Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.

AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

In-silico investigation of RPS6KB1 associated cancer inhibitor: a drug repurposing study.

The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments.Communicated by Ramaswamy H. Sarma.

PPM1H is down-regulated by ATF6 and dephosphorylates p-RPS6KB1 to inhibit progression of hepatocellular carcinoma.

We have shown previously that polymorphism of activating transcription factor 6 (ATF6) is associated with susceptibility to hepatocellular carcinoma (HCC). Therefore, genes down-regulated by ATF6 might play a tumor-suppressing role. In the present study, we identified that expression of protein phosphatase magnesium- or manganous-dependent 1H (PPM1H) mRNA and protein can be inhibited by ATF6 in hepatoma cells and mice with liver Atf6 knockdown. Tumor tissues from 134 HCC patients were analyzed by immunohistochemistry, and PPM1H exhibited higher expression levels in adjacent para-cancer tissues than in HCC tissues. Therefore, patients with higher expression of PPM1H had a better prognosis. PPM1H inhibited proliferation, migration, and invasion of hepatoma cells. In addition, PPM1H inhibited induced HCC nodule formation as well as tumor xenograft growth in diethylnitrosamine/CCl4-induced HCC mouse model and nude mouse tumorigenicity assay, respectively. A 3D model of PPM1H was obtained by homology multi-template modeling, and ribosomal protein S6 kinase B1 (RPS6KB1) in the bone morphogenetic protein (BMP)/transforming growth factor ß (TGF-ß) pathway was screened out as the potential substrate of PPM1H by Rosetta. PPM1H could directly dephosphorylate p-RPS6KB1. To conclude, we discovered RPS6KB1 as a new PPM1H dephosphorylation substrate. PPM1H exhibited a suppressive effect on HCC progression by dephosphorylating p-RPS6KB1.

Negative regulation of HBG1/2 expression through S6K by long noncoding RNA NR_120526.

Background: High levels of fetal hemoglobin (HbF) may alleviate clinical symptoms in patients with ß-thalassemia. A previous study showed that the long noncoding RNA NR_120526 (lncRNA NR_120526) might be involved in regulating HbF levels (HBG1/2 gene expression). However, the function and mechanism by which NR_120526 regulates HbF expression remains unknown. Here, we investigated the effect of NR_120526 on HbF and its mechanism so as to provide an experimental basis for treating patients with ß-thalassemia. Methods: Chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) assay, database query, and bioinformatics analysis were performed to explore the proteins that specifically bind to NR_120526 and their interactions. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) were used to determine whether NR_120526 directly regulates the expression of HBG1/2. The NR_120526 gene was knocked out (KO) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in K562 cells. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the messenger RNA (mRNA) and protein expressions of HBG1/2, ribosomal protein S6 kinase B1 (RPS6KB1, S6K), and Ras homologous family member A (RhoA), respectively. Results: We found that NR_120526 interacts with ILF2, ILF3, and S6K. However, ILF2/ILF3 bound to NR_120526 did not interact with HBG1/2, suggesting that NR_120526 may regulate HBG1/2 expression indirectly. The qRT-PCR results showed no statistical difference in the mRNA expression levels of HBG1/2, S6K, and RhoA between the NR_120526-KO group and negative control (NC) group (P>0.05). However, Western blot results showed a significant increase in the protein levels of HBG1/2, S6K, and RhoA in the KO group (P<0.05). It was found that NR_120526 inhibited S6K, thereby downregulating RhoA and leading to decreased HBG1/2 expression. Conclusions: LncRNA NR_120526 negatively regulates the expression of HBG1/2 through S6K. These new findings provide mechanistic insights into the regulation of HbF and offer potential therapeutic targets for precision medicine in patients with ß-thalassemia.

Prognostic and survival impact of BCL9 and RPS6KB1 copy number variation detected from circulating free DNA in hepatocellular carcinoma.

BACKGROUND: Circulating cell-free DNA (cfDNA) is a noninvasive substitute to liver biopsy for hepatocellular carcinoma (HCC) molecular profiling. This study aimed to use cfDNA to investigate copy number variation (CNV) in the BCL9 and RPS6KB1 genes and its impact on prognosis in HCC. METHODS: Real-Time Polymerase Chain Reaction was used to determine the CNV and cfDNA integrity index in 100 HCC patients. RESULTS: CNV gain in BCL9 and RPS6KB1 genes was detected in 14% and 24% of patients, respectively. Gain in CNV of BCL9 associated with risk of HCC in alcohol drinkers and hepatitis C seropositivity. In patients with RPS6KB1 gain, HCC risk increased with a high body mass index, smoking, schistosomiasis, and Barcelona clinical liver cancer stage (BCLC) A. Gain in both genes showed a high risk of HCC with elevated liver enzymes, Schistosomiasis, BCLC C, and PS > 1. The integrity of cfDNA was higher in patients with CNV gain in RPS6KB1 than those harboring CNV gain in BCL9. Lastly, BCL9 gain and BCL9 + RPS6KB1 gain led to higher mortality rates and reduced survival times. CONCLUSION: cfDNA was used to detect BCL9 and RPS6KB1 CNVs, which influence prognosis and can be used as independent predictors of HCC patient survival.

Preliminary Study on Human Adipose Stem Cells Promoting Skin Wound Healing through Notch Signaling Pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) have been documented as possible candidates for wound healing treatment because their use could reinforce the regenerative capacity of many tissues. Human adipose stem cells (hADSCs) have the advantages of easy access, large quantity and easy operation. They can be fully applied in the treatment of skin wounds. OBJECTIVE: In this study, we aim to explore the roles and potential mechanisms of hADSCs in cutaneous wound healing. METHODS: hADSCs were obtained from human subcutaneous fat. Adipocytes and osteocytes differentiated from hADSCs were determined by staining with Oil Red O and alkaline phosphatase (ALP), respectively. We assessed the effects of hADSCs and hADSC conditional medium (CM) on wound healing in an injury model of mice. Then, we investigated the biological effects of hADSCs on human keratinocytes HaCAT cells in vitro. RESULTS: The results showed that hADSCs could be successfully differentiated into osteogenic and lipogenic cells. hADSCs and hADSCs-CM significantly promote skin wound healing in vivo. hADSCs significantly promoted HaCAT cell proliferation and migration by activating the Notch signaling pathway and activated the AKT signaling pathway by Rps6kb1 kinase in HaCAT cells. In addition, we found that hADSCs-mediated activation of Rps6kb1/AKT signaling was dependent on the Notch signaling pathway. CONCLUSION: We demonstrated that hADSCs can promote skin cell-HaCAT cell proliferation and migration via the Notch pathway, suggesting that hADSCs may provide an alternative therapeutic approach for the treatment of skin injury.

MiR-33-5p alleviates spinal cord injury in rats and protects PC12 cells from lipopolysaccharide-induced apoptosis.

MicroRNAs (miRNAs) exert critical effects in spinal cord injury (SCI). The miR-33-5p level is found to be lower in rats with SCI compared with that in control (untreated) and sham-operated (laminectomy but no contusion) rats. Therefore, we investigated the biological functions of miR-33-5p and related mechanisms in SCI pathogenesis and development. An in vivo SCI model and a lipopolysaccharide (LPS)-induced cell model of SCI were established. A downregulated level of miR-33-5p in experimental SCI and in LPS-treated PC12 cells was revealed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). MiR-33-5p upregulation alleviated the leakage of the blood-spinal cord barrier (BSCB) induced by SCI and improved the neurological functions of SCI rats, as evidenced by the Basso, Beattie, and Bresnahan (BBB) scores and Evans blue staining. The regulatory relationship between miR-33-5p and Rps6kb1 was verified by luciferase reporter assays, which demonstrated that miR-33-5p bound to the Rps6kb1 3'UTR. Moreover, as MTT assays and flow cytometry showed, the suppressive effects of miR-33-5p upregulation on cell apoptosis were attenuated by Rps6kb1 upregulation. In conclusion, miR-33-5p ameliorates SCI in rats and inhibits the LPS-induced apoptosis of PC12 cells.

MiRNA-30e downregulation increases cancer cell proliferation, invasion and tumor growth through targeting RPS6KB1.

Human esophagus carcinoma (EC) is one of the most common malignant tumors, especially in Africa and Asia including China. In EC initiation and progression, genetic and epigenetic aberrations have been reported to play a major role, but the underlying molecular mechanisms are largely unknown. In this study, the miR-30e levels were analyzed in human EC tissues and TCGA databases, and the results demonstrated that miR-30e expression in EC tissues was significantly decreased compared to adjacent normal tissues. To further investigate the role of miR-30e in cancer cells, we found that forced expression of miR-30e dramatically inhibited cell proliferation, invasion, tube formation, and colony formation of cancer cells. To determine the underlying mechanism of miR-30e, we found that RPS6KB1 was a direct target of miR-30e by binding to its 3'-UTR, which was verified by luciferase activity assay using reporters with wild-type miR-30e and its seed sequence mutant constructs and Western blotting assay. In vivo experiment showed that miR-30e overexpression significantly inhibited tumor growth and decreased RPS6KB1 expression in xenografts. In EC, high expression of RPS6KB1 in tumor tissues indicated poor prognosis of patients with less survival rate. High levels of RPS6KB1 and low levels of miR-30e closely correlated poor survival of patients with several other types of cancer. These findings show that miR-30e and its target RPS6KB1 are important in cancer development and clinical outcomes, and miR-30e/RPS6KB1 is a potential future therapeutic pathway for EC intervention.

TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.

Upregulation of MTOR, RPS6KB1, and EIF4EBP1 in the whole blood samples of Iranian patients with multiple sclerosis compared to healthy controls.

Various genetic and epigenetic mechanisms have been suggested to play roles as the underlying pathophysiology of Multiple Sclerosis (MS). Changes in different parts of the mTOR signaling pathway are among the potential suggested mechanisms based on the specific roles of this pathway in CNS. MTOR, RPS6KB1, and EIFEBP1 genes are among important genes in the mTOR pathway, responsible for the proper function of acting proteins in this signaling pathway. This study aimed to investigate the relative expression levels of these genes in the blood samples of relapsing-remitting MS (RRMS) patients compared to healthy controls. In this case-control study blood samples were collected from 30 newly diagnosed RRMS patients and 30 age and sex-matched healthy controls. mRNA level of MTOR, RPS6KB1, and EIFEBP1 genes were assessed using Real-Time PCR. The expression of MTOR, RPS6KB1, and EIF4EBP1 genes was up regulated in MS patients compared to healthy controls (p < 0.001 for all mentioned genes). Considering gender differences, expression of the mentioned genes was increased among female patients (all P < 0.001). However, no statistically significant changes were observed among male patients. Based on the receiver operating characteristic, MTOR gene had the highest diagnostic value followed by EIF4EBP1 and RPS6KB1 genes in differentiating RRMS patients from controls. In conclusion, we found the simultaneous upregulation of MTOR, RPS6KB1, and EIF4EBP1 genes among RRMS patients. MTOR showed to have the highest diagnostic value compared to other 2 genes in differentiating RRMS patients. Further studies evaluating the importance of these findings from pharmacological and prognostic perspectives are necessary.

Regulation of Mumps Virus Replication and Transcription by Kinase RPS6KB1.

Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.

Distinct Roles of mTOR Targets S6K1 and S6K2 in Breast Cancer.

The mechanistic target of rapamycin (mTOR) is a master regulator of protein translation, metabolism, cell growth and proliferation. It forms two complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 is frequently deregulated in many cancers, including breast cancer, and is an important target for cancer therapy. The immunosuppressant drug rapamycin and its analogs that inhibit mTOR are currently being evaluated for their potential as anti-cancer agents, albeit with limited efficacy. mTORC1 mediates its function via its downstream targets 40S ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). There are two homologs of S6K: S6K1 and S6K2. Most of the earlier studies focused on S6K1 rather than S6K2. Because of their high degree of structural homology, it was generally believed that they behave similarly. Recent studies suggest that while they may share some functions, they may also exhibit distinct or even opposite functions. Both homologs have been implicated in breast cancer, although how they contribute to breast cancer may differ. The purpose of this review article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer.

Cycloastragenol ameliorates experimental heart damage in rats by promoting myocardial autophagy via inhibition of AKT1-RPS6KB1 signaling.

Cycloastragenol, a naturally occurring compound in Astragali Radix, has been demonstrated to possess various pharmacological actions including anti-aging, anti-inflammation, anti-fibrosis, antibacterial, liver and endothelium protection. However, whether cycloastragenol ameliorates heart failure remains unclear. Isoproterenol administration to rats triggered classic cardiac damage, as demonstrated by objective parameters of cardiac dysfunction. The treatment of cycloastragenol improved deranged cardiac parameters in the isoproterenol-induced heart damage model in a dose-dependent manner. At the same time, cycloastragenol markedly ameliorated cardiac histological changes and down-regulated serum levels of various neuroendocrine factors including norepinephrine, aldosterone, brain natriuretic peptide, endothelin 1, angiotensin II and so on. Moreover, the expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in rat heart were also inhibited by cycloastragenol. Mechanistically, augmenting autophagy of myocardial cells via the inhibition of AKT1-RPS6KB1 signaling contributed to the improvement of isoproterenol-induced rat heart failure by cycloastragenol. These results suggest that cycloastragenol ameliorates cardiac dysfunction and remodeling through promoting autophagy in myocardial cells and suppressing MMP-2 and MMP-9 expressions, indicating that it could be a drug candidate for patients with congestive heart failure.

Suppression of ribosomal protein RPS6KB1 by Nexrutine increases sensitivity of prostate tumors to radiation.

Radiation therapy (XRT) is a standard treatment for prostate cancer (PCa). Although dose escalation increases local control, toxicity hampers further escalation. Broader improvement will be possible by the addition of adjuvant therapies, which can synergize with radiation and thus improve efficacy. We have identified a natural compound (Nexrutine, Nx) that inhibits the survival and growth of PCa cells in combination with radiation. Combination studies demonstrated strong interaction between Nx and radiation both in vitro in multiple PCa cell lines and in the Transgenic adenocarcinoma of mouse prostate (TRAMP) model. Nx potentiated growth inhibitory effects of IR by down regulating ribosomal protein S6K (RPS6KB1), CyclinD1, Chk1 and HIF-1 α and prolonging G2/M checkpoint block. RPS6KB1 is upregulated in prostate cancers and its expression is correlated with tumor grade. Knockdown of RPS6KB1 in PCa cells increased their sensitivity toward radiation-induced survival inhibition. Overall, we provide scientific evidence (i) in support of Nx as an adjuvant in PCa patients receiving XRT (ii) suggesting that RPS6KB1 is an important player in Nx-mediated combinatorial benefits and emphasizes that RPS6KB1 is a novel target for PCa treatment. These data underscore the need to test the agent in additional preclinical models to validate these observations.

EVA1A inhibits GBM cell proliferation by inducing autophagy and apoptosis.

Eva-1 homolog A (EVA1A) is a novel lysosome and endoplasmic reticulum-associated protein involved in autophagy and apoptosis. In this study, we constructed a recombinant adenovirus 5-EVA1A vector (Ad5-EVA1A) to overexpress EVA1A in glioblastoma (GBM) cell lines and evaluated its anti-tumor activities in vitro and in vivo. We found that overexpression of EVA1A in three GBM cell lines (U251, U87 and SHG44) resulted in a suppression of tumor cell growth via activation of autophagy and induction of cell apoptosis in a dose- and time-dependent manner. EVA1A-mediated autophagy was associated with inactivation of the mTOR/RPS6KB1 signaling pathway. Furthermore in vivo, overexpression of EVA1A successfully inhibited tumor growth in NOD/SCID mice. Our data suggest that EVA1A-induced autophagy and apoptosis play a role in suppressing the development of GBM and their up-regulation may be an effective method for treating this form of cancer.

Polymorphisms of RPS6KB1 and CD86 associates with susceptibility to multiple sclerosis in Iranian population.

OBJECTIVE:  Multiple sclerosis (MS) is the most prevalent disorder of nervous system inflammation which involves demyelination of spinal cord; this process depends on both environmental and genetic susceptibility factors. In the present study, we examined the association between two SNPs in RPS6KB1 (rs180515) and CD86 (rs9282641) with MS in Iranian population. RPS6KB1gene encodes p70S6K1 protein which plays a key role in mTOR signaling pathway, while CD86 gene codes a membrane protein type I which belongs to immunoglobulin super family act on co-stimulation signaling pathway. METHODS: In this case-control study 130 patients with MS and 128 matched healthy controls were enrolled, genomic DNA was isolated and genotyping was performed using mismatched PCR-RFLP. The results were finally analyzed using SPSS. RESULTS: Our results showed significant difference in allelic frequency of SNP rs180515 among cases and controls (P = 0.004). For this variation, AA genotype was shown to have protective effect (P = 0.016 and OR = 0.6), while GG genotype was a susceptive genotype to MS (P = 0.04 and OR = 2.2). Allelic frequency of SNP rs9282641 also showed significant difference between cases and controls (P = 0.006). For this SNP, AG genotype had predisposing effect (P = 0.04, OR = 2.3), and GG genotype showed protective (P = 0.01, OR = 0.411). CONCLUSION: We successfully replicated the association of two novel SNPs introduced by a GWAS study, and MS in the Iranian population. This result can open ways for better understanding the mechanisms involved in MS.

Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells.

Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.

A disputed evidence on obesity: comparison of the effects of Rcan2(-/-) and Rps6kb1(-/-) mutations on growth and body weight in C57BL/6J mice.

It is widely accepted that body weight and adipose mass are tightly regulated by homeostatic mechanisms, in which leptin plays a critical role through hypothalamic pathways, and obesity is a result of homeostatic disorder. However, in C57BL/6J mice, we found that Rcan2 increases food intake and plays an important role in the development of age- and diet-induced obesity through a leptin-independent mechanism. RCAN2 was initially identified as a thyroid hormone (T3)-responsive gene in human fibroblasts. Expression of RCAN2 is regulated by T3 through the PI3K-Akt/PKB-mTOR-Rps6kb1 signaling pathway. Intriguingly, both Rcan2(-/-) and Rps6kb1(-/-) mutations were reported to result in lean phenotypes in mice. In this study we compared the effects of these two mutations on growth and body weight in C57BL/6J mice. We observed reduced body weight and lower fat mass in both Rcan2(-/-) and Rps6kb1(-/-) mice compared to the wild-type mice, and we reported other differences unique to either the Rcan2(-/-) or Rps6kb1(-/-) mice. Firstly, loss of Rcan2 does not directly alter body length; however, Rcan2(-/-) mice exhibit reduced food intake. In contrast, Rps6kb1(-/-) mice exhibit abnormal embryonic development, which leads to smaller body size and reduced food intake in adulthood. Secondly, when fed a normal chow diet, Rcan2(-/-) mice weigh significantly more than Rps6kb1(-/-) mice, but both Rcan2(-/-) and Rps6kb1(-/-) mice develop similar amounts of epididymal fat. On a high-fat diet, Rcan2(-/-) mice gain body weight and fat mass at slower rates than Rps6kb1(-/-) mice. Finally, using the double-knockout mice (Rcan2(-/-) Rps6kb1(-/-)), we demonstrate that concurrent loss of Rcan2 and Rps6kb1 has an additive effect on body weight reduction in C57BL/6J mice. Our data suggest that Rcan2 and Rps6kb1 mutations both affect growth and body weight of mice, though likely through different mechanisms.

TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy.

Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed Tnfaip3-deficient naïve CD4 T cells in vitro. We demonstrated that Tnfaip3-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that Tnfaip3-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in Tnfaip3-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. Tnfaip3-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of Tnfaip3-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by Atg5 deficiency. Thus, enhanced MTOR activity regulates the survival of Tnfaip3-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells.

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.