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AIMS/HYPOTHESIS: Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models. METHODS: Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 μmol/l MGO. The glyoxalase-1 inhibitor S-p-bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks. RESULTS: MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance. CONCLUSIONS/INTERPRETATION: MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.

Recent studies have indicated that endoplasmic reticulum stress, the unfolded protein response activation and altered GRP78 expression can play an important role in a variety of tumors development and progression. Very recently we reported for the first time that GRP78 is increased in endometrial tumors. However, whether GRP78 could play a role in the growth and/or invasiveness of endometrial cancer cells is still unknown. Here we report that the silencing of GRP78 expression affects both cell growth and invasiveness of Ishikawa and AN3CA cells, analyzed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell migration assay, respectively. At variance with Ishikawa cells, AN3CA cells showed, besides an endoplasmic reticulum, also a plasma membrane GRP78 localization, evidenced by both immunofluorescence and cell membrane biotinylation experiments. Intriguingly, flow cytometry experiments showed that the treatment with a specific antibody targeting GRP78 C-terminal domain caused apoptosis in AN3CA but not in Ishikawa cells. Induction of apoptosis in AN3CA cells was not mediated by the p53 pathway activation but was rather associated to reduced AKT phosphorylation. Interestingly, immunofluorescence analysis evidenced that endometrioid adenocarcinoma tissues displayed, similarly to AN3CA cells, also a GRP78 plasma membrane localization. These data suggest that GRP78 and its plasma membrane localization, might play a role in endometrial cancer development and progression and might constitute a novel target for the treatment of endometrial cancer.

Resin-based dental restorative materials release residual monomers that may affect the vitality of pulp cells. The purpose of this study was to evaluate the cytotoxic effect of two light-cured restorative materials with and without bis-GMA resin, respectively (Clearfil Majesty Posterior and Clearfil Majesty Flow) and a self-curing one (Clearfil DC Core Automix) when applied to the fibroblast cell line NIH-3T3. Samples of the materials were light-cured and placed directly in contact to cells for 24, 48, 72 and 96h. Cytotoxicity was evaluated by measuring cell death by flow cytometry, cell proliferation by proliferation curves analysis and morphological changes by optical microscopy analysis. All the composite materials tested caused a decrease in cell proliferation, albeit at different degrees. However, only Clearfil DC Core Automix induced cell death, very likely by increasing apoptosis. Morphological alteration of treated cells was also evident, particularly in the Clearfil DC Core Automix-treated cells. The different cytotoxic effects of dental composites should be considered when selecting an appropriate resin-based dental restorative material for operative restorations. Copyright © 2011 John Wiley & Sons, Ltd.

Diabetes is a major risk factor for cardiovascular disease, and recent advances in research indicate that a detailed understanding of the pathophysiology of its effects is mandatory to reduce diabetes-related mortality and morbidity. Advanced Glycation End Products (AGEs) play a central role in the genesis and progression of complications of both type 1 and type 2 diabetes mellitus, and have been found to be important even in non-diabetic patients as a marker of cardiovascular disease. AGEs have a profound impact on patient's prognosis regardless of the glycemic control, and therefore pharmacologic approaches against AGEs accumulation have been proposed over the years to treat cardiovascular diseases, parallel to a more detailed understanding of AGEs pathophysiology. Compounds with anti-AGEs effects are currently under investigation in both pre-clinical and clinical scenarios, and many of the drugs previously used to treat specific diseases have been found to have AGE-inhibitory effects. Some products are still in "bench evaluation", whereas others have been already investigated in clinical trials with conflicting evidences. This review aims at summarizing the mechanisms of AGEs formation and accumulation, and the most relevant issues in pre-clinical and clinical experiences in anti-AGEs treatment in cardiovascular research.

Perturbing the endoplasmic reticulum. homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum. Ca2+ adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNR)/stress-activated protein kinase and nuclear factor-kappaB (NF-kappaB) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappaB signaling pathway was assessed by measuring the level of inhibitory subunit IkappaBalpha, DNA binding, and transcriptional activity of NF-kappaB. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit IkappaBalpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNT receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappaB, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

Chronic exposure to polychlorinated biphenyls (PCBs), a class of ubiquitous environmental toxicants, causes neurocognitive anomalies. The transcription factor repressor element 1-silencing transcription factor (REST) plays a critical role in neuronal phenotype elaboration in both neural progenitor cells and non-neuronal cells. Here, we investigated the possible relationship between PCBs and REST in neuroblastoma SH-SY5Y cells. In these cells, chronic exposure to the PCB mixture Aroclor 1254 (A-1254; 5-30 ¼g/ml) caused dose-dependent cell death via the induction of calpain but not caspase-3. Intriguingly, this effect was prevented by the calpain inhibitor calpeptin. Furthermore, A-1254 enhanced REST mRNA and protein expression levels after both 24 and 48 h. REST down-regulation by small interfering RNA prevented A-1254-induced cell death. In addition, A-1254 enhanced the binding of REST to the synapsin 1 gene promoter, and synapsin 1 knockdown potentiated A-1254-induced cell death. A-1254 (10 ¼g/ml) also increased the expression of the two REST cofactors, the REST corepressor and the mammalian SIN3 homolog A transcription regulator. Moreover, the PCB mixture decreased acetylation of the histone proteins H3 and H4. It is noteworthy that the histone deacetylase inhibitor trichostatin A prevented such decreases and reduced the A-1254-induced neurotoxic effect. Collectively, these results suggest that A-1254 exerts its toxic effect via REST by down-regulating synapsin 1 and decreasing H3 and H4 acetylation.

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y(2) purinoceptor activation provoked a transient increase of [Ca(2+)](i), followed by a decreasing sustained phase. The alpha and beta1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca(2+)](i) level and increased the peak of Ca(2+) entry of the P2Y(2)-provoked Ca(2+)transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn(2+) and Ba(2+) uptake were not changed by Gö 6976. Similarly, the Na(+)/Ca(2+) exchanger was not implicated, since the rate of decrement to the basal [Ca(2+)](i) level was equally decreased in physiological and Na(+)-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic-endoplasmic reticulum Ca(2+)ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca(2+)](i) level after P2Y(2) stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca(2+) transients caused by P2Y(2) stimulation.

Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y(+)LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y(+)L, and b(0,+) systems, as assessed by L-arginine uptake and kinetics, inhibition of L-arginine transport by N-ethylmaleimide and neutral amino acids, and L-cystine transport studies. y(+)L and y(+) systems account for the highest transport rate (with y(+)L > y+) and b(0,+) for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y(+)LAT1, y(+)LAT2, rBAT, and b(0,+)AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y(+)LAT1, and rBAT/b(0,+)AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term L-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.

Cnidocytes are the most specialized cells of cnidarians and the structural morphology of their capsules, the cnidocysts, has been recognized as an important taxonomic character within the phylum Cnidaria. Nevertheless, this character has been almost completely neglected by specialists of alcyonarians (Octocorallia), due mainly to the difficulties inherent in the examination of soft tissues lacking calcareous sclerites and the supposed homogeneity of the Octocorallia cnidome. Two different types of cnidocysts, holotrichous isorhizas and atrichous isorhizas, are described here for the first time from the Mediterranean red coral Corallium rubrum (L.) (Octocorallia). Proposed evolutionary trends of anthozoan cnidocysts are also discussed.

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm(3) in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (K(d) approximately 1 microg/ml) and low (K(d) approximately 50 to 60 microg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.

The endoplasmic reticulum (ER) is a complex and multifunctional organelle. It is the intracellular compartment of protein folding, a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. The ER is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity and responds by activating signal transduction pathways, known as unfolded protein response (UPR). Three major classes of transducer are known, inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), which sense with their endoluminal domain the state of protein folding, although the exact mechanism(s) involved is not entirely clear. Depending on whether the homeostatic response of the UPR is successful in restoring an equilibrium between ER load and protein folding or not, the two possible outcomes of the UPR so far considered have been life or death. Indeed, recent efforts have been devoted to understand the life/death switch mechanisms. However, recent data suggest that what appears to be a pure binary decision may in fact be more complex, and survival may be achieved at the expenses of luxury cell functions, such as expression of differentiation genes.

Activation of the Ras-Raf–extracellular signal–regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation by this pathway is poorly understood. In a systemof two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA–polyoma–middle T [PC EIA + Py] and PC EIA–v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py–middle T, evident toward serum-deprivation– and H2O2-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)– dependent, as shown by pharmacologicalMEK inhibition. TheMEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by senescence-associated β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

Conditions perturbing the homeostasis of the endoplasmic reticulum (ER) cause accumulation of unfolded proteins and trigger ER stress. In PC Cl3 thyroid cells, thapsigargin and tunicamycin interfered with the folding of thyroglobulin, causing accumulation of this very large secretory glycoprotein in the ER. Consequently, mRNAs encoding BiP and XBP-1 were induced and spliced, respectively. In the absence of apoptosis, differentiation of PC Cl3 cells was inhibited. mRNA and protein levels of the thyroid-specific genes encoding thyroglobulin, thyroperoxidase and the sodium/iodide symporter and of the genes encoding the thyroid transcription factors TTF-1, TTF-2 and Pax-8 were dramatically downregulated. These effects were, at least in part, transcriptional. Moreover, they were selective and temporally distinct from the general and transient PERK-dependent translational inhibition. Thyroid dedifferentiation was accompanied by changes in the organization of the polarized epithelial monolayer. Downregulation of the mRNA encoding E-cadherin, and upregulation of the mRNAs encoding vimentin, α-smooth muscle actin, α(1)(I) collagen and SNAI1/SIP1, together with formation of actin stress fibers and loss of trans-epithelial resistance were found, confirming an epithelial-mesenchymal transition (EMT). The thyroid-specific and epithelial dedifferentiation by thapsigargin or tunicamycin were completely prevented by the PP2 inhibitor of Src-family kinases and by stable expression of a dominant-negative Src. Together, these data indicate that ER stress induces dedifferentiation and an EMT-like phenotype in thyroid cells through a Src-mediated signaling pathway.

Thyroid cells transformed by the Kirsten-ras oncogene become tumorigenic in syngeneic animals. Their growth is no longer dependent on TSH but becomes dependent on serum. Combining morphological and biochemical evidence, we show that serum withdrawal induces apoptotic cell death in Kirsten and Harvey-ras transformed thyroid cell. On the other hand, neither serum nor TSH withdrawal induce apoptosis in differentiated FRTL-5 cells. The induction of apoptosis by serum withdrawal is rapid and not triggered at a specific phase of the cell cycle. We suggest that induction of apoptosis following growth factor deprivation is an additional important characteristic, besides TSH-independence for growth and dedifferentiation, of the thyroid transformed phenotype.

Multiple lines of evidence suggest that metformin, an antidiabetic drug, exerts anti-tumorigenic effects in different types of cancer. Metformin has been reported to affect cancer cells’ metabolism and proliferation mainly through the activation of AMP-activated protein kinase (AMPK). Here, we show that metformin inhibits, indeed, endometrial cancer cells’ growth and induces apoptosis. More importantly, we report that metformin affects two important pro-survival pathways, such as the Unfolded Protein Response (UPR), following endoplasmic reticulum stress, and the WNT/β-catenin pathway. GRP78, a key protein in the pro-survival arm of the UPR, was indeed downregulated, while GADD153/CHOP, a transcription factor that mediates the pro-apoptotic response of the UPR, was upregulated at both the mRNA and protein level. Furthermore, metformin dramatically inhibited β-catenin mRNA and protein expression. This was paralleled by a reduction in β-catenin transcriptional activity, since metformin inhibited the activity of a TCF/LEF-luciferase promoter. Intriguingly, compound C, a well-known inhibitor of AMPK, was unable to prevent all these effects, suggesting that metformin might inhibit endometrial cancer cells’ growth and survival through the modulation of specific branches of the UPR and the inhibition of the Wnt/β-catenin pathway in an AMPK-independent manner. Our findings may provide new insights on the mechanisms of action of metformin and refine the use of this drug in the treatment of endometrial cancer.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia, responsible for the onset of several long-term complications. Recent evidence suggests that cognitive dysfunction represents an emerging complication of DM, but the underlying molecular mechanisms are still obscure. Dopamine (DA), a neurotransmitter essentially known for its relevance in the regulation of behavior and movement, modulates cognitive function, too. Interestingly, alterations of the dopaminergic system have been observed in DM. This review aims to offer a comprehensive overview of the most relevant experimental results assessing DA’s role in cognitive function, highlighting the presence of dopaminergic dysfunction in DM and supporting a role for glucotoxicity in DM-associated dopaminergic dysfunction and cognitive impairment. Several studies confirm a role for DA in cognition both in animal models and in humans. Similarly, significant alterations of the dopaminergic system have been observed in animal models of experimental diabetes and in diabetic patients, too. Evidence is accumulating that advanced glycation end products (AGEs) and their precursor methylglyoxal (MGO) are associated with cognitive impairment and alterations of the dopaminergic system. Further research is needed to clarify the molecular mechanisms linking DM-associated dopaminergic dysfunction and cognitive impairment and to assess the deleterious impact of glucotoxicity.

The endoplasmic reticulum stress and the unfolded protein response are triggered following an imbalance between protein load and protein folding. Until recently, two possible outcomes of the unfolded protein response have been considered: life or death. We sought to substantiate a third alternative, dedifferentiation, mesenchymal shift, and activation of the antioxidant response by using typical endocrine cells, i.e. thyroid cells. The thyroid is a unique system both of endoplasmic reticulum stress (a single protein, thyroglobulin represents the majority of proteins synthesized in the endoplasmic reticulum by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism of decline/loss of function leading to a deficit of thyroid hormones formation.

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.

Objectives: Endometrial cancer is the most common malignancy of the female genital tract. However, in spite of a huge advance in our understanding of endometrial cancer biology, therapeutic modalities haven't significantly changed over the past 40 years. The activation of the Unfolded Protein Response (UPR) and GRP78 increase following Endoplasmic Reticulum (ER) stress have been recently identified as mechanisms favoring growth, invasion and resistance to therapy of different types of cancer. However, a possible role of ER stress and GRP78 in endometrial cancer has never been investigated. Methods: Tissue specimens from normal and neoplastic endometrium were analyzed for the expression of the ER stress markers GRP78, ATF6 and CHOP by Real-Time RT-PCR. In addition, GRP78 protein expression and localization were evaluated by Western blot and immunohistochemistry, respectively. The effect of GRP78 knock down on cell growth of Ishikawa cells was analyzed by proliferation curve analysis. Results: In this analysis, the expression levels of GRP78, ATF6 and CHOP mRNAs were significantly increased in specimens of endometrioid endometrial carcinomas. GRP78 and ATF6 protein expression levels were also increased in specimens of endometrial adenocarcinomas. GRP78 knock down caused a decrease of Ishikawa cells' growth. Conclusions: The increased expression of ER stress markers in endometrioid endometrial carcinomas suggests a role for ER stress, the UPR and, possibly, GRP78 in endometrial cancer. Whether these mechanisms have a substantial function in the pathogenesis of malignant transformation of human endometrium is still under investigation in our laboratory. © 2012 Elsevier Inc. All rights reserved.

AIMS/HYPOTHESIS: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis. METHODS: We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence. RESULTS: Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose. CONCLUSIONS/INTERPRETATION: Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.

Chronic exposure to polychlorinated biphenyls (PCBs), a class of ubiquitous environmental toxicants, causes neurocognitive anomalies. The transcription factor repressor element 1-silencing transcription factor (REST) plays a critical role in neuronal phenotype elaboration in both neural progenitor cells and non-neuronal cells. Here, we investigated the possible relationship between PCBs and REST in neuroblastoma SH-SY5Y cells. In these cells, chronic exposure to the PCB mixture Aroclor 1254 (A-1254; 5-30 μg/ml) caused dose-dependent cell death via the induction of calpain but not caspase-3. Intriguingly, this effect was prevented by the calpain inhibitor calpeptin. Furthermore, A-1254 enhanced REST mRNA and protein expression levels after both 24 and 48 h. REST down-regulation by small interfering RNA prevented A-1254-induced cell death. In addition, A-1254 enhanced the binding of REST to the synapsin 1 gene promoter, and synapsin 1 knockdown potentiated A-1254-induced cell death. A-1254 (10 μg/ml) also increased the expression of the two REST cofactors, the REST corepressor and the mammalian SIN3 homolog A transcription regulator. Moreover, the PCB mixture decreased acetylation of the histone proteins H3 and H4. It is noteworthy that the histone deacetylase inhibitor trichostatin A prevented such decreases and reduced the A-1254-induced neurotoxic effect. Collectively, these results suggest that A-1254 exerts its toxic effect via REST by down-regulating synapsin 1 and decreasing H3 and H4 acetylation.

Objective To investigate immunologic parameters and endoplasmic reticulum (ER) stress associated with unexplained infertility. Design Case-control study. Setting Academic center. Patient(s) Women with no fertility problems (FS) (n = 13), women with recurrent miscarriage (RM) (n = 15) and women with repeated in vitro fertilization failure (RIF) (n = 15). Intervention(s) Endometrial biopsy and collection of peripheral blood during the midsecretory phase of menstrual cycle. Main Outcome Measure(s) Leptin, resistin, soluble tumor necrosis factor receptor (sTNF-R), myeloperoxidase (MPO), soluble intercellular adhesion molecule 1 (sICAM-1), and interleukin 22 (IL-22) concentration in peripheral blood, endometrial CD3 + , CD4 + , CD5 + , CD8 + , and FoxP3 + T lymphocytes, and endometrial expression of HSPA5, a specific marker of ER stress. Result(s) We found an increase of proinflammatory molecules such as resistin, leptin, and IL-22 in both RM and RIF patients; sTNF-R and MPO only in RIF patients when compared with the FS women. We also found in endometria of infertile women a statistically significant increase of CD3 + , CD4 + , CD8 + in both RM and RIF patients and CD5 + in RM patients when compared with FS women. This was paralleled by a statistically significant reduction of infiltrating FoxP3 + regulatory T cells. Finally, endometrial HSPA5 expression levels were statistically significantly up-regulated in both RM and RIF patients. Conclusion(s) Women with RM and RIF showed an increase of circulating proinflammatory cytokines, altered endometrial T lymphocytes subsets, and signs of endometrial ER stress.

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15) mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15)). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15) cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15). These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A. , and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.

AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.