Phosphorylation Of P70 S6 Kinase Research Articles

Abstract 2948: Synergistic effect of combining Akt inhibitor SC66 with anticancer drugs cisplatin and paclitaxel in chemoresistant ovarian cancer cells

Abstract Background and aims Akt plays a central role in regulating cell growth and cell cycle progression and is regarded as a promising therapeutic target. We examined whether Akt inhibition by SC66 is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with chemotherapy drugs. Methods Using eight human ovarian cancer cell lines, we determined the effect of SC66 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, western blot, and apoptosis assays. We evaluated the association between phospho-Akt/mTOR, COL11A1 and SC66 sensitivity. We also determined the effect of SC66 on tumor growth using mice inoculated with human ovarian cancer cells. Results Cell sensitivity to SC66 was inversely correlated with the expressions of phosphorylation of Akt and COL11A1, and cisplatin resistance. SC66 inhibited the phosphorylation of downstream 4E-BP1 and p70S6 kinase. SC66 also attenuated the expression of TWIST1 and Mcl-1, important factors in invasiveness and anti-apoptosis. SC66 sensitizes chemo-resistant cells to cisplatin and paclitaxel treatment, and enhanced apoptosis in the chemo-resistant cells. In addition, SC66 inhibited COL11A1 expression via decreasing the binding activity of c/EBPβ and NF-YA to the COL11A1 promoter, which was associated with chemoresistance and cell invasiveness respectively. In vivo, treatment with SC66 enhanced the therapeutic efficacy of cisplatin and paclitaxel in mouse xenograft. Conclusions These results indicate the SC66 could have therapeutic efficacy in human ovarian cancers. Citation Format: Yi-Hui Wu, Cheng-Yang Chou. Synergistic effect of combining Akt inhibitor SC66 with anticancer drugs cisplatin and paclitaxel in chemoresistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2948.

Phosphorylated P70 S6 Kinase and S6 Ribosomal Protein Value as Diagnostic Marker of Antibody-mediated Rejection in Lung Allografts: A Multicenter Study

Antibody-mediated rejection (AMR) is emerging as an important cause of solid organ transplantation failure. The role of AMR in lung allografts is a source of ongoing investigation, as well as the identification of more sensitive and accurate diagnostic features. The mammalian target of rapamycin (mTOR) pathway, in particular phosphorylated P70 S6 kinase (S6K) and S6 ribosomal protein (S6RP), is reported to be associated with AMR in heart and kidney transplantation. The main goal of the study was to evaluate S6K and S6RP expression in lung transplanted (LT) patients with AMR from different LT centers.

Increased Phosphorylation of p70S6 Kinase is Associated With CMV Reactivation in Lung Transplant Patients

Increased Phosphorylation of p70S6 Kinase is Associated With CMV Reactivation in Lung Transplant Patients

The miR-139-5p regulates proliferation of supratentorial paediatric low-grade gliomas by targeting the PI3K/AKT/mTORC1 signalling.

Paediatric low-grade gliomas (pLGGs) are a heterogeneous group of brain tumours associated with a high overall survival: however, they are prone to recur and supratentorial lesions are difficult to resect, being associated with high percentage of disease recurrence. Our aim was to shed light on the biology of pLGGs. We performed microRNA profiling on 45 fresh-frozen grade I tumour samples of various histological classes, resected from patients aged ≤16 years. We identified 93 microRNAs specifically dysregulated in tumours as compared to non-neoplastic brain tissue. Pathway analysis of the microRNAs signature revealed PI3K/AKT signalling as one of the centrally enriched oncogenic signalling. To date, activation of the PI3K/AKT pathway in pLGGs has been reported, although activation mechanisms have not been fully investigated yet. One of the most markedly down-regulated microRNAs in our supratentorial pLGGs cohort was miR-139-5p, whose targets include the gene encoding the PI3K's (phosphatidylinositol 3-kinase) catalytic unit, PIK3CA. We investigated the role of miR-139-5p in regulating PI3K/AKT signalling by the use of human cell cultures derived from supratentorial pLGGs. MiR-139-5p overexpression inhibited pLGG cell proliferation and decreased the phosphorylation of PI3K target AKT and phosphorylated-p70 S6 kinase (p-p70 S6K), a hallmark of PI3K/AKT/mTORC1 signalling activation. The effect of miR-139-5p was mediated by PI3K inhibition, as suggested by the decrease in proliferation and phosphorylation of AKT and p70 S6K after treatment with the direct PI3K inhibitor LY294002. These findings provide the first evidence that down-regulation of miR-139-5p in supratentorial pLGG drives cell proliferation by derepressing PI3K/AKT signalling.

Abstract P3-06-08: Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2

Abstract Breast cancer is a major cause of cancer-related death and there is a need for novel therapies with increased efficacy and decreased toxicity. The small molecule ONC201 was initially identified as a TRAIL pathway inducer. The compound has entered early phase clinical trials and is being tested in a range of solid tumors and hematological malignancies. Our previously published data demonstrate that ONC201 has potent anti-proliferative and pro-apoptotic effects in a broad range of breast cancer subtypes through TRAIL-dependent and TRAIL-independent mechanisms. Analogs of ONC201 with a shared pharmacophore have been developed and form the novel “imipridone” class. When compared with ONC201, imipridones ONC206, ONC212, and ONC213 showed increased potency of anti-proliferative or pro-apoptotic effects in breast cancer cells. We were interested in further defining the previously unstudied anti-proliferative effects of the imipridone compounds. Single agent efficacy of potent imipridone ONC212 in a xenograft model of TNBC was observed in the absence of apoptosis induction. This indicates that the anti-proliferative actions of the compound are sufficient for an in vivo anti-tumor effect. Our lab has previously shown that ONC201 activates an ATF4-dependent integrated stress response (ISR), essential for apoptosis induction in colon cancer cells. In contrast, in breast cancer cells, although ATF4 knockdown did not block cell death induced by ONC201 it did partially abrogate the anti-proliferative effects of the compound. The mammalian target of rapamycin complex 1 (mTORC1) is a well-known regulator of cellular growth and proliferation. mTORC1 signaling is inactivated in breast cancer cells following treatment with ONC201 and its analogs ONC212 and ONC213, regardless of whether the cells undergo apoptosis. Knockdown of ATF4 abrogated ONC201-mediated inhibition of p70 S6 kinase and ribosomal protein S6 phosphorylation, linking ISR induction to mTORC1 inhibition. We hypothesized that sestrin2, an endogenous mTORC1 inhibitor known to be upregulated following cellular stress, might represent a link between induction of ATF4 and inhibition of mTORC1. Treatment with ONC201 and its analogs ONC212 and ONC213 in multiple breast cancer cell lines resulted in sestrin2 upregulation. This was blocked by ATF4 knockdown. Furthermore, knockdown of sestrin2 abrogated the effects of ONC201 on mTORC1 signaling in breast cancers from multiple molecular subtypes. Previous mechanistic studies have focused exclusively on the relevance of ATF4 in the pro-apoptotic effects of ONC201. The novel findings described here help to elucidate the mechanism behind the potent and understudied anti-proliferative effects of the imipridones. Our findings also strengthen the preclinical rationale for testing of imipridone compounds against breast cancers regardless of molecular subtype. Citation Format: Ralff MD, Kline CLB, El-Deiry WS. Imipridone compounds inhibit breast cancer mTORC1 signaling through integrated stress response-mediated upregulation of endogenous mTORC1 inhibitor sestrin2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-08.

Leucyl-tRNA synthetase is required for the myogenic differentiation of C2C12 myoblasts, but not for hypertrophy or metabolic alteration of myotubes

Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 (Igf2) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR–IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes.

Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes

ABSTRACT Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Bufadienolides from Venenum Bufonis Inhibit mTOR‐Mediated Cyclin D1 and Retinoblastoma Protein Leading to Arrest of Cell Cycle in Cancer Cells

Objective Bufadienolides, the main components in Venenum Bufonis secreted from toads, have been proved to be with significant anticancer activity aside from the positive inotropic action as cardenolides. Here an underlying anticancer mechanism was further elucidated for an injection made from Venenum Bufonis containing nine bufadienolides. Methods One solution reagent and cell cycle analyses were for determining effect of bufadienolides on cancer cells. Western blotting was used for protein expression. Results Bufadienolides inhibit cell proliferation and arrest cells in G1 phase. Bufadienolides also inhibit the mammalian target of rapamycin (mTOR) signaling pathway, which is evidenced by the data that bufadienolides inhibit type I insulin-like growth factor- (IGF-1-) activated phosphorylation of mTOR by a concentration- and time-dependent way, as well as phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Subsequent results indicated that cyclin D1 expression and phosphorylation of retinoblastoma protein (Rb)—two characterized regulators in cell cycle of G1—are also inhibited and the process is dependent on mTOR pathway. Conclusion Bufadienolides inhibit proliferation partially due to arresting cell cycle in G1 phase, which is mediated by inhibiting mTOR-cyclin D1/Rb signal pathway.

Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling.

The antiparkinsonian ropinirole and pramipexole are D3 receptor- (D3R-) preferring dopaminergic (DA) agonists used as adjunctive therapeutics for the treatment resistant depression (TRD). While the exact antidepressant mechanism of action remains uncertain, a role for D3R in the restoration of impaired neuroplasticity occurring in TRD has been proposed. Since D3R agonists are highly expressed on DA neurons in humans, we studied the effect of ropinirole and pramipexole on structural plasticity using a translational model of human-inducible pluripotent stem cells (hiPSCs). Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084 and by the mTOR pathway kinase inhibitors LY294002 and rapamycin. All treatments were also effective in attenuating the D3R-dependent increase of p70S6-kinase phosphorylation. Immunoneutralisation of BDNF, inhibition of TrkB receptors, and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity, suggesting a critical interaction between BDNF and D3R signaling pathways. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpin their reliability for characterization of pharmacological agents acting via dopaminergic mechanisms.

Amyloid β-Derived Diffusible Ligands (ADDLs) Induce Abnormal Autophagy Associated with Aβ Aggregation Degree.

Autophagy is disturbed in Alzheimer's disease (AD) and maintaining normal autophagy homeostasis is a new therapeutic strategy for AD treatment. Amyloid β-derived diffusible ligands (ADDLs), the most toxic species of which are oligomeric forms of amyloid β peptide (Aβ) that originate from amyloid β precursor protein (APP) via autophagy; however, whether ADDLs are involved in autophagy-related AD pathogenesis remains unclear. In this study, we primarily defined the specific subsets of ADDLs, A-0, A-12, A-24, and A-48, which were generated from ADDL aggregation mixtures at different time courses of assembly. The secondary structures of ADDL subsets were detected by circular dichroism (CD). Neuronal or non-neuronal cells were exposed to the subsets of ADDLs in vitro, and then, autophagic markers were detected. Our results first showed that exogenous or endogenous LC3 puncta (autophagosomes) were induced in the cytoplasm of cells exposed to ADDLs and that the LC3 puncta were the strongest with A-24 exposure. Then, the CD spectroscopy data also indicated that the proportion of α-helices decreased, whereas the proportion of β-strands and β-turns increased during ADDL assembly from 0 to 24h. In addition, the quantitative Western blot data demonstrated that the ratio of LC3B-II/I was significantly increased, and SQSTM1/p62 decreased over time. Finally, our results indicated that the level of phosphorylated p70 S6 kinase (p-p70 S6 kinase), which is a substrate protein in the MTOR pathway, and the ratio of p-p70 S6 kinase/p70 S6 kinase significantly decreased following A-24 exposure. Taken together, our data suggest that ADDL-induced abnormal autophagy is correlated with Aβ aggregation degree and the MTOR pathway, which might contribute to ADDL-induced AD pathogenesis.

Suppression by HSP90 inhibitors of BMP‑4‑stimulated osteoprotegerin synthesis in osteoblasts: Attenuation of p70 S6 kinase.

Heat shock protein 90 (HSP90) is an ATP‑dependent ubiquitous molecular chaperon which is important in cell homeostasis. The authors previously demonstrated that bone morphogenetic protein (BMP)‑4 stimulates osteoprotegerin (OPG) production in osteoblast‑like MC3T3‑E1 cells, and that p70 S6 kinase positively regulates the OPG synthesis by BMP‑4. The present study investigated the involvement of HSP90 in the BMP‑4‑stimulated OPG synthesis and the mechanism in MC3T3‑E1 cells. HSP90 inhibitors, 17‑allylamino‑17demethoxy‑geldanamycin (17‑AAG), 17‑dimethylamino‑ethylamino‑17‑demethoxy‑geldanamycin (17‑DMAG) and geldanamycin significantly suppressed the BMP‑4‑stimulated OPG release. Geldanamycin markedly reduced the BMP‑4‑induced mRNA expression of OPG. 17‑AAG and 17‑DMAG significantly attenuated the phosphorylation of p70 S6 kinase induced by BMP‑4 without affecting the BMP‑4‑induced phosphorylation of mothers against decapentaplegic homolog 1/5. The results suggest that HSP90 inhibitors suppress the BMP‑4‑stimulated OPG synthesis in osteoblasts, and that their suppressive effects are exerted through downregulating p70 S6 kinase.

Leucine supplementation after mechanical stimulation activates protein synthesis via L-type amino acid transporter 1 in vitro.

Branched-chain amino acid supplements consumed following exercise are widely used to increase muscle mass. Although both exercise (ie, mechanical stimulation) and branched-chain amino acid leucine supplementation have been reported to stimulate muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway independently, the mechanisms underlying their synergistic effects are largely unknown. Utilizing cultured differentiated C2C12 myotubes, we established a combination treatment model in which the cells were subjected to cyclic uniaxial mechanical stretching (4 h, 15%, 1 Hz) followed by stimulation with 2 mM leucine for 45 min. Phosphorylation of p70 S6 kinase (p70S6K), an mTOR-regulated marker of protein translation initiation, was significantly increased following mechanical stretching alone but returned to the baseline after 4 h. Leucine supplementation further increased p70S6K phosphorylation, with a greater increase observed in the stretched cells than in the non-stretched cells. Notably, the expression of L-type amino acid transporter 1 (LAT1), a stimulator of the mTOR pathway, was also increased by mechanical stretching, and siRNA-mediated knockdown partially attenuated leucine-induced p70S6K phosphorylation. These results suggest that mechanical stretching promotes LAT1 expression and, consequently, amino acid uptake, leading to enhanced leucine-induced activation of protein synthesis. LAT1 has been demonstrated to be a point of crosstalk between exercise- and nutrition-induced skeletal muscle growth.

EGF regulation of proximal tubule cell proliferation and VEGF-A secretion

Proximal tubule cell (PTC) proliferation is critical for tubular regeneration and recovery from acute kidney injury. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF‐A) are important for the maintenance of tubulointerstitial integrity and can stimulate PTC proliferation. We utilized HK‐2 cells, an immortalized human PTC line, to characterize the EGF‐dependent regulation of VEGF‐A secretion and proliferation in PTCs. We demonstrate that EGF stimulates VEGF‐A secretion via the EGF receptor (EGFR) and stimulates cell proliferation via activation of the VEGF receptor, VEGFR‐2. EGFR activation promotes MAPK (ERK1/2) activation and HIF‐1α expression, which are required for basal and EGF‐stimulated VEGF‐A secretion. EGF also stimulates the phosphorylation of P70S6 kinase (P70S6K), the downstream target of mTORC1. Rapamycin decreased basal and EGF stimulated HIF‐1α and enhanced MAPK (ERK1/2) activation, while MAPK (ERK/12) inhibition downregulated HIF‐1α expression and the phosphorylation of p70S6K. EGF stimulation of p70S6K was also independent of p‐AKT. Inhibition of the mTORC1 pathway with rapamycin abolished phosphorylation of p70S6K but had no effect on VEGF‐A secretion, indicating that EGF‐stimulated VEGF‐A secretion did not require mTORC1 pathway activation. We demonstrate evidence of a complex crosstalk between the MAPK/ERK and mTORC1 pathways, wherein MAPK (ERK1/2) activation stimulates p‐P70S6K, while p‐P70S6K activation seems to inhibit MAPK (ERK1/2) in EGF‐treated HK‐2 cells. Our results suggest that EGF stimulates MAPK (ERK1/2) in HK‐2 cells, which in turn increases HIF‐1α expression and VEGF‐A secretion, indicating that VEGF‐A mediates EGF‐stimulated cell proliferation as an autocrine proximal tubular epithelial cell growth factor.

RET Signaling in Prostate Cancer.

Purpose: Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its coreceptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Because both GDNF and GFRα1 are secreted by nerves, we examined the role of RET signaling in prostate cancer.Experimental Design: Expression of RET, GDNF, and/or GFRα1 was assessed. The impact of RET signaling on proliferation, invasion and soft agar colony formation, perineural invasion, and growth in vivo was determined. Cellular signaling downstream of RET was examined by Western blotting.Results: RET is expressed in all prostate cancer cell lines. GFRα1 is only expressed in 22Rv1 cells, which is the only line that responds to exogenous GDNF. In contrast, all cell lines respond to GDNF plus GFRα1. Conditioned medium from dorsal root ganglia contains secreted GFRα1 and promotes transformation-related phenotypes, which can be blocked by anti-GFRα1 antibody. Perineural invasion in the dorsal root ganglion assay is inhibited by anti-GFRα antibody and RET knockdown. In vivo, knockdown of RET inhibits tumor growth. RET signaling activates ERK or AKT signaling depending on context, but phosphorylation of p70S6 kinase is markedly increased in all cases. Knockdown of p70S6 kinase markedly decreases RET induced transformed phenotypes. Finally, RET is expressed in 18% of adenocarcinomas and all three small-cell carcinomas examined.Conclusions: RET promotes transformation associated phenotypes, including perineural invasion in prostate cancer via activation of p70S6 kinase. GFRα1, which is secreted by nerves, is a limiting factor for RET signaling, creating a perineural niche where RET signaling can occur. Clin Cancer Res; 23(16); 4885-96. ©2017 AACR.

Effect of acute treadmill exercise on cisplatin-induced muscle atrophy in the mouse.

Cisplatin, a platinum-based anti-cancer drug, is one of the most effective broad-spectrum anti-cancer agents used against various cancers. It has been recently suggested that low skeletal muscle mass is predictive of mortality in patients with cancer. Although several molecules produced by the actual tumor itself contribute to skeletal muscle impairment, we recently suggested that the administration of cisplatin could increase levels of muscle RING finger-1 (MuRF1) and atrogin-1, possibly leading to muscle atrophy in the mouse. Exercise is an important factor that induces muscle protein synthesis and muscle hypertrophy by enhancing the positive effects of the Akt/mTOR/p70S6 kinase pathway. In the present study, we therefore investigated the effect of treadmill exercise on cisplatin-induced muscle atrophy. C57BL/6J mice were treated with cisplatin (3mg/kg, i.p.) or saline for four consecutive days. On day 4, the quadriceps and gastrocnemius muscles were isolated from the mice. The animals in the treadmill exercise groups were forced to run on a motorized treadmill for 20min once a day for 9days. In addition to muscle mass, the decrease in myofiber diameter associated with cisplatin administration was significantly restored by treadmill exercise. This exercise also significantly attenuated cisplatin-induced upregulation of MuRF1 and atrogin-1 in quadriceps and gastrocnemius muscle. The decreased Akt, p70S6 kinase, and Foxo3a phosphorylation observed with cisplatin treatment was significantly recovered by treadmill exercise in both the muscles. In the present study, myostatin (Mstn) gene expression, upregulated by cisplatin administration, was also attenuated by treadmill exercise. These findings suggest that treadmill exercise could attenuate cisplatin-induced muscle atrophy, at least partially, and could improve prognosis.

Effect of endurance training and branched-chain amino acids on the signaling for muscle protein synthesis in CKD model rats fed a low-protein diet.

A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched-chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately one-half of the animals in each group was subjected to treadmill exercise (15 m/min, 1 h/day, 5 days/wk). After 7 wk, renal function was measured, and soleus muscles were collected to evaluate muscle protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified.

Role of follistatin in muscle and bone alterations induced by gravity change in mice.

Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin-related factors in the hypergravity-induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity-enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor-κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change.

Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

A novel, de novo mutation in the PRKAG2 gene: infantile-onset phenotype and the signaling pathway involved.

PRKAG2 encodes the γ2-subunit isoform of 5'-AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in the regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload, ventricular preexcitation, and hypertrophy. We identified a novel, de novo PRKAG2 mutation (K475E) in a neonate with prenatal onset of HCM. We aimed to investigate the cellular impact, signaling pathways involved, and therapeutic options for K475E mutation using cells stably expressing human wild-type (WT) or the K475E mutant. In human embryonic kidney-293 cells, the K475E mutation induced a marked increase in the basal phosphorylation of T172 and AMPK activity, reduced sensitivity to AMP in allosteric activation, and a loss of response to phenformin. In H9c2 cardiomyocytes, the K475E mutation induced inhibition of AMPK and reduced the response to phenformin and increases in the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Primary fibroblasts from the patient with the K475E mutation also showed marked increases in the phosphorylation of p70S6K and 4E-BP1 compared with those from age-matched, nondiseased controls. Moreover, overexpression of K475E induced hypertrophy in H9c2 cells, which was effectively reversed by treatment with rapamycin. Taken together, we have identified a novel, de novo infantile-onset PRKAG2 mutation causing HCM. Our study suggests the K475E mutation induces alteration in basal AMPK activity and results in a hypertrophy phenotype involving the mechanistic target of rapamycin signaling pathway, which can be reversed with rapamycin.NEW & NOTEWORTHY We identified a novel, de novo PRKAG2 mutation (K475E) in the cystathionine β-synthase 3 repeat, a region critical for AMP binding but with no previous reported mutation. Our data suggest the mutation affects AMP-activated protein kinase activity, activates cell growth pathways, and results in cardiac hypertrophy, which can be reversed with rapamycin.

(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts.

Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.