huei Lin, Yu-Rung Hsu and Ya-Yun Shih contributed equally to this work.     

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin-separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2-M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1-securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.     

AMPK represses TOP mRNA translation but not global protein synthesis in liver

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5′-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1α. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or β-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.     

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.