Activation Of p70S6K Research Articles

YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.

Mild Osmotic Stress Alters mTOR Pathway Signaling

Increased osmotic stress reduces cell volume, thus disrupting homeostatic processes. This could impact signaling that promotes muscle growth and maintenance at rest and in response to mechanical stimuli. To date, the detrimental effect of osmotic stress on muscle protein turnover has been established in in vitro and in situ models, but the effects on muscular anabolic signaling in humans are unknown. Purpose: To examine the effect of fluid restriction (FR)-induced mild osmotic stress (plasma osmolality ≥295.0 mmol×kg−1) on muscular anabolic signaling in response to resistance exercise (RE) in trained young men. Hypothesis: We hypothesized that FR would alter the Akt/mTOR signaling. Method: RE-trained men (n=11, 21±1yr, 175.9±6.2cm, 79.2±12.3kg, 18.4±6.7% fat) completed 2 identical RE bouts either with (FR) or without (CON) fluid restriction from 24 hrs before to 3 hrs after RE. RE consisted of bilateral leg press and knee extensions (5 sets x 10 repetitions at 80% of 1 repetition maximum). The two conditions were 2-3 wks apart. Plasma osmolality (PO) was measured before (PRE), and 5 min, 15 min, 30 min, 1 hr, and 3 hrs after RE. Muscle samples were collected at PRE and 1 and 3 hrs after RE. Western blots were used to analyze contents of proteins in the mTOR signaling pathway: Akt, p-AktS473, mTOR, p-mTORS2448, p70S6K, p-p70S6KT389, rpS6, p-rpS6S240/244, eEF2, and p-eEF2T56 and normalized to Ponceau S staining (total protein). Results: Increased PO was successfully induced in FR vs. CON (condition effect, p<0.0001; 299±2 vs 286±1 mmol/kg). P-AktS473 significantly (p≤0.05) increased from PRE to 1 and 3 hrs in CON, and from PRE to 1h with a return to PRE by 3h in FR (condition x time). P-AktS473 was higher at 1h and lower at 3h in FR versus CON. P-mTORS2448 increased from PRE to 1 and 3 hrs regardless of condition (main time). P-p70S6KT389 was greater in FR than CON regardless of time point (main condition). P-rpS6S240/244 increased from PRE to 1 and 3 hrs for CON and FR, and was greater for FR than CON at PRE (condition x time). Conclusion: Baseline anabolic signaling appeared elevated when individuals were subjected to mild osmotic stress. After RE, signaling through mTOR was elevated as expected when plasma was iso-osmotic, although anticipated time effects for p-p70S6K and p-eEF2 were not detected. Upstream of mTOR, activation of Akt was stronger yet shorter in duration with mild osmotic stress. Downstream of mTOR, p70S6K and rpS6 activation were greater with mild osmotic stress. While these results suggest increased anabolic signaling with mild osmotic stress, concomitant catabolic signaling should be assessed. Together, the present results and future measures will inform how conditions that confer osmotic stress, such as dehydration, may alter intramuscular protein homeostasis at rest and in response to an anabolic stimulus. This study was funded by National Strength and Conditioning Association Foundations. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

β-Hydroxy-β-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes.

β-Hydroxy-β-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.

Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

Acute effect of electrical stimulation on muscle protein synthesis and break-down in the soleus muscle of hindlimb unloaded rats.

Electrical stimulation (ES) is effective for disuse-induced muscle atrophy. However, the acute effect of ES on muscle protein synthesis (MPS) and muscle protein breakdown (MPB) remains unclear. We investigated the effect of a single-session ES treatment on mTORC1 signaling, MPS, and MPB in the soleus muscle of 2-week hindlimb unloaded rats. Sprague Dawley rats (n = 12 male) were randomly divided into control (CON) and hindlimb unloaded (HU) groups. After 2 weeks, the right soleus muscle was percutaneously stimulated and underwent supramaximal isometric contractions. The left soleus muscle served as an internal control. We collected soleus muscle samples 6 h after ES. Two weeks of HU decreased p70S6K and S6rp activation, downstream factors for mTORC1 signaling, and SUnSET method-assessed MPS, but increased the LC3-II/I ratio, an indicator of autophagy. ES on disused muscle successfully activated mTORC1 signaling but did not affect MPS. Contrary, ES decreased ubiquitinated proteins expression and LC3B-II/I ratio. HU might affect mTORC1 activation and MPS differently in response to acute ES possibly due to excessive ROS production caused by ES. Our findings suggest that ES applied to disused skeletal muscles may suppress MPB, but its effect on MPS appears to be attenuated.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer

BackgroundProtein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC).MethodsPolyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients.ResultsWe showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC.ConclusionsOur finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC.CUkGCcvqWD-y5KjYaB3kRRVideo

Interleukin-33 facilitates liver regeneration through serotonin-involved gut-liver axis.

Insufficient liver regeneration causes post-hepatectomy liver failure and small-for-size syndrome. Identifying therapeutic targets to enhance hepatic regenerative capacity remains urgent. Recently, increased IL-33 was observed in patients undergoing liver resection and in mice after partial hepatectomy (PHx). The present study aims to investigate the role of IL-33 in liver regeneration after PHx and to elucidate its underlying mechanisms. We performed PHx in IL-33 -/- , suppression of tumorigenicity 2 (ST2) -/- , and wild-type control mice, and found deficiency of IL-33 or its receptor ST2 delayed liver regeneration. The insufficient liver regeneration could be normalized in IL-33 -/- but not ST2 -/- mice by recombinant murine IL-33 administration. Furthermore, we observed an increased level of serotonin in portal blood from wild-type mice, but not IL-33 -/- or ST2 -/- mice, after PHx. ST2 deficiency specifically in enterochromaffin cells recapitulated the phenotype of delayed liver regeneration observed in ST2 -/- mice. Moreover, the impeded liver regeneration in IL-33 -/- and ST2 -/- mice was restored to normal levels by the treatment with (±)-2,5-dimethoxy-4-iodoamphetamine, which is an agonist of the 5-hydroxytrytamine receptor (HTR)2A. Notably, in vitro experiments demonstrated that serotonin/HTR2A-induced hepatocyte proliferation is dependent on p70S6K activation. Our study identified that IL-33 is pro-regenerative in a noninjurious model of liver resection. The underlying mechanism involved IL-33/ST2-induced increase of serotonin release from enterochromaffin cells to portal blood and subsequent HTR2A/p70S6K activation in hepatocytes by serotonin. The findings implicate the potential of targeting the IL-33/ST2/serotonin pathway to reduce the risk of post-hepatectomy liver failure and small-for-size syndrome.

Abstract 1078: Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines

Abstract KRAS is the most frequently mutated oncogene in lung adenocarcinoma (50%) and it is associated with a poor response to antineoplastic treatments, as well as poorer survival rates. KRAS activates the MAPK pathway, promoting cell survival and increasing protein synthesis. Novel inhibitors of KRAS G12C mutation (sotorasib) have demonstrated short-lasting responses due to resistance mediated by the AKT-mTOR-P70S6K pathway. In this context, metformin is a promising candidate to avoid this inconvenience by inhibiting mTOR and P70S6K. Therefore, this project aimed to explore the effects of the combination of sotorasib and metformin on cytotoxicity, apoptosis, and the activity of the mTOR pathway in lung cancer cell lines with or without KRAS mutations. Methods: We performed dose-effect curves to determine the IC50 concentration of sotorasib, and IC10 of metformin. Then, these doses were used to treat lung cancer cell lines A549 (KRAS G12S), H522 (without KRAS), and H23 (KRAS G12C). Cellular cytotoxicity was evaluated by MTT assay, apoptosis induction through flow cytometry with Annexin V and 7AAD, and the activity of P70S6K and AKT proteins by western blot. Main outcomes and measures: cell survival, apoptosis, expression, and phosphorylation of intermediaries belonging to the mTOR pathway. Results: The combined treatment showed a synergic effect on cytotoxicity, increased apoptosis induction, and notable inhibition of AKT and P70S6K1 proteins, compared to sotorasib alone or to control groups in all the cell lines, but predominantly in KRAS-mutated cells (H23 and A549). Conclusions: The combination of metformin with sotorasib showed synergic effects on cytotoxicity, increased apoptosis induction, and a remarkable inhibition of downstream proteins involved in the signaling of growth factor receptors in all tested cells. Moreover, these results suggest a sensitizing effect of metformin to sotorasib treatment in cells without KRAS mutations, such as H522. Citation Format: Norma Yanet Hernandez-Pedro, Pedro Barrios-Bernal, José Lucio-Lozada, Maritza Ramos-Ramirez, Oscar Arrieta. Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1078.

Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. eEF2k impairs the ability of eEF2 to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hindlimb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P<0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated:total eEF2 was robustly elevated after 3-day HS, which was completely reversed by BAPTA-AM and decreased by 1.7-fold (P<0.05) by nifedipine. Transfection of C2C12 with pCMV-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. Importantly, pharmacological enhancement of eEF2 phosphorylation induced P-p70S6k (T389) upregulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway is upregulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on p70S6k activity as well as protein expression of key atrophy biomarkers, MAFbx/atrogin-1 and MuRF-1.

LPA/LPAR1 signaling induces PGAM1 expression via AKT/mTOR/HIF-1α pathway and increases aerobic glycolysis, contributing to keratinocyte proliferation

AimsPsoriasis is a chronic inflammatory skin disease and lysophosphatidic acid (LPA) has recently been reported to contribute to its pathogenesis through keratinocyte proliferation. In this study, we investigated the metabolic changes in the LPA-induced keratinocyte hyperproliferation and underlying mechanisms. Main methodsHaCaT or HEKa cells were treated with LPA in the presence or absence of various chemical inhibitors. The glycolysis stress was measured using the Seahorse extracellular flux analyzer. Gene knockdown by siRNA transfection was used to investigate the role of LPAR1, PGAM1, and HIF-1α in LPA-induced metabolic changes. We confirmed the expression of PGAM1 and HIF-1α in imiquimod (IMQ)-induced psoriasis-like BALB/c mice. Key findingsLPA increased aerobic glycolysis; however, treatment with ki16425, or LPAR1 knockdown inhibited LPA-induced glycolysis in HaCaT cells. LPA increased the expression of glycolytic enzyme PGAM1 through LPAR1. PGAM1 knockdown in HaCaT cells suppressed LPA-induced cell proliferation, changes in cell cycle factor expression, and inhibited LPA-induced aerobic glycolysis. LPA sequentially activated AKT, mTOR, its downstream target, p70 S6K, and increased HIF-1α expression through LPAR1. HIF-1α knockdown inhibited LPA-induced PGAM1 expression and aerobic glycolysis. A6730 also decreased LPA-induced activation of mTOR and p70 S6K and LPA-induced increases in HIF-1α and PGAM1 expression. Ki16425 suppressed the increased expression of PGAM1 and HIF-1α in the lesions of IMQ-induced psoriasis-like mice. In primary keratinocytes, LPA/LPAR1 signaling also induced AKT-mediated aerobic glycolysis. SignificanceCollectively, the results demonstrated that LPA induces aerobic glycolysis via AKT/mTOR/HIF-1α-dependent PGAM1 expression during keratinocytes proliferation and this might be one of the mechanisms of psoriasis development.

Suppression of SREBP-1 Expression by Simvastatin Decreases Visfatin-Induced Chemoresistance to Sunitinib in Human Renal Carcinoma 786-O Cells.

The resistance of renal cell carcinoma (RCC) to sunitinib impedes the success of chemotherapy in cancer treatment. Although several sunitinib resistance mechanisms have been proposed, little is known concerning the impact of obesity and adipokines in RCC cells. The upregulation of sterol-regulatory element-binding protein-1 (SREBP-1) has been reported to modulate the progression of tumor cells. The present study investigated the effect of visfatin on sunitinib-induced cytotoxicity in RCC cells through SREBP-1 expression. We found that visfatin-induced Akt and p70S6K activation increased SREBP-1 expression in 786-O cells. The visfatin-induced SREBP-1 mRNA and protein levels were attenuated through the inactivation of Akt and p70S6K by pharmacological inhibitors. In addition, the SREBP-1 knockdown using siRNA enhanced the cytotoxic effects of sunitinib. Our results also revealed the roles of simvastatin in attenuating the effects of visfatin on 786-O cells by inhibiting the production of reactive oxygen species. In particular, simvastatin co-treatment increased the cell cytotoxicity of sunitinib in visfatin-treated 786-O cells, which were associated with down-regulation of SREBP-1 expression. Our results suggest an important role of SREBP-1 in visfatin-induced drug resistance of RCC cells to sunitinib. The cytotoxic mechanism of simvastatin on RCC cells may provide a new strategy to improve therapeutic outcomes for the RCC treatment.

Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

Insulin-like growth factor-1 inhibits apoptosis of rat gastric smooth muscle cells under high glucose condition via adenosine monophosphate-activated protein kinase (AMPK) pathway.

Diabetic gastroparesis (DGP) is a common chronic complication of diabetes characterized by decreased gastric motility, and an effective number of gastric smooth muscle cells (GSMCs) ensures gastric motility. A previous study documented that apoptosis was present in gastric smooth muscles in rats with DGP and adenosine monophosphate-activated protein kinase (AMPK) was an important factor of apoptosis of rat GSMCs cultured under high glucose conditions. This study aimed to explore the effect of insulin-like growth factor-1 (IGF-1) on apoptosis of high glucose cultured rat GSMCs after silencing of AMPK and elucidate the underlying mechanism. A total of 120 rats were divided into normal control (NC, n = 20), diabetic gastroparesis (DGP, n = 50) and DGP + IGF-1 (n = 50) groups. After establishing the rat model of DGP, rats in the DGP+IGF-1 group received an intraperitoneal injection of IGF-1 at a dose of 1.5 μg/kg/d for 10 weeks. The level of AMPK activity, liver kinase B1 (LKB1) activity, and calcium/calmodulin-dependent protein kinase b (CaMKKb) expression in rat gastric smooth muscle tissues was detected by Western blot analysis. Apoptosis in rat gastric smooth muscle tissues was detected by TUNEL assay. We also cultured rat GSMCs in vitro under high glucose (HG) condition (35 mM), incubated cells with IGF-1, and silenced AMPK with siRNA. The cells were divided into HG, HG + IGF-1, HG + siRNA, and HG + siRNA + IGF-1 groups. The apoptosis rates of rat GSMCs after silencing AMPK were detected by TUNEL assay and flow cytometry, and apoptosis-related protein expression in rat GSMCs was detected by Western blot. IGF-1 decreased LKB1 activity, CaMKKb expression, AMPK activity, and inhibited apoptosis in rat gastric smooth muscle tissues. Compared with rat GSMCs cultured in vitro under HG conditions, apoptosis rates were reduced after treatment with IGF-1 and AMPK silencing (both p < 0.01). Apoptosis rates were higher in the HG + siRNA group compared with the HG + IGF-1 group (p < 0.05). IGF-1 down-regulated the expression of calcium/calmodulin-dependent kinase II (CaMKII) and p53, up-regulated the expression of p21, PLC-b3, PI3K p110 Ser1070, and the activities of Akt, p70S6K, mTORC1, and mTORC2. IGF-1 also up-regulated Bcl-2 expression and down-regulated the expression of BAX and Caspase-3. IGF-1 can inhibit the apoptosis of rat GSMCs under high glucose conditions, its mechanism may be related to the regulation of expression and activity of p53, PI3K, TSC-2, Akt, mTOR, 4E-BP1, p70S6K, p21, CaMKII, and PLC-b3 in rat GSMCs acting through AMPK pathway.

Polycystin-1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model.

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus

BackgroundIQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. MethodsWe generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. ResultsWe found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1–mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor–guanine nucleotide exchange factor) inactive IQSEC3 mutant. ConclusionsOur results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.

Investigation of the effects of mTOR inhibitors rapamycin and everolimus in combination with carboplatin on canine malignant melanoma cells

BackgroundMalignant melanoma in dogs is considered to be largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that contribute to tumour progression despite aggressive local and systemic therapy. Upregulation of the PI3K/AKT/mTOR pathway has been observed in canine malignant melanoma and may represent a potential target for therapy. Rapamycin (sirolimus) and everolimus are commercially available small molecule inhibitors that target mTOR and therefore may have anticancer activity in canine melanoma. It was hypothesized that there is synergism between rapamycin or everolimus and platinum chemotherapy, and that combination drug treatment would inhibit target/downstream proteins involved in cell viability/proliferation and increase cell death in canine melanoma cells. It was further hypothesized that rapamycin or everolimus would impact metabolism by reducing glycolysis in these cells. Four canine melanoma cell lines were treated in vitro with rapamycin and everolimus as sole treatment or combined with carboplatin. Cell viability, apoptosis, target modulation, and glycolytic metabolism were evaluated by crystal violet colourimetric assay, Annexin V/PI flow cytometry, western blotting, and Seahorse bioanalyzer, respectively.ResultsWhen combined with carboplatin chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis was noted at 72 h after treatment compared to the vehicle control. Levels of phosphorylated mTOR were reduced by rapamycin and everolimus in all four cell lines, but activation of the downstream protein p70S6K was not consistently reduced by treatment in two of the cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma cells, indicating reduced cancer cell glycolytic activity.ConclusionsInhibition of mTOR by rapalogs, such as rapamycin and everolimus combined with carboplatin chemotherapy may have activity in canine melanoma. Future mechanistic investigation is warranted, including in vivo assessment of this combination therapy.

Bisdemethoxycurcumin Promotes Apoptosis and Inhibits the Epithelial-Mesenchymal Transition through the Inhibition of the G-Protein-Coupled Receptor 161/Mammalian Target of Rapamycin Signaling Pathway in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the leading causes of cancer death in the world and lacks an effective targeted therapy. G-protein-coupled receptor 161 (GPR161) has been demonstrated to perform the functional regulations on TNBC progression and might be a potential new target for TNBC therapy. This study showed the effects of bisdemethoxycurcumin (BDMC) on GPR161 regulation, indicating that BDMC effectively inhibited GPR161 expression and downregulated GPR161-driven signaling. BDMC showed the potent inhibitory effects on TNBC proliferation through suppressing GPR161-mediated mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase (p70S6K) activation. Besides, in this study, we discover the mechanism of GPR161-driven TNBC metastasis, linking to GPR161-mediated twist-related protein 1 (Twist1)/matrix metallopeptidase 9 (MMP9) contributing to the epithelial-mesenchymal transition (EMT). BDMC effectively repressed GPR161-mediated TNBC metastasis via inhibiting Twist1/MMP9-induced EMT. The three-dimensional invasion assay also showed that BDMC significantly inhibited TNBC invasion. The combination treatment of BDMC and rapamycin enhanced the inhibition of TNBC proliferation and metastasis through increasing the blockage of mTOR activation. Furthermore, this study also observed that BDMC activated the caspase 3/9 signaling pathway to induce TNBC apoptosis. Therefore, BDMC could be applicable to anticancer therapy, especially targeting on the GPR161-driven cancer type.

Azolla leaf meal at 5% of the diet improves growth performance, intestinal morphology and p70S6K1 activation, and affects cecal microbiota in broiler chicken

With growing concern about including unconventional dietary protein sources in poultry diets to substitute the protein sources that are essential for human consumption such as soybean meal, Azolla leaf meal (ALM) has grown in popularity. In our prior experiment, ALM was used at inclusion rates of 5 and 10%. Five per cent inclusion of ALM increased broiler chicken growth performance, the concentration of cecal propionic acid, and activation of skeletal muscle p70S6 Kinase1 (p70S6K1) without having detrimental effects on the meat quality. Those results prompted us to further evaluate the effect of the same inclusion rates of ALM on phase feeding and intestine and liver health of the broiler chicks. The current study hypothesis is that dietary ALM positively affects phase feeding, intestinal morphology and p70S6K1 activation, cecal microbial gene expression, and improves the liver energy status. For this, we enrolled 135 one-day-old broiler chicks and collected growth performance data (starter, grower, and finisher stages) and samples of the gastrointestinal tract to analyse the morphology of the villi, immune-related organs, mucin, and abundance of intestinal p70S6K1. Cecal bacterial species were analysed using qPCR and liver samples were collected to analyse adenosine monophosphate (AMP) and ATP content and selected oxidative stress biomarkers. ALM increased BW and feed intake during the starter and grower phases but did not affect the feed conversion ratio. Liver oxidative stress and AMP: ATP ratio increased in chickens fed on a diet containing 10% ALM (AZ10; P < 0.05). Jejunum villi length and abundance of duodenal neutral mucin increased but villi of the ileum decreased in chickens fed on a diet containing 5% ALM (AZ5), while lymphoid follicle areas of the cecal tonsils decreased with both doses of ALM. Activation of p70S6K1 increased with AZ10 in the duodenum and AZ5 in the jejunum. In the gut, the family of Enterobacteriaceae decreased with both ALM doses. In conclusion, our results indicate an overall positive effect of dietary inclusion of ALM in the broiler chicken diet via its positive effect on intestinal morphology and function; however, a negative effect on the liver was observed with 10% ALM.

Paclitaxel Inhibits Synoviocyte Migration and Inflammatory Mediator Production in Rheumatoid Arthritis

Activated fibroblast-like synoviocytes (FLSs) play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). It is urgent to develop new drugs that can effectively inhibit the abnormal activation of RA-FLS. In our study, the RA-FLS cell line, MH7A, and mice with collagen-induced arthritis (CIA) were used to evaluate the effect of paclitaxel (PTX). Based on the results, PTX inhibited the migration of RA-FLS in a dose-dependent manner and significantly reduced the spontaneous expression of IL-6, IL-8, and RANKL mRNA and TNF-α-induced transcription of the IL-1 β, IL-8, MMP-8, and MMP-9 genes. However, PTX had no significant effect on apoptosis in RA-FLS. Mechanistic studies revealed that PTX significantly inhibited the TNF-α-induced phosphorylation of ERK1/2 and JNK in the mitogen-activated protein kinase (MAPK) pathway and suppressed the TNF-α-induced activation of AKT, p70S6K, 4EBP1, and HIF-1α in the AKT/mTOR pathway. Moreover, PTX alleviated synovitis and bone destruction in CIA mice. In conclusion, PTX inhibits the migration and inflammatory mediator production of RA-FLS by targeting the MAPK and AKT/mTOR signaling pathways, which provides an experimental basis for the potential application in the treatment of RA.