Decreased Akt Phosphorylation Research Articles

Effect of oxymatrine on liver gluconeogenesis is associated with the regulation of PEPCK and G6Pase expression and AKT phosphorylation.

An increase in liver gluconeogenesis is an important pathological phenomenon in type 2 diabetes mellitus (T2DM) and oxymatrine is an effective natural drug used for T2DM treatment. The present study aimed to explore the effect of oxymatrine on gluconeogenesis and elucidate the underlying mechanism. Male Sprague-Dawley rats were treated with a high-fat diet and streptozotocin for 4 weeks to induce T2DM, and HepG2 cells were treated with 55 mM glucose to simulate T2DM in vitro. T2DM rats were treated with oxymatrine (10 or 20 mg/kg weight) or metformin for 4 weeks, and HepG2 cells were treated with oxymatrine (0.1 or 1 µM), metformin (0.1 µM), or oxymatrine combined with MK-2206 (AKT inhibitor) for 24 h. Fasting blood glucose and insulin sensitivity of rats were measured to evaluate insulin resistance. Glucose production and uptake ability were measured to evaluate gluconeogenesis in HepG2 cells, and the expression of related genes was detected to explore the molecular mechanism. Additionally, the body weight, liver weight and liver index were measured and hematoxylin and eosin staining was performed to evaluate the effects of the disease. The fasting glucose levels of T2DM rats was 16.5 mmol/l, whereas in the control rats, it was 6.1 mmol/l. Decreased insulin sensitivity (K-value, 0.2), body weight loss (weight, 300 g), liver weight gain, liver index increase (value, 48) and morphological changes were observed in T2DM rats, accompanied by reduced AKT phosphorylation, and upregulated expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). High-glucose treatment significantly increased glucose production and decreased glucose uptake in HepG2 cells, concomitant with a decrease in AKT phosphorylation and increase of PEPCK and G6Pase expression. In vivo, oxymatrine dose-dependently increased the sensitivity of T2DM rats to insulin, increased AKT phosphorylation and decreased PEPCK and G6Pase expression in the liver, and reversed the liver morphological changes. In vitro, oxymatrine dose-dependently increased AKT phosphorylation and glucose uptake of HepG2 cells subjected to high-glucose treatment, which was accompanied by inhibition of the expression of the gluconeogenesis-related genes, PEPCK and G6Pase. MK-2206 significantly inhibited the protective effects of oxymatrine in high-glucose-treated cells. These data indicated that oxymatrine can effectively prevent insulin resistance and gluconeogenesis, and its mechanism may be at least partly associated with the regulation of PEPCK and G6Pase expression and AKT phosphorylation in the liver.

Serum‐ and glucocorticoid‐inducible kinase 1 promotes insulin resistance in adipocytes via degradation of insulin receptor substrate 1

Accumulating evidence indicates that serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a role in the development of metabolic syndrome via a poorly understood mechanism. This study aimed to investigate the direct effect of SGK1 on insulin sensitivity in adipose tissue. We ectopically expressed or silenced SGK1 in adipocytes via lentiviral transfection, measured glucose uptake and evaluated insulin signalling using western blotting. In vivo insulin resistance was measured at the whole-body and adipose tissue levels in db/db mice treated with an inhibitor of SGK1. After 8 weeks of SGK1 inhibitor treatment, the serum insulin level andhomeostasis model assessment of insulin resistance index were significantly decreased, and AKT phosphorylation in adipose tissue was enhanced in db/db mice. Overexpression of constitutively active SGK1 in adipocytes in vitro decreased AKT phosphorylation and insulin-stimulated glucose uptake. Dexamethasone and oleic acid increased SGK1 expression and decreased AKT phosphorylation and insulin receptor substrate expression in adipocytes. Administration of an inhibitor of SGK1 or Lv-shSGK1 reversed the suppression of insulin signalling induced by dexamethasone and oleic acid. SGK1 overexpression increased FoxO1 phosphorylation, and administration of Lv-shSGK1 reversed an increase in FoxO1 phosphorylation induced by dexamethasone and oleic acid. Thus, SGK1 mediates the effect of glucocorticoids and high-fat feeding and induces insulin resistance in adipocytes. Our data suggest that SGK1 is a possible therapeutic target for metabolic syndrome and related complications.

Roles of ERK/Akt signals in mitochondria-dependent and endoplasmic reticulum stress-triggered neuronal cell apoptosis induced by 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a major active metabolite of bisphenol A

Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toxicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoxicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5–15 μM) exhibited greater neuronal cytotoxicity than BPA (50–100 μM). The mitochondria-dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the increase in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 μM)-induced Neuro-2a cell death. Exposure of Neuro-2a cells to MBP (10 μM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-box binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 μM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pretreatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoxicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.

Galectin-14 Promotes Trophoblast Migration and Invasion by Upregulating the Expression of MMP-9 and N-Cadherin.

Galectin-14 is specifically expressed in placental trophoblasts, and its expression is reduced in trophoblasts retrieved from the cervix of women destined to develop early pregnancy loss. However, the roles of galectin-14 in regulating trophoblasts and in the pathogenesis of pregnancy complication have never been investigated. In the current research, we aimed to investigate the roles of galectin-14 in the regulation of trophoblasts. Tissues of the placenta and villi were collected. Primary trophoblasts and human trophoblast cell line HTR-8/SVneo were used. Western blotting and RT-PCR were used to quantify gene expression. The siRNA-mediated galectin-14 knockdown and lentivirus-mediated overexpression were performed to manipulate the gene expression in trophoblasts. Transwell migration and invasion assays were used to evaluate cell migration and invasion capacity. Gelatin zymography was used to determine the gelatinase activity. Galectin-14 was significantly decreased in the villi of early pregnancy loss and the placenta of preeclampsia. Knockdown of galectin-14 in primary trophoblasts inhibited cell migration and invasion, downregulated the expression of matrix metalloproteinase (MMP)-9 and N-cadherin, the activity of MMP-9, and decreased the phosphorylation of Akt. Meanwhile, the overexpression of galectin-14 in HTR-8/SVneo promoted cell migration and invasion, upregulated the expression of MMP-9 and N-cadherin, the activity of MMP-9, and increased the phosphorylation of Akt. Increased Akt phosphorylation promoted cell migration and invasion and upregulated the expression and activity of MMP-9, while decreased Akt phosphorylation inhibited cell migration and invasion and downregulated the expression and activity of MMP-9. Thus, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation. The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.

Host versus cell-dependent effects of β-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is significantly overexpressed (>4-fold) in PCa cells relative to βarr2. In this study, we investigated the effect of βarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in β-arrestin depletion of βarr1 in TRAMP mice (TRAMP/βarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/βarr2-/- animals. Prostate tissues from TRAMP/βarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of βarr1 in TRAMP-C1 (TRAMP-C1-βarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of βarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-βarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-βarr1-GFP and MDA PCa 2b-βarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of βarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

H2O2 mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.

Abstract PO041: Everolimus and MK-2206 reverse P4-resistant endometrial hyperplasia in Mig-6 mutant mice

Abstract Endometrial cancer is the most frequent type of gynecological tumor in the United States. Hysterectomy, the surgical removal of the uterus, is the main therapy for endometrial cancer, but this is not an option for women seeking to preserve their fertility. In cases of early-stage endometrial cancer and endometrial hyperplasia, hormonal therapy is a vital nonsurgical option. However, progesterone (P4) resistance robs women of the option to choose hormone therapy to preserve their uterus. Therefore, developing nonsurgical treatments for endometrial cancer remains an essential goal for women who wish to maintain endometrial functions and women in high-risk surgical conditions. Mitogen-inducible gene 6 (MIG-6) acts as a tumor suppressor during endometrial tumorigenesis and as a P4 target gene in humans and mice. We developed mice with the conditional knockout of Mig-6 in all uterine compartments (Pgrcre/+Mig-6f/f; Mig-6KO) and endometrial epithelial cell-specific Mig-6 knockout (Sprr2fcre/+Mig-6f/f; Mig-6Ep-KO) mice. P4 treatment abated the development of endometrial hyperplasia and restored morphological and histological characteristics of the uterus of Mig-6Ep-KO mice, but Mig-6Ep-KO mice showed P4 resistance. P4 treatment reduced cell proliferation which was accompanied by decreased AKT signaling. In order to investigate the inhibition effect of AKT/mTOR signaling as non-surgical treatment on P4-resistant endometrial hyperplasia, we treated Mig-6KO and Mig-6Ep-KO mice with either: 1) vehicle (PBS); 2) MK-2206 (360 mg/kg, once a week); or 3) everolimus (10mg/kg daily) at 10 weeks old (n=6/treatment/genotype) for 1 month. MK-2206 has completed phase II trials and inhibits AKT phosphorylation. Everolimus is FDA-approved mTOR inhibitor. The vehicle group of Mig-6KO and Mig-6Ep-KO mice manifested the endometrial hyperplasia phenotype. In contrast, IHC analysis showed that the groups receiving MK-2206 or everolimus experienced significantly decreased AKT phosphorylation and phosphorylated S6 levels, respectively, and decreased epithelial proliferation, resulting in restoration of normal uterine histology. These data suggest that everolimus and MK-2206 may be new non-surgical treatment options for P4-resistant endometrial hyperplasia. This information may help guide more effective non-surgical interventions in the future. The project described was supported by Grant Number P50CA098258 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Citation Format: Tae Hoon Kim, Jae-Wook Jeong. Everolimus and MK-2206 reverse P4-resistant endometrial hyperplasia in Mig-6 mutant mice [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO041.

The kynurenine derivative 3-HAA sensitizes hepatocellular carcinoma to sorafenib by upregulating phosphatases.

Objectives: Sorafenib is the only FDA-approved first-line target drug for HCC patients. However, sorafenib merely confers 3-5 months of survival benefit with less than 30% of HCC patients sensitive to sorafenib therapy. Thus, it's necessary to develop a sensitizer for hepatocellular carcinoma (HCC) to sorafenib. Methods: The principal component analysis, gene ontology, and KEGG analysis are utilized following RNA-sequencing. The mass spectrometry analysis following immunoprecipitation is performed to discover the phosphatase targets. Most importantly, both the cell line-derived xenograft (CDX) and the patient-derived xenograft (PDX) mouse model are used to determine the effect of 3-HAA on sorafenib-resistant HCC in vivo. Results: In nude mice carrying HCC xenograft, tumor growth is inhibited by sorafenib or 3-HAA alone. When used in combination, the treatment particularly prevents the xenograft from growing. Combined treatment also suppresses the growth of sorafenib-resistant (≥30mg/kg) PDXs. In a set of mechanistic experiments, we find enhanced AKT activation and decreased apoptotic cells in de novo and acquired sorafenib-resistant HCC cells and tissues. 3-HAA decreases AKT phosphorylation and increases the apoptosis of HCC in both cultured cells and mouse xenografts by upregulation of phosphatases PPP1R15A/DUSP6. PPP1R15A/PPP1α directly reduces Akt phosphorylation while DUSP6 decreases Akt activity through inhibiting PDK1. The AKT activator abolishes 3-HAA inhibition of HCC growth in vitro and in mice. Conclusion: This study demonstrates that 3-HAA sensitizes HCC cells to sorafenib by upregulation of phosphatases, suggesting it as a promising molecule for HCC therapy.

METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial\u2013mesenchymal transition of renal tubular cells in diabetic kidney disease

Histone deacetylase 5 (HDAC5) belongs to class II HDAC subfamily and is reported to be increased in the kidneys of diabetic patients and animals. However, little is known about its function and the exact mechanism in diabetic kidney disease (DKD). Here, we found that HDAC5 was located in renal glomeruli and tubular cells, and significantly upregulated in diabetic mice and UUO mice, especially in renal tubular cells and interstitium. Knockdown of HDAC5 ameliorated high glucose-induced epithelial–mesenchymal transition (EMT) of HK2 cells, indicated in the increased E-cadherin and decreased α-SMA, via the downregulation of TGF-β1. Furthermore, HDAC5 expression was regulated by PI3K/Akt signaling pathway and inhibition of PI3K/Akt pathway by LY294002 treatment or Akt phosphorylation mutation reduced HDAC5 and TGF-β1 expression in vitro high glucose-cultured HK2 cells. Again, high glucose stimulation downregulated total m6A RNA methylation level of HK2 cells. Then, m6A demethylase inhibitor MA2 treatment decreased Akt phosphorylation, HDAC5, and TGF-β1 expression in high glucose-cultured HK2 cells. In addition, m6A modification-associated methylase METTL3 and METTL14 were decreased by high glucose at the levels of mRNA and protein. METTL14 not METTL3 overexpression led to PI3K/Akt pathway inactivation in high glucose-treated HK2 cells by enhancing PTEN, followed by HDAC5 and TGF-β1 expression downregulation. Finally, in vivo HDACs inhibitor TSA treatment alleviated extracellular matrix accumulation in kidneys of diabetic mice, accompanied with HDAC5, TGF-β1, and α-SMA expression downregulation. These above data suggest that METTL14-regulated PI3K/Akt signaling pathway via PTEN affected HDAC5-mediated EMT of renal tubular cells in diabetic kidney disease.

Silymarin Inhibits Glutamate Release and Prevents against Kainic Acid-Induced Excitotoxic Injury in Rats.

Silymarin, a polyphenoic flavonoid derived from the seeds of milk thistle (Silybum marianum), exhibits neuroprotective effects. In this study, we used a model of rat cerebrocortical synaptosomes to investigate whether silymarin affects the release of glutamate, an essential neurotransmitter involved in excitotoxicity. Its possible neuroprotective effect on a rat model of kainic acid (KA)-induced excitotoxicity was also investigated. In rat cortical synaptosomes, silymarin reduced glutamate release and calcium elevation evoked by the K+ channel blocker 4-aminopyridine but did not affect glutamate release caused by the Na+ channel activator veratridine or the synaptosomal membrane potential. Decreased glutamate release by silymarin was prevented by removal of extracellular calcium and blocking of N- and P/Q-type Ca2+ channel or extracellular signal-regulated kinase 1/2 (ERK1/2) but not by blocking of intracellular Ca2+ release. Immunoblotting assay results revealed that silymarin reduced 4-aminopyridine-induced phosphorylation of ERK1/2. Moreover, systemic treatment of rats with silymarin (50 or 100 mg/kg) 30 min before systemic KA (15 mg/kg) administration attenuated KA-induced seizures, glutamate concentration elevation, neuronal damage, glial activation, and heat shock protein 70 expression as well as upregulated KA-induced decrease in Akt phosphorylation in the rat hippocampus. Taken together, the present study demonstrated that silymarin depressed synaptosomal glutamate release by suppressing voltage-dependent Ca2+ entry and ERK1/2 activity and effectively prevented KA-induced in vivo excitotoxicity.

DDRE-34. TRIBBLES-1 (TRIB1) PSEUDOKINASE PROMOTES SURVIVAL OF GBM CELLS BY DECREASING RT/TMZ INDUCED APOPTOSIS

Abstract INTRODUCTION Glioblastoma (GBM) is the most aggressive form of glioma with a mean survival of 12 to 15 months after diagnosis. The current therapy involves administration of radiation therapy and temozolomide (TMZ). Due to poor survival outcome, new druggable therapeutic targets need to be identified for drug development. TRIB1 is a Ser/Thr pseudokinase that acts as a scaffold protein to mediate proteasomal degradation of its substrate proteins in the cell. TRIB1 has been associated with tumorigenesis and therapy resistance of several cancers, including HCC, NSCLC among others. In the present study we show that TRIB1 can promote GBM by increasing survival of GBM cells and reducing RT/TMZ-induced cell death. MATERIALS AND METHODS We identified TRIB1 by statistical association (Cox regression analysis) of the patient-derived methylation data generated using 450K and EPIC methylation array. Functional validation of TRIB1 was done in vitro by overexpression and knockdown approaches. Doxycycline inducible system was used for TRIB1 knockdown. Stable cell lines were generated by puromycin selection. Western blotting was utilized to detect protein levels. RESULTS The global methylation analysis on a Utrecht GBM cohort showed that TRIB1 promoter methylation was associated with better OS of GBM patients (HR = 0.81 (0.62–1.05); p = 0.11). We also observed that TRIB1 overexpression caused a decrease in apoptosis of patient-derived GBM cell lines after RT/TMZ treatment. Additionally, an increase in the phosphorylation of ERK and Akt was also noted. However, TRIB1 knockdown only led to decreased Akt phosphorylation. CONCLUSION TRIB1 promotes GBM cell survival by upregulating survival signaling pathways like ERK and Akt, which leads to decreased effects of RT/TMZ therapy. Targeting TRIB1 would reduce oncogenic signaling in these cells and therefore may sensitize them towards RT/TMZ therapy.

Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation.

Does osteoprotegerin (OPG) promote human endometrial stromal decidualization? OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation. OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive. We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs. We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT-PCR (qRT-PCR). Immunofluorescence staining and western blot, and qRT-PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT-PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization. OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P < 0.05) and prolactin (PRL) (P < 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P < 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P < 0.05). N/A. In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration. OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL. This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.

Astaxanthin Suppresses PM2.5-Induced Neuroinflammation by Regulating Akt Phosphorylation in BV-2 Microglial Cells.

Air pollution has become one of the most serious issues for human health and has been shown to be particularly concerning for neural and cognitive health. Recent studies suggest that fine particulate matter of less than 2.5 (PM2.5), common in air pollution, can reach the brain, potentially resulting in the development and acceleration of various neurological disorders including Alzheimer’s disease, Parkinson’s disease, and other forms of dementia, but the underlying pathological mechanisms are not clear. Astaxanthin is a red-colored phytonutrient carotenoid that has been known for anti-inflammatory and neuroprotective effects. In this study, we demonstrated that exposure to PM2.5 increases the neuroinflammation, the expression of proinflammatory M1, and disease-associated microglia (DAM) signature markers in microglial cells, and that treatment with astaxanthin can prevent the neurotoxic effects of this exposure through anti-inflammatory properties. Diesel particulate matter (Sigma-Aldrich) was used as a fine particulate matter 2.5 in the present study. Cultured rat glial cells and BV-2 microglial cells were treated with various concentrations of PM2.5, and then the expression of various inflammatory mediators and signaling pathways were measured using qRT-PCR and Western blot. Astaxanthin was then added and assayed as above to evaluate its effects on microglial changes, inflammation, and toxicity induced by PM2.5. PM2.5 increased the production of nitric oxide and reactive oxygen species and upregulated the transcription of various proinflammatory markers including Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), inducible nitric oxide synthase (iNOS), triggering receptor expressed on myeloid cells 2 (TREM2), Toll-like receptor 2/4 (TLR2/4), and cyclooxygenase-2 (COX-2) in BV-2 microglial cells. However, the mRNA expression of IL-10 and arginase-1 decreased following PM2.5 treatment. PM2.5 treatment increased c-Jun N-terminal kinases (JNK) phosphorylation and decreased Akt phosphorylation. Astaxanthin attenuated these PM2.5-induced responses, reducing transcription of the proinflammatory markers iNOS and heme oxygenase-1 (HO-1), which prevented neuronal cell death. Our results indicate that PM2.5 exposure reformulates microglia via proinflammatory M1 and DAM phenotype, leading to neurotoxicity, and the fact that astaxanthin treatment can prevent neurotoxicity by inhibiting transition to the proinflammatory M1 and DAM phenotypes. These results demonstrate that PM2.5 exposure can induce brain damage through the change of proinflammatory M1 and DAM signatures in the microglial cells, as well as the fact that astaxanthin can have a potential beneficial effect on PM2.5 exposure of the brain.

Overexpression of Smad7 in hypothalamic POMC neurons disrupts glucose balance by attenuating central insulin signaling

ObjectiveAlthough the hypothalamus is crucial for peripheral metabolism control, the signals in specific neurons involved remain poorly understood. The aim of our current study was to explore the role of the hypothalamic gene mothers against decapentaplegic homolog 7 (Smad7) in peripheral glucose disorders. MethodsWe studied glucose metabolism in high-fat diet (HFD)-fed mice and middle-aged mice with Cre-mediated recombination causing 1) overexpression of Smad7 in hypothalamic proopiomelanocortin (POMC) neurons, 2) deletion of Smad7 in POMC neurons, and 3) overexpression of protein kinase B (AKT) in arcuate nucleus (ARC) in Smad7 overexpressed mice. Intracerebroventricular (ICV) cannulation of insulin was used to test the hypothalamic insulin sensitivity in the mice. Hypothalamic primary neurons were used to investigate the mechanism of Smad7 regulating hypothalamic insulin signaling. ResultsWe found that Smad7 expression was increased in POMC neurons in the hypothalamic ARC of HFD-fed or middle-aged mice. Furthermore, overexpression of Smad7 in POMC neurons disrupted the glucose balance, and deletion of Smad7 in POMC neurons prevented diet- or age-induced glucose disorders, which was likely to be independent of changes in body weight or food intake. Moreover, the effect of Smad7 was reversed by overexpression of AKT in the ARC. Finally, Smad7 decreased AKT phosphorylation by activating protein phosphatase 1c in hypothalamic primary neurons. ConclusionsOur results demonstrated that an excess of central Smad7 in POMC neurons disrupts glucose balance by attenuating hypothalamic insulin signaling. In addition, we found that this regulation was mediated by the activity of protein phosphatase 1c.

Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis

ObjectiveMicroRNAs (miRNAs) may be viable targets for treating renal interstitial fibrosis (RIF). Fuzheng Huayu recipe (FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF. MethodsRIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content, Masson’s staining and type I collagen expression were used to evaluate renal collagen deposition. Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog (PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II (Ang-II) to induce epithelial-to-mesenchymal transition (EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), while E-cadherin and α-smooth muscle actin (α-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation. ResultsFZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited α-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells. ConclusionmiR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.

Abstract 1430: CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells

Abstract Background-The canonical PI3K/Akt pathway is hyperactive in a variety of cancers including ovarian cancer (OVCa). Using baculovirus-expressed, FPLC-purified enzymes, we previously demonstrated that Calcium/Calmodulin-dependent CaM Kinase Kinase 2 (CaMKK2) activates the oncogene Akt by direct phosphorylation of its primary activation site T308. In OVCAR-3 and other OVCa cells, RNAi-mediated knockdown of CaMKK2 decreased basal Akt phosphorylation at T308 in a Phosphoinositide-Dependent Kinase-1 (PDK1)-independent manner. CaMKK2 knockdown also decreased Akt phosphorylation at its secondary activation site, S473 by an apparently indirect mechanism. Results- In this study we show that CaMKK2 mediates downstream effects of epidermal growth factor (EGF) on the Akt cascade in OVCa cells. Treatment of OVCAR3 cells with EGF produced a prominent enhancement of p-Akt T308 which was reduced ~2-fold, by either CaMKK2 or PDK1 knockdowns. Knockdown of CaMKK2 but not PDK1 reduced p-Akt S473 after EGF treatment. Similar results were obtained with Insulin-like growth factor 1 (IGF1). To discern how CaMKK2 is engaged by EGF action we examined whether, in vitro, CaMKK2 phosphorylation of WT Akt or a synthetic peptide encompassing T308 is stimulated by Calcium/CaM and/or Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). CaMKK2 activity was enhanced by Calcium/CaM but not by soluble PIP3. CaMKK2 did not bind to a dot blot of various phosphoinositides, although strong binding to PIP3 was observed with PDK1. Alternatively, Akt regulation by CaMKK2 may occur via a Calcium/CaM-dependent pathway. Increased p-Akt T308 and S473 upon treatment of cells with the calcium-ionophore, ionomycin, was significantly reduced by CaMKK2 knockdown. EGF stimulation of Akt phosphorylation at both T308 and S473 was significantly reduced by the intracellular calcium chelator, BAPTA-AM and by the CaM inhibitor, W7. We also probed the mechanism by which optimal cellular Akt phosphorylation at S473 requires CaMKK2. mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PKcs) are considered candidate kinases for p-Akt S473. CaMKK2 knockdown regulated p-Akt S473 in M059J cells, a DNA-PKcs null cell line similarly to that observed with OVCAR3 cells, suggesting that mTORC2 may be regulated by CaMKK2. Using an MS/MS Protein-protein interaction (PPI) screen we observed CaMMK2 interaction with multiple ribosomal proteins an observation consistent with suggested mTORC2 ribosomal regulation of Akt S473 phosphorylation. Conclusions-This study documents Akt activating phosphorylation by CaMKK2 in response to EGF in a Calcium/CaM-dependent, PI3K/PIP3 independent, manner. Besides directly phosphorylating Akt at T308, CaMKK2 regulates p-Akt S473 potentially through the regulation of mTORC2 activity. Our results highlight the importance of CaMKK2 activated by growth factor signals as a parallel system to the oncogenic PI3K/PDK1 pathway. Citation Format: Shuhang Dai, Angela M. Gocher, Arthur M. Edelman. CaMKK2 regulates EGF-dependent activation of oncogenic Akt in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1430.

Dihydroartemisinin inhibits the migration of esophageal cancer cells by inducing autophagy.

Esophageal cancer (EC) is a complex gastrointestinal malignancy and its global incidence rate ranks 7th among all cancer types. Due to its aggressive nature and the potential for early metastasis, the survival rates of patients with EC are poor. Dihydroartemisinin (DHA) is the primary active derivative of artemisinin, and, as well as its use as an anti-malarial, DHA has also exhibited antitumor activity in various cancer models, such as cholangiocarcinoma, head and neck carcinoma, and hepatocellular carcinoma cells. However, the molecular mechanisms underlying the antitumor effect of DHA in the treatment of EC remains poorly understood. The results of the present study demonstrated that DHA significantly inhibited the migration of TE-1 and Eca-109 EC cells in a dose-dependent manner by activating autophagy. DHA treatment also significantly reversed epithelial-mesenchymal transition (EMT) by downregulating the EMT-associated markers, N-cadherin and vimentin, and upregulating the expression of E-cadherin. Mechanistically, DHA treatment decreased Akt phosphorylation and inhibited the Akt/mTOR signaling pathway, leading to the activation of autophagy. The levels of the autophagy-associated proteins were suppressed and DHA-mediated inhibition of migration in EC cells was reversed when an active form of Akt was overexpressed. In conclusion, the present study demonstrated the potential value of DHA in the treatment of EC, and revealed the underlying mechanism by which FDHA inhibits cellular migration.

Metformin and Everolimus: A Promising Combination for Neuroendocrine Tumors Treatment.

Introduction: Treatment options for neuroendocrine tumors (NETs) are rarely curative, as NETs frequently show resistance to medical therapy. The use of everolimus, an mTOR inhibitor, is limited by the development of resistance, probably due to the activation of Akt signaling. In this context, the antidiabetic drug metformin is able to inhibit mTOR, providing a rationale for the use of metformin and everolimus in combination. Methods: We investigated the effects of the metformin and everolimus combination on NET cell proliferation, apoptosis, colony formation, cell viability, NET spheroids growth and the involvement of the Akt and mTOR pathways, and also developed everolimus-resistant NET cells to further study this combination. Results: Metformin and everolimus in combination are more effective than monotherapy in inhibiting pancreatic NET (PAN-NET) cell proliferation (−71% ± 13%, p < 0.0001 vs. basal), whereas no additive effects were observed on pulmonary neuroendocrine tumor (PNT) cell proliferation. The combinatorial treatment is more effective than monotherapy in inhibiting colony formation, cell viability, NET spheroids growth rate and mTOR phosphorylation in both NET cell lines. In a PAN-NET cell line, metformin did not affect Akt phosphorylation; conversely, it significantly decreased Akt phosphorylation in a PNT cell line. Using everolimus-resistant NET cells, we confirmed that metformin maintained its effects, acting by two different pathways: Akt-dependent or independent, depending on the cell type, with both leading to mTOR suppression. Conclusions: Considering the promising effects of the everolimus and metformin combination in NET cells, our results provide a rationale for its use in NET patients.

Lignan from Alnus japonica Inhibits Adipocyte Differentiation via Cell Cycle and FOXO1 Regulation.

In the present study, we isolated a lignan ((−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol, DFS) from Alnus japonica and evaluated its antiobesity potential in vitro. We also determined its mechanism of action in a mouse pre-adipocyte 3T3-L1 cell line. DFS dose- and day-dependently inhibited adipogenesis by downregulation of adipogenic factors and lipid metabolism-regulating factors during adipocyte differentiation. In particular, DFS suppressed cell cycle-regulating factors and induced G0/G1 cell cycle arrest, implying that it had an inhibitory effect on mitotic clonal expansion which occurred at an early stage of adipogenesis. DFS also suppressed adipogenesis through decreasing Akt phosphorylation and increasing the level of Forkhead box protein-O1 (FOXO1). These results suggest that DFS may be a pharmacological candidate for the development of antiobesity, therapeutic, and nutraceutical products.

Tripartite motif 10 regulates cardiac hypertrophy by targeting the PTEN/AKT pathway.

The pathogenesis of cardiac hypertrophy is tightly associated with activation of intracellular hypertrophic signalling pathways, which leads to the synthesis of various proteins. Tripartite motif 10 (TRIM10) is an E3 ligase with important functions in protein quality control. However, its role in cardiac hypertrophy was unclear. In this study, neonatal rat cardiomyocytes (NRCMs) and TRIM10‐knockout mice were subjected to phenylephrine (PE) stimulation or transverse aortic constriction (TAC) to induce cardiac hypertrophy in vitro and in vivo, respectively. Trim10 expression was significantly increased in hypertrophied murine hearts and PE‐stimulated NRCMs. Knockdown of TRIM10 in NRCMs alleviated PE‐induced changes in the size of cardiomyocytes and hypertrophy gene expression, whereas TRIM10 overexpression aggravated these changes. These results were further verified in TRIM10‐knockout mice. Mechanistically, we found that TRIM10 knockout or knockdown decreased AKT phosphorylation. Furthermore, we found that TRIM10 knockout or knockdown increased ubiquitination of phosphatase and tensin homolog (PTEN), which negatively regulated AKT activation. The results of this study reveal the involvement of TRIM10 in pathological cardiac hypertrophy, which may occur by prompting of PTEN ubiquitination and subsequent activation of AKT signalling. Therefore, TRIM10 may be a promising target for treatment of cardiac hypertrophy.