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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.

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.

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.

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.

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.

The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.