Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway.

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.

Cell fate following ER stress: just a matter of "quo ante" recovery or death?

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.

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity.

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.

Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells.

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.

The expression of the sarco/endoplasmic reticulum Ca2+-ATPases in thyroid and its down-regulation following neoplastic transformation.

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.

Graves' disease: a host defense mechanism gone awry.

In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.

Thyroglobulin repression of thyroid transcription factor 1 (TTF-1) gene expression is mediated by decreased DNA binding of nuclear factor I proteins which control constitutive TTF-1 expression.

Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp -264 and -153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp -264 to -153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.

Follicular thyroglobulin (TG) suppression of thyroid-restricted genes involves the apical membrane asialoglycoprotein receptor and TG phosphorylation.

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.

Thyroglobulin regulates follicular function and heterogeneity by suppressing thyroid-specific gene expression.

Thyroglobulin (TG) is the primary synthetic product of the thyroid and the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and lysosomal degradation can each modulate thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (the TSHR), increases thyroid hormone levels by upregulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of the thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex (MHC) class I gene expression, which is also regulated by TTF-1 and Pax-8 in the thyroid, is simultaneously decreased; this maintains self tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. We now show that follicular TG, 27S > 19S > 12S, counter-regulates TSH-increased thyroid-specific gene transcription by suppressing the expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS and TSHR genes, but increases class I expression. TG action involves an apical membrane TG-binding protein; however, it acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the start of TTF-1 transcription. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS and/or TSHR gene expression. TG activity is not duplicated by thyroid hormones or iodide. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism which regulates follicular function, regulates thyroid hormone secretion, and contributes to follicular heterogeneity.

In vivo expression of thyroid transcription factor-1 RNA and its relation to thyroid function and follicular heterogeneity: identification of follicular thyroglobulin as a feedback suppressor of thyroid transcription factor-1 RNA levels and thyroglobulin synthesis.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

Perturbation of cellular calcium delays the secretion and alters the glycosylation of thyroglobulin in FRTL-5 cells.

Treatment of FRTL-5 cells with a Ca2+ ionophore, A23187, or a specific inhibitor of the endoplasmic reticulum Ca2+ ATPases, thapsigargin, delayed thyroglobulin secretion. The secreted thyroglobulin showed an increased electrophoretic mobility and a reduced sensitivity to neuraminidase. Only thyroglobulin that was still in the endoplasmic reticulum was sensitive to the Ca(2+)-perturbant drugs as shown by experiments in which the drugs were added at different times during a chase. Analysis of the carbohydrate chains by BioGel P4 showed that thyroglobulin secreted in the presence of the Ca(2+)-perturbants displayed an increased ratio high mannose/complex chains.

Serum withdrawal induces apoptotic cell death in Ki-ras transformed but not in normal differentiated thyroid cells.

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.

Differential expression of the asialoglycoprotein receptor in discrete brain areas, in kidney and thyroid.

The asyaloglycoprotein receptor is a dimer formed by two polypeptide chains abundantly expressed in the liver (RHL-1 and RHL-2). Using specific primers for the two polypeptide chains we measured, by semiquantitative reverse PCR (RT-PCR), the corresponding mRNAs in different rat tissues. We found that both RHL-1 and RHL-2 mRNAs are expressed in the liver, kidney, brain and thyroid. Under the same conditions we did not detect any specific mRNA in the spleen. In the brain these sequences are expressed along a posterior-anterior gradient. Cerebellum and brainstem display the highest expression of the brain RHL-1 and RHL-2 mRNAs. Tissues and regional distribution of this receptor suggest that other body districts besides liver may participate in the clearance of serum glycoproteins.