mTORC1 Signaling Pathway Research Articles

Zein-Derived Peptides from Corn Promote the Proliferation of C2C12 Myoblasts via Crosstalk of mTORC1 and mTORC2 Signaling Pathways.

Dietary protein supplementation has emerged as a promising strategy in combating sarcopenia. Furthermore, searching for alternatives of animal proteins has been a hot topic. The present study aimed to investigate the effects of zein peptides on C2C12 myoblasts and explore their potential molecular mechanisms. The proliferative, cell cycle, and anti-apoptotic activities of zein peptides were evaluated. Peptidomics analysis and transcriptome sequencing were employed to explore the structure-activity relationship and underlying molecular mechanisms. The results indicated that zein peptides (0.05-0.2 mg/mL) exerted a significant proliferation-promoting impact on C2C12 cells, via increasing cell viability by 33.37 to 42.39%. Furthermore, zein peptides significantly increased S phase proportion and decreased the apoptosis rate from 34.08% (model group) to 28.96% in C2C12 cells. In addition, zein peptides exhibited a pronounced anti-apoptotic effect on C2C12 cells. Zein peptides are abundant in branch-chain amino acids, especially leucine. Transcriptomics analysis revealed that zein peptides can promote proliferation, accelerate cell cycle, and improve protein synthesis of muscle cells through mTORC1 and mTORC2 signaling pathways.

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/β-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Imidazole propionate ameliorates atopic dermatitis-like skin lesions by inhibiting mitochondrial ROS and mTORC2.

Imidazole propionate (IMP) is a histidine metabolite produced by some gut microorganisms in the human colon. Increased levels of IMP are associated with intestinal inflammation and the development and progression of cardiovascular disease and diabetes. However, the anti-inflammatory activity of IMP has not been investigated. This study aimed to elucidate the role of IMP in treating atopic dermatitis (AD). To understand how IMP mediates immunosuppression in AD, IMP was intraperitoneally injected into a Dermatophagoides farinae extract (DFE)/1-chloro-2,4 dinitrochlorobenzene (DNCB)-induced AD-like skin lesions mouse model. We also characterized the anti-inflammatory mechanism of IMP by inducing an AD response in keratinocytes through TNF-α/IFN-γ or IL-4 stimulation. Contrary to the prevailing view that IMP is an unhealthy microbial metabolite, we found that IMP-treated AD-like skin lesions mice showed significant improvement in their clinical symptoms, including ear thickness, epidermal and dermal thickness, and IgE levels. Furthermore, IMP antagonized the expansion of myeloid (neutrophils, macrophages, eosinophils, and mast cells) and Th cells (Th1, Th2, and Th17) in mouse skin and prevented mitochondrial reactive oxygen species production by inhibiting mitochondrial energy production. Interestingly, we found that IMP inhibited AD by reducing glucose uptake in cells to suppress proinflammatory cytokines and chemokines in an AD-like in vitro model, sequentially downregulating the PI3K and mTORC2 signaling pathways centered on Akt, and upregulating DDIT4 and AMPK. Our results suggest that IMP exerts anti-inflammatory effects through the metabolic reprogramming of skin inflammation, making it a promising therapeutic candidate for AD and related skin diseases.

Identification of SNPs and candidate genes associated with growth using GWAS and transcriptome analysis in Coilia nasus

The improvement of growth trait can foster the sustained and stable development of the aquaculture industry and is generally regarded as a primary objective in the breeding projects for economic fish. Coilia nasus, a migratory fish species naturally inhabiting in the middle and lower reaches of the Yangtze River and offshore waters of China, exhibits significant culturing potential and economic value. However, due to genetic degradation, there is a pronounced trend towards miniaturization and exaggerated growth variations within the cultured population. In order to facilitate the healthy development of the aquaculture industry of C. nasus, whole-genome association analysis (GWAS) and transcriptome analysis (RNA-seq) have been employed to identify molecular markers associated with growth traits and elucidate genetic foundations. A total of 234 individuals of C. nasus were subjected to whole-genome resequencing for genotyping, resulting in the identification of 2,306,254 high-quality SNPs. Fourteen SNPs were proved to have suggestive association with body length (BL) or body weight (BW), with the explained phenotypic variation ranging from 2.21% to 2.71%. We obtained two shared significant SNP peaks for BL and BW on LG6 and LG9, and bmp8a and uqcrfs1 were identified as their causal genes. Comparative transcriptome analysis based on extreme body size obtained 54 and 20 overlapping differentially expressed genes (DEGs) between different sexes in brain and muscle, respectively. Combining the results of GWAS and RNA-seq, we identified five candidate genes and one pathway associated with growth, including uqcrfs1, bmp8a, gabrb4, tshz3a, ahcyl1, and the mTORC1 signaling pathway. Our research findings provide molecular markers for genetic selection in C. nasus growth, and contribute to understand the molecular mechanisms of population growth variations.

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours

BackgroundPaired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated.MethodsPRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1.ResultsHigh expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1–TOP2A interaction.ConclusionTargeting the PRRX1–TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

Machine learning developed an intratumor heterogeneity signature for predicting prognosis and immunotherapy benefits in skin cutaneous melanoma.

Intratumor heterogeneity (ITH) is defined as differences in molecular and phenotypic profiles between different tumor cells and immune cells within a tumor. ITH was involved in the cancer progression, aggressiveness, therapy resistance and cancer recurrence. Integrative machine learning procedure including 10 methods was conducted to develop an ITH-related signature (IRS) in The Cancer Genome Atlas (TCGA), GSE54467, GSE59455 and GSE65904 cohort. Several scores, including tumor immune dysfunction and exclusion (TIDE) score, tumor mutation burden (TMB) score and immunophenoscore (IPS), were used to evaluate the role of IRS in predicting immunotherapy benefits. Two immunotherapy datasets (GSE91061 and GSE78220) were utilized to the role of IRS in predicting immunotherapy benefits of skin cutaneous melanoma (SKCM) patients. The optimal prognostic IRS constructed by Lasso method acted as an independent risk factor and had a stable and powerful performance in predicting the overall survival rate in SKCM, with the area under the curve of 2-, 3- and 4-year receiver operating characteristic curve being 0.722, 0.722 and 0.737 in TCGA cohort. We also constructed a nomogram and the actual 1-, 3- and 5-year survival times were highly consistent with the predicted survival times. SKCM patients with low IRS scores had a lower TIDE score, lower immune escape score and higher TMB score, higher PD1&CTLA4 IPS. Moreover, SKCM patients with low IRS scores had a lower gene sets score involved in DNA repair, angiogenesis, glycolysis, hypoxia, IL2-STAT5 signaling, MTORC1 signaling, NOTCH signaling and P53 pathway. The current study constructed a novel IRS in SKCM using 10 machine learning methods. This IRS acted as an indicator for predicting the prognosis and immunotherapy benefits of SKCM patients.

Exploiting epigenetic targets to overcome taxane resistance in prostate cancer

The development of taxane resistance remains a major challenge for castration resistant prostate cancer (CR-PCa), despite the effectiveness of taxanes in prolonging patient survival. To uncover novel targets, we performed an epigenetic drug screen on taxane (docetaxel and cabazitaxel) resistant CR-PCa cells. We identified BRPF reader proteins, along with several epigenetic groups (CBP/p300, Menin-MLL, PRMT5 and SIRT1) that act as targets effectively reversing the resistance mediated by ABCB1. Targeting BRPFs specifically resulted in the resensitization of resistant cells, while no such effect was observed on the sensitive compartment. These cells were successfully arrested at the G2/M phase of cell cycle and underwent apoptosis upon BRPF inhibition, confirming the restoration of taxane susceptibility. Pharmacological inhibition of BRPFs reduced ABCB1 activity, indicating that BRPFs may be involved in an efflux-related mechanism. Indeed, ChIP-qPCR analysis confirmed binding of BRPF1 to the ABCB1 promoter suggesting direct regulation of the ABCB1 gene at the transcriptional level. RNA-seq analysis revealed that BRPF1 knockdown affects the genes enriched in mTORC1 and UPR signaling pathways, revealing potential mechanisms underlying its functional impact, which is further supported by the enhancement of taxane response through the combined inhibition of ABCB1 and mTOR pathways, providing evidence for the involvement of multiple BRPF1-regulated pathways. Beyond clinical attributes (Gleason score, tumor stage, therapy outcome, recurrence), metastatic PCa databases further supported the significance of BRPF1 in taxane resistance, as evidenced by its upregulation in taxane-exposed PCa patients.

TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway

Diabetic cardiomyopathy (DCM) is a major complication of diabetes. Transient receptor potential melastatin 2 (TRPM2) activity increases in diabetic oxidative stress state, and it is involved in myocardial damage and repair. We explore the protective effect of TRPM2 knockdown on the progression of DCM. A type 2 diabetes animal model was established in C57BL/6N mice by long-term high-fat diet (HFD) feeding combined with a single injection of 100-mg/kg streptozotocin (STZ). Genetic knockdown of TRPM2 in heart was accomplished by the intravenous injection via the tail vein of adeno-associated virus type 9 carrying TRPM2 shRNA. Neonatal rat ventricular myocytes was exposed to 45 mM of high-glucose (HG) stimulation for 72 h in vitro to mimic the in vivo conditions. Western blot, real-time quantitative PCR (RT-qPCR), immunohistochemistry and fluorescence, electron, CCK-8, and flow cytometry were used to evaluate the phenotype of cardiac inflammation, fibrosis, apoptosis, and autophagy. Mice with HFD/STZ-induced diabetes exhibited systolic and diastolic dysfunction, as demonstrated by increased myocardial apoptosis and autophagy inhibition in the heart. Compared to control group, the protein expression of TRPM2, bax, cleaved caspase-3, and P62 was significantly elevated, and the protein expression of bcl-2 and LC3-II was significantly decreased in the myocardial tissues of the HFD/STZ-induced diabetes group. Knockdown of TRPM2 significantly reversed the HFD/STZ-induced myocardial apoptosis and autophagy inhibition. TRPM2 silencing attenuated HG-induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the MEK/ERK mTORC1 signaling pathway. TRPM2 knockdown attenuates hyperglycemia-induced myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice or HG-stimulated cardiomyocytes via regulating the MEK/ERK and mTORC1 signaling pathway.

Mechanism about LMP1 of EB Virus Promoting Plasma Blast Differentiation of DLBCL Cell via mTORC1

To investigate possible mechanism on protien LMP1 expressed by EBV inducing plasmablast differentiation of DLBCL cell via the mTORC1 pathway. The expression levels of LMP1 protein, CD38 and the phosphorylation levels of p70S6K in EBV+ and EBV- DLBCL cell lines were detected by Western blot. Cell lines overexpressing LMP1 gene stablely were constructed and LMP1 gene was silenced by RNAi. The expression of LMP1 gene was verified by RT-qPCR. The expression levels of LMP1 and CD38 and the phosphorylation levels of p70S6K in each group were detected by Western blot. Compared with EBV-DLBCL cells, the expression of LMP1 was detected on EBV +DLBCL cells (P =0.0008), EBV +DLBCL cells had higher phosphorylation levels of p70S6K (P =0.0072) and expression levels of CD38(P =0.0091). Compared with vector group, the cells of LMP1OE group had higher expression levels of LMP1 and CD38 (P =0.0353; P <0.0001), meanwhile molecular p70S6K was phosphorylated much more(P =0.0065); expression of LMP1 mRNA was verified(P <0.0001). Compared with si-NC group, expression level of LMP1 protein(P =0.0129) was not detected and phosphorylated p70S6K disappeared of LMP1KO group (P =0.0228); meanwhile, expression of CD38 decreased,although there was no significant difference (P =0.2377). LMP1 promotes DLBCL cells plasmablast differentiation via activating mTORC1 signal pathway.

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis)

Purpose Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). Methods OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. Results Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. Conclusions These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

SPOP negatively regulates mTORC1 activity by ubiquitinating Sec13

The mammalian target of rapamycin complex1 (mTORC1) can response to amino acid to regulate metabolism and cell growth. GATOR2 act as important role in amino acid mediated mTORC1 signaling pathway by repressing GTPase activity (GAP) of GATOR1. However, it is still unclear how GATOR2 regulates mTORC1 signaling pathway. Here, we found that K63-ubiquitination of Sce13, one component of GATOR2, suppresses the mTORC1 activity by lessening the inter-interaction of GATOR2. Mechanistically, the ubiquitination of Sec13 was mediated by SPOP. Subsequently, the ubiquitination of Sec13 attenuated its interaction with the other component of GATOR2, thus suppressing the activity of mTORC1. Importantly, the deficiency of SPOP promoted the faster proliferation and migration of breast cancer cells, which was attenuated by knocking down of Sec13. Therefore, SPOP can act as a tumor suppressor gene by negatively regulating mTORC1 signaling pathway.

Reduced Branched-Chain Amino Acid Intake Improved High-Fat Diet-Induced Nonalcoholic Fatty Pancreas Disease in Mice.

To explore the effects of branched-chain amino acids (BCAAs) on nonalcoholic fatty pancreas disease (NAFPD) and its possible mechanism in high-fat diet (HFD) induced mice. Pancreatic morphology and lipid infiltration was assessed by hematoxylin-eosin staining and immunohistochemistry, and lipid levels in the pancreas were determined using colorimetric enzymatic method. Relevant mechanism was investigated using western blotting and biochemical test. In HFD-fed mice, dietary BCAAs restriction could attenuate body weight increase, improve glucose metabolism, and reduce excessive lipid accumulation in the pancreas. Furthermore, expression of AMPKα and downstream uncoupling protein 1 were upregulated, while genes related to mammalian target of rapamycin complex 1 (mTORC1) signal pathway and lipid de novo synthesis were suppressed in HFD-BCAA restriction group compared with HFD and HFD-high BCAAs fed mice. In addition, BCAA restriction upregulated expression of BCAAs related metabolic enzymes including PPM1K and BCKDHA, and decreased the levels of BCAAs and branched chain keto acid in the pancreas. However, there was no difference in levels of lipid content in the pancreas and gene expression of AMPKα and mTORC1 between HFD and HFD-high BCAAs groups. Branched-chain amino acid restriction ameliorated HFD-induced NAFPD in mice by activation of AMPKα pathway and suppression of mTORC1 pathway.

OTUD3 suppresses the mTORC1 signaling by deubiquitinating KPTN.

Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitin-proteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3K-AKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors.

Novel two-tiered screening approach identifies synergistic combinations of natural compounds for prostate cancer prevention and treatment.

Prostate cancer (PCa) is the second most common cancer type among American men and it is estimated that in 2023, 34,700 men will die from PCa. Since it can take a considerable amount of time for the disease to progress to clinically evident cancer, there is ample opportunity for effective chemopreventive strategies to be applied for the successful management of PCa progression. In the current study, we have developed a two-tiered metabolomics-based screen to identify synergistic combinations of phytochemicals for PCa chemoprevention. This involves an initial screen for ATP depletion in PCa cells followed by a targeted screen for blocking glutamine uptake in the same cells. One of the phytochemical combinations (enoxolone [ENO] + silibinin [SIL]), identified via this screen, was examined for effects on PCa cell survival, oncogenic signaling and tumor growth in vivo. This combination was found to synergistically reduce cell survival, colony formation and cell cycle progression of PCa cell lines to a greater extent than either agent alone. The combination of ENO and SIL also synergistically reduced tumor growth when administered ad libitum through the diet in a HMVP2 allograft PCa tumor model. Treatment with the combination also significantly reduced STAT3 and mTORC1 signaling pathways in mouse and human PCa cells while significantly reducing levels of critical cell cycle regulatory proteins, contributing to the synergistic inhibition of tumor growth observed. Collectively, the current results demonstrate a novel approach to identifying synergistic combinations of phytochemicals for chemoprevention of PCa and possibly other cancers.

THADA inhibits autophagy and increases 5-FU sensitivity in gastric cancer cells via the PI3K/AKT/mTOR signaling pathway.

5-Fluorouracil (5-FU) is currently the main drug used in chemotherapy for gastric cancer (GC). The main clinical problems of 5-FU therapy are insensitivity and acquired resistance to 5-FU. The mechanism of GC cell resistance to 5-FU is currently unknown. This study employed next-generation sequencing (NGS) to analyze the differentially expressed genes (DEGs) in chemotherapy-sensitive and non-sensitive GC tissues. In addition, a bioinformatics analysis was conducted using the GC dataset of GEO, and further validated and explored through in vitro experiments. Thyroid adenoma-associated gene (THADA) was highly expressed in GC tissues from chemotherapy-sensitive patients and was an independent prognostic factor in GC patients receiving postoperative 5-FU adjuvant chemotherapy. Notably, heightened THADA expression in GC cells was associated with the down-regulation of autophagy-related proteins (LC-3, ATG13, ULK1, and TFEB). Furthermore, the PI3K/AKT/mTOR signaling pathway and mTORC1 signaling pathway were remarkably increased in patients with elevated THADA expression. THADA expression was associated with mTOR, the core protein of the mTOR signaling pathway, and related proteins involved in regulating the mTORC1 signaling pathway (mLST8, RHEB, and TSC2). THADA exhibited inhibitory effects on autophagy and augmented the sensitivity of GC cells to 5-FU through the PI3K/AKT/mTOR signaling pathway. The findings suggest that THADA may be involved in the regulatory mechanism of GC cell sensitivity to 5-FU. Consequently, the detection of THADA in tumor tissues may bring clinical benefits, specifically for 5-FU-related chemotherapy administered to GC patients with elevated THADA expression.

Systems Pharmacology Strategy for the Investigation of Action Mechanisms of Qin Herb Libanotis Buchtormensis (Fisch.) DC. in Bone Diseases.

Qin medicines are medicinal plants growing in habitat around the peak of Qinling Mountain. Their unique curative effects on bone metabolic diseases and pain diseases have been favoured by the local people in clinical trials for thousands of years. Libanotis buchtormensis (Fisch.) DC. (LBD), is one of the popular Qin herbs, which has been widely used for the treatment of various diseases, such as osteoporosis, rheumatic, and cardiovascular diseases. However, due to the multiple compounds in LBD, the underlying molecular mechanisms of LBD remain unclear. This study aimed to systemically investigate the underlying mechanisms of LBD against bone diseases. In this study, a systems pharmacology platform included the potential active compound screening, target fishing, and network pharmacological analysis was employed to decipher the action mechanisms of LBD. As a result, 12 potential active compounds and 108 targets were obtained. Furthermore, compound-target network and target-pathway network analysis showed that multi-components interacted with multi-targets and multi-pathways, i.e., MARK signalling pathway, mTORC1 signalling pathway, etc., involved in the regulation of the immune system and circulatory system. These results suggested the mechanisms of the therapeutic effects of LBD on various diseases through most compounds targeted by multiple targets. In conclusion, we successfully predicted the LBD bioactive compounds and potential targets, implying that LBD could be applied as a novel therapeutic herb in osteoporosis, rheumatic, and cardiovascular diseases. This work provides insight into the therapeutic mechanisms of LBD for treating various diseases.

Naked cuticle homolog 2 controls the differentiation of osteoblasts and osteoclasts and ameliorates bone loss in ovariectomized mice

Naked cuticle homolog 2 (NKD2) has been recognized as an antagonist of Wnt/β-catenin signaling and a tumor suppressor. The role of NKD2 in osteoblast and osteoclast differentiation and the mechanism are not fully understood. In this study, we identified the up-regulation of NKD2 during osteoblast and adipocyte differentiation. Functional experiments revealed that NKD2 stimulated osteoblast differentiation and suppressed adipocyte formation. Furthermore, NKD2 down-regulated the expression of receptor activator of nuclear factor-κB ligand in bone marrow mesenchymal stem cells and inhibited osteoclast formation from osteoclast precursor cells. Mechanistic investigations revealed that the regulation of osteoblast and adipocyte differentiation by NKD2 involved Wnt/β-catenin and tuberous sclerosis complex subunit 1 (TSC1)/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways. Unlike in undifferentiated mesenchymal cells where NKD2 promoted Dishevelled-1 degradation, in the cells differentiating toward osteoblasts or adipocytes NKD2 down-regulated secreted frizzled related protein 1/4 expression and failed to destabilize Dishevelled-1, thereby activating Wnt/β-catenin signaling. Moreover, NKD2 bound to TSC1 and inhibited mTORC1 signaling. Further investigation uncovered an interplay between TSC1/mTORC1 and Wnt/β-catenin signaling pathways. Finally, transplantation of NKD2-overexpressing bone marrow mesenchymal stem cells into the marrow of mice increased osteoblasts, reduced osteoclasts and marrow fat, and partially prevented bone loss in ovariectomized mice. This study provides evidence that NKD2 in mesenchymal stem/progenitor cells reciprocally regulates the differentiation of osteoblasts and adipocytes by modulating Wnt/β-catenin and mTORC1 pathways and inhibits osteoclast formation by down-regulating receptor activator of nuclear factor-κB ligand. It suggests that NKD2 up-regulation may ameliorate postmenopausal bone loss.

Hyperactive mTORC1 in striatum dysregulates dopamine receptor expression and odor preference behavior.

Mechanistic target of rapamycin (mTOR) plays an important role in brain development and synaptic plasticity. Dysregulation of the mTOR pathway is observed in various human central nervous system diseases, including tuberous sclerosis complex, autism spectrum disorder (ASD), and neurodegenerative diseases, including Parkinson's disease and Huntington's disease. Numerous studies focused on the effects of hyperactivation of mTOR on cortical excitatory neurons, while only a few studies focused on inhibitory neurons. Here we generated transgenic mice in which mTORC1 signaling is hyperactivated in inhibitory neurons in the striatum, while cortical neurons left unaffected. The hyperactivation of mTORC1 signaling increased GABAergic inhibitory neurons in the striatum. The transgenic mice exhibited the upregulation of dopamine receptor D1 and the downregulation of dopamine receptor D2 in medium spiny neurons in the ventral striatum. Finally, the transgenic mice demonstrated impaired motor learning and dysregulated olfactory preference behavior, though the basic function of olfaction was preserved. These findings reveal that the mTORC1 signaling pathway plays an essential role in the development and function of the striatal inhibitory neurons and suggest the critical involvement of the mTORC1 pathway in the locomotor abnormalities in neurodegenerative diseases and the sensory defects in ASD.

Identification of (E)-1-((1H-indol-3-yl)methylene)-4-substitute-thiosemicarbazones as potential anti-hepatic fibrosis agents

Liver fibrosis remains a global health challenge due to its rapidly rising prevalence and limited treatment options. The orphan nuclear receptor Nur77 has been implicated in regulation of autophagy and liver fibrosis. Targeting Nur77-mediated autophagic flux may thus be a new promising strategy against hepatic fibrosis. In this study, we synthesized four types of Nur77-based thiourea derivatives to determine their anti-hepatic fibrosis activity. Among the synthesized thiourea derivatives, 9e was the most potent inhibitor of hepatic stellate cells (HSCs) proliferation and activation. This compound could directly bind to Nur77 and inhibit TGF-β1-induced α-SMA and COLA1 expression in a Nur77-dependent manner. In vivo, 9e significantly reduced CCl4-mediated hepatic inflammation response and extracellular matrix (ECM) production, revealing that 9e is capable of blocking the progression of hepatic fibrosis. Mechanistically, 9e induced Nur77 expression and enhanced autophagic flux by inhibiting the mTORC1 signaling pathway in vitro and in vivo. Thus, the Nur77-targeted lead 9e may serve as a promising candidate for treatment of chronic liver fibrosis.