Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.     

Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel-staining analysis were used. Our results showed that chronic pretreatment with beta-estradiol 3-benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed-lineage kinase 3 (MLK3), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non-nuclear components, c-Jun and Bcl-2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3-kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERalpha and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet-stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERalpha interacts with PI3K to activate Akt1, which may inhibit the MLK3-MKK4/7-JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.     

Inhibition of SH2-domain containing inositol phosphatase 2 (SHIP2) in insulin producing INS1E cells improves insulin signal transduction and induces proliferation

Inhibition of the lipid phosphatase SH2-domain containing inositol phosphatase 2 (SHIP2) in L6-C10 muscle cells, in 3T3-L1 adipocytes and in the liver of db/db mice has been shown to ameliorate insulin signal transduction and established SHIP2 as a negative regulator of insulin action. Here we show that SHIP2 inhibition in INS1E insulinoma cells increased Akt, glycogen synthase kinase 3 and extracellular signal-regulated kinases 1 and 2 phosphorylation. SHIP2 inhibition did not prevent palmitate-induced apoptosis, but increased cell proliferation. Our data raise the interesting possibility that SHIP2 inhibition exerts proliferative effects in beta-cells and further support the attractiveness of a specific inhibition of SHIP2 for the treatment of type 2 diabetes.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

Oxidative stress and the etiology of insulin resistance and type 2 diabetes

The condition of oxidative stress arises when oxidant production exceeds antioxidant activity in cells and plasma. The overabundance of oxidants is mechanistically connected to the multifactorial etiology of insulin resistance, primarily in skeletal muscle tissue, and the subsequent development of type 2 diabetes. Two important mechanisms for this oxidant excess are (1) the mitochondrial overproduction of hydrogen peroxide and superoxide ion under conditions of energy surplus and (2) the enhanced activation of cellular NADPH oxidase via angiotensin II receptors. Several recent studies are reviewed that support the concept that direct exposure of mammalian skeletal muscle to an oxidant stress (including hydrogen peroxide) results in stimulation of the serine kinase p38 mitogen-activated protein kinase (p38 MAPK), and that the engagement of this stress-activated p38 MAPK signaling is mechanistically associated with diminished insulin-dependent stimulation of insulin signaling elements and glucose transport activity. The beneficial interactions between the antioxidant α-lipoic acid and the advanced glycation end-product inhibitor pyridoxamine that ameliorate oxidant stress-associated defects in whole-body and skeletal-muscle insulin action in the obese Zucker rat, a model of prediabetes, are also addressed. Overall, this review highlights the importance of oxidative stress in the development of insulin resistance in mammalian skeletal muscle tissue, at least in part via a p38-MAPK-dependent mechanism, and indicates that interventions that reduce this oxidative stress and oxidative damage can improve insulin action in insulin-resistant animal models. Strategies to prevent and ameliorate oxidative stress remain important in the overall treatment of insulin resistance and type 2 diabetes.     

2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, enhances the adhesion of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes

2-Arachidonoylglycerol (2-AG), an endogenous cannabionoid receptor (CB1 and CB2) ligand, enhanced the adhesion of HL-60 cells differentiated into macrophage-like cells to fibronectin and the vascular cell adhesion molecule-1. The CB2 receptor, Gi/Go, intracellular free Ca(2+) and phosphatidylinositol 3-kinase were shown to be involved in 2-AG-induced augmented cell adhesion. 2-AG also enhanced the adhesion of human monocytic leukemia U937 cells and peripheral blood monocytes. These results strongly suggest that 2-AG plays some essential role in inflammatory reactions and immune responses by inducing robust adhesion to extracellular matrix proteins and adhesion molecules in several types of inflammatory cells and immune-competent cells.     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

Alpha-V-dependent outside-in signaling is required for the regulation of CD44 surface expression, MMP-2 secretion, and cell migration by osteopontin in human melanoma cells

The level of integrin alpha(v)beta3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp(60c-Src) kinase activity associated with integrin alpha(v)beta3 in melanoma cells (M21) treated with soluble OPN. pp(60c-Src) kinase activity was not observed in melanoma cells expressing alpha(v) that lacks the cytoplasmic domain (alpha(v)995). Results of the current study demonstrate that the amino acid sequence '995RPPQEEQERE1004' in the beta-turn of alpha(v) chain is required for the interaction of pp(60c-Src). Our results suggest that the beta-turn of alpha(v) chain may be indispensable for alpha(v)-associated signaling complex formation and outside-in signaling. To further analyze the alpha(v)beta3 signaling in melanoma cells, we over expressed OPN in M21 cells (M21/OPN). CD44 surface expression and MMP-2 activity in the conditioned medium were increased to a greater extent in M21/OPN cells as compared with M21 or alpha(v)995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to alpha(v) and MMP-2. Our findings suggest that the increase in MMP-2 activity is integrin-dependent as MMP-2 activity is reduced in cells treated with an inhibitor to alpha(v) or in alpha(v)995 cells expressing mutant alpha(v).     

Spatiotemporal control of cyclic AMP immunomodulation through the PKA-Csk inhibitory pathway is achieved by anchoring to an Ezrin-EBP50-PAG scaffold in effector T cells

A variety of immunoregulatory signals to effector T cells from monocytes, macrophages and regulatory T cells act through cyclic adenosine monophosphate. In the effector T cell, the protein kinase A (PKA) type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal target for PKA phosphorylation is C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The A kinase anchoring protein Ezrin colocalizes PKA and Csk by forming a supramolecular signaling complex consisting of PKA, Ezrin, Ezrin/radixin/moesin (ERM) binding protein of 50 kDa (EBP50), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (GEMs) (PAG) and Csk.     

Inhibition of mTOR down-regulates scavenger receptor,class B,type I (SR-BI) expression,reduces endothelial cell migration and impairs nitric oxide production

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.     

Conjugated linoleic acid-induced apoptosis in mouse mammary tumor cells is mediated by both G protein coupled receptor-dependent activation of the AMP-activated protein kinase pathway and by oxidative stress

Conjugated linoleic acid (CLA) has shown chemopreventive activity in several tumorigenesis models, in part through induction of apoptosis. We previously demonstrated that the t10,c12 isomer of CLA induced apoptosis of TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum (ER) stress pathways, and that the AMP-activated protein kinase (AMPK) played a critical role in the apoptotic effect. In the current study, we focused on the upstream pathways by which AMPK was activated, and additionally evaluated the contributing role of oxidative stress to apoptosis. CLA-induced activation of AMPK and/or induction of apoptosis were inhibited by infection of TM4t cells with an adenovirus expressing a peptide which blocks the interaction between the G protein coupled receptor (GPCR) and Gα_q, by the phospholipase C (PLC) inhibitor U73122, by the inositol trisphosphate (IP₃) receptor inhibitor 2-APB, by the calcium/calmodulin-dependent protein kinase kinase α (CaMKK) inhibitor STO-609 and by the intracellular Ca²⁺ chelator BAPTA-AM. This suggests that t10,c12-CLA may exert its apoptotic effect by stimulating GPCR through Gα_q signaling, activation of phosphatidylinositol-PLC, followed by binding of the PLC-generated IP₃ to its receptor on the ER, triggering Ca²⁺ release from the ER and finally stimulating the CaMKK–AMPK pathway. t10,c12-CLA also increased oxidative stress and lipid peroxidation, and antioxidants blocked its apoptotic effect, as well as the CLA-induced activation of p38 MAPK, a downstream effector of AMPK. Together these data elucidate two major pathways by which t10,c12-CLA induces apoptosis, and suggest a point of intersection of the two pathways both upstream and downstream of AMPK.     

Moderate superoxide production is an early promoter of mitochondrial biogenesis in differentiating N2a neuroblastoma cells

Reactive oxygen species (ROS) have been widely considered as harmful for cell development and as promoters of cell aging by increasing oxidative stress. However, ROS have an important role in cell signaling and they have been demonstrated to be beneficial by triggering hormetic signals, which could protect the organism from later insults. In the present study, N2a murine neuroblastoma cells were used as a paradigm of cell-specific (neural) differentiation partly mediated by ROS. Differentiation was triggered by the established treatments of serum starvation, forskolin or dibutyryl cyclic AMP. A marked differentiation, expressed as the development of neurites, was detected by fixation and staining with coomassie brilliant blue after 48 h treatment. This was accompanied by an increase in mitochondrial mass detected by mitotracker green staining, an increased expression of the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1-alpha (PGC-1α) and succinate dehydrogenase activity as detected by MTT. In line with these results, an increase in free radicals, specifically superoxide anion, was detected in differentiating cells by flow cytometry. Superoxide scavenging by MnTBAP and MAPK inhibition by PD98059 partially reversed differentiation and mitochondrial biogenesis. In this way, we demonstrated that mitochondrial biogenesis and differentiation are mediated by superoxide and MAPK cues. Our data suggest that differentiation and mitochondrial biogenesis in N2a cells are part of a hormetic response which is triggered by a modest increase of superoxide anion concentration within the mitochondria.     

Mitigation of radiation injury by selective stimulation of the LPA2 receptor

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA₂ receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA₂ receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24 h after radiation exposure. Our findings suggest that by specifically activating LPA₂ receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.