mTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1- pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.     

Sorting through the extensive and confusing roles of sortilin in metabolic disease

Sortilin is a post-Golgi trafficking receptor homologous to the yeast vacuolar protein sorting receptor 10 (VPS10). The VPS10 motif on sortilin is a 10-bladed β-propeller structure capable of binding more than 50 proteins, covering a wide range of biological functions including lipid and lipoprotein metabolism, neuronal growth and death, inflammation, and lysosomal degradation. Sortilin has a complex cellular trafficking itinerary, where it functions as a receptor in the trans-Golgi network, endosomes, secretory vesicles, multivesicular bodies, and at the cell surface. In addition, sortilin is associated with hypercholesterolemia, Alzheimer’s disease, prion diseases, Parkinson’s disease, and inflammation syndromes. The 1p13.3 locus containing SORT1, the gene encoding sortilin, carries the strongest association with LDL-C of all loci in human genome-wide association studies. However, the mechanism by which sortilin influences LDL-C is unclear. Here, we review the role sortilin plays in cardiovascular and metabolic diseases and describe in detail the large and often contradictory literature on the role of sortilin in the regulation of LDL-C levels.     

C9orf72: At the intersection of lysosome cell biology and neurodegenerative disease

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith‐Magenis chromosome region 8 (SMCR8) and WD repeat‐containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72‐containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.      

Multisite Phosphorylation of S6K1 Directs a Kinase Phospho-code that Determines Substrate Selection

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Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.     

Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as mTORC1/mTORC2 dual inhibitors

New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives, were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target compounds were synthesized and structural optimization on the structure of the lead compound based on cytotoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the compound 12q showed excellent activity with an IC₅₀ value of 54 nM in the same level of the positive control BEZ235 with IC₅₀ value of 55 nM under the same test conditions. The western blot and cell cycle results demonstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR active site.