Defective Thyroglobulin: Cell Biology of Disease.

The primary functional units of the thyroid gland are follicles of various sizes comprised of a monolayer of epithelial cells (thyrocytes) surrounding an apical extracellular cavity known as the follicle lumen. In the normal thyroid gland, the follicle lumen is filled with secreted protein (referred to as colloid), comprised nearly exclusively of thyroglobulin with a half-life ranging from days to weeks. At the cellular boundary of the follicle lumen, secreted thyroglobulin becomes iodinated, resulting from the coordinated activities of enzymes localized to the thyrocyte apical plasma membrane. Thyroglobulin appearance in evolution is essentially synchronous with the appearance of the follicular architecture of the vertebrate thyroid gland. Thyroglobulin is the most highly expressed thyroid gene and represents the most abundantly expressed thyroid protein. Wildtype thyroglobulin protein is a large and complex glycoprotein that folds in the endoplasmic reticulum, leading to homodimerization and export via the classical secretory pathway to the follicle lumen. However, of the hundreds of human thyroglobulin genetic variants, most exhibit increased susceptibility to misfolding with defective export from the endoplasmic reticulum, triggering hypothyroidism as well as thyroidal endoplasmic reticulum stress. The human disease of hypothyroidism with defective thyroglobulin (either homozygous, or compound heterozygous) can be experimentally modeled in thyrocyte cell culture, or in whole animals, such as mice that are readily amenable to genetic manipulation. From a combination of approaches, it can be demonstrated that in the setting of thyroglobulin misfolding, thyrocytes under chronic continuous ER stress exhibit increased susceptibility to cell death, with interesting cell biological and pathophysiological consequences.

Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype.

Transient covalent interactions of newly synthesized thyroglobulin with oxidoreductases of the endoplasmic reticulum.

Newly synthesized thyroglobulin (Tg), the thyroid prohormone, forms detectable high molecular weight mixed disulfide adducts: until now, only Tg "adduct B" was identified as primarily engaging the endoplasmic reticulum oxidoreductases ERp57 and protein disulfide isomerase. Here, we demonstrate that the faster migrating Tg adduct C primarily engages the CaBP1/P5 oxidoreductase, whereas the slower migrating Tg adduct A primarily engages ERp72. Upon siRNA-mediated knockdown of CaBP1/P5 or ERp72, adducts C or A, respectively, are decreased. Within the three Tg adduct bands that do not exhibit a precursor-product relationship, Tg exhibits distinct oxidation patterns. We present evidence suggesting that disulfide maturation occurs within Tg monomers engaged in each of the adduct bands. Moreover, the same Tg substrate molecules can form simultaneous mixed disulfides with both CaBP1/P5 and protein disulfide isomerase, although these are generally viewed as components of distinct oxidoreductase-chaperone protein complexes. Such substrate-oxidoreductase combinations offer Tg the potential for simultaneous oxidative maturation along different parallel tracks leading to the native state.

GRP78 mediates cell growth and invasiveness in endometrial cancer.

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.

Cytotoxicity of dental resin composites: an in vitro evaluation.

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 96 h. 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.

Increased hexosamine biosynthetic pathway flux alters cell-cell adhesion in INS-1E cells and murine islets.

PURPOSE: In type 2 Diabetes, ß-cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (dedifferentiation, decline of glucose-stimulated insulin secretion). Apoptosis and dysfunction are caused, at least in part, by glucotoxicity, in which increased flux of glucose in the hexosamine biosynthetic pathway plays a role. In this study, we sought to clarify whether increased hexosamine biosynthetic pathway flux affects another important aspect of ß-cell physiology, that is ß-cell-ß-cell homotypic interactions. METHODS: We used INS-1E cells and murine islets. The expression and cellular distribution of E-cadherin and ß-catenin was evaluated by immunofluorescence, immunohistochemistry and western blot. Cell-cell adhesion was examined by the hanging-drop aggregation assay, islet architecture by isolation and microscopic observation. RESULTS: E-cadherin expression was not changed by increased hexosamine biosynthetic pathway flux, however, there was a decrease of cell surface, and an increase in intracellular E-cadherin. Moreover, intracellular E-cadherin delocalized, at least in part, from the Golgi complex to the endoplasmic reticulum. Beta-catenin was found to parallel the E-cadherin redistribution, showing a dislocation from the plasmamembrane to the cytosol. These changes had as a phenotypic consequence a decreased ability of INS-1E to aggregate. Finally, in ex vivo experiments, glucosamine was able to alter islet structure and to decrease surface abundandance of E-cadherin and ß-catenin. CONCLUSION: Increased hexosamine biosynthetic pathway flux alters E-cadherin cellular localization both in INS-1E cells and murine islets and affects cell-cell adhesion and islet morphology. These changes are likely caused by alterations of E-cadherin function, highlighting a new potential target to counteract the consequences of glucotoxicity on ß-cells.

Maturation of thyroglobulin protein region I.

In vertebrates, the thyroglobulin (Tg) gene product must be exported to the lumen of thyroid follicles for thyroid hormone synthesis. In toto, Tg is composed of multiple type-1 repeats connected by linker and hinge (altogether considered as "region I," nearly 1,200 residues); regions II-III (~720 residues); and cholinesterase-like (ChEL) domain (~570 residues). Regions II-III and ChEL rapidly acquire competence for secretion, yet regions I-II-III require 20 min to become a partially mature disulfide isomer; stabilization of a fully oxidized form requires ChEL. Transition from partially mature to mature Tg occurs as a discrete "jump" in mobility by nonreducing SDS-PAGE, suggesting formation of at most a few final pairings of Cys residues that may be separated by significant intervening primary sequence. Using two independent approaches, we have investigated which portion of Tg is engaged in this late stage of its maturation. First, we demonstrate that this event is linked to oxidation involving region I. Introduction of the Tg-C1245R mutation in the hinge (identical to that causing human goitrous hypothyroidism) inhibits this maturation, although the Cys-1245 partner remains unidentified. Second, we find that Tg truncated after its fourth type-1 repeat is a fully independent secretory protein. Together, the data indicate that final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I.

Endoplasmic reticulum stress is activated in endometrial adenocarcinoma.

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.

Cis and trans actions of the cholinesterase-like domain within the thyroglobulin dimer.

Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the approximately 570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal alpha-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients.

The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone.

Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein ("secretory ChEL") was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism.

The Pervasive Effects of ER Stress on a Typical Endocrine Cell: Dedifferentiation, Mesenchymal Shift and Antioxidant Response in the Thyrocyte.

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.

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase.

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.

The sarcoplasmic-endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells.

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.

Effects of extracellular nucleotides in the thyroid: P2Y2 receptor-mediated ERK1/2 activation and c-Fos induction in PC Cl3 cells.

Aim of the present paper was to investigate the signaling pathways of P2Y2 in rat thyroid PC Cl3 cell line and its effects on proliferation. This study demonstrates that P2Y2 activation provoked: (a) a cytosol-to-membrane translocation of PKC-alpha, -betaI and -epsilon; (b) the phosphorylation of the extra cellular signal-regulated kinases 1 and 2 (ERK1/2); (c) the expression of c-Fos protein; (d) no effects on the G1/S progression and overall cell proliferation. The P2Y2-stimulated ERK1/2 phosphorylation was: (a) completely blocked by PD098059, a mitogen-activated protein kinase (MEK) inhibitor or by W-7, a Ca2+-calmodulin (CaM) antagonist; (b) reduced by GF109203X, inhibitor of PKCs, or AG1478, inhibitor of EGFR tyrosine kinase, or LY294002/wortmannin, inhibitors of phosphoinositide 3-kinases, or cytochalasin D, inhibitor of actin microfilament bundles polymerization. The c-Fos induction was greatly diminished by Go6976 or PD098059, and completely abolished when combined. In conclusion, data indicate that the P2Y2-induced phosphorylation of ERK1/2 and the induction of c-Fos are due to the operation of CaM, with PKC, PI3K, EGFR and receptor endocytosis mechanisms endorsing the signalling. On the other hand, no mitogenic effects of P2Y2 are whatsoever noticed in PC Cl3 cells.

Thyroglobulin From Molecular and Cellular Biology to Clinical Endocrinology.

Thyroglobulin (Tg) is a vertebrate secretory protein synthesized in the thyrocyte endoplasmic reticulum (ER), where it acquires N-linked glycosylation and conformational maturation (including formation of many disulfide bonds), leading to homodimerization. Its primary functions include iodide storage and thyroid hormonogenesis. Tg consists largely of repeating domains, and many tyrosyl residues in these domains become iodinated to form monoiodo- and diiodotyrosine, whereas only a small portion of Tg structure is dedicated to hormone formation. Interestingly, evolutionary ancestors, dependent upon thyroid hormone for development, synthesize thyroid hormones without the complete Tg protein architecture. Nevertheless, in all vertebrates, Tg follows a strict pattern of region I, II-III, and the cholinesterase-like (ChEL) domain. In vertebrates, Tg first undergoes intracellular transport through the secretory pathway, which requires the assistance of thyrocyte ER chaperones and oxidoreductases, as well as coordination of distinct regions of Tg, to achieve a native conformation. Curiously, regions II-III and ChEL behave as fully independent folding units that could function as successful secretory proteins by themselves. However, the large Tg region I (bearing the primary T4-forming site) is incompetent by itself for intracellular transport, requiring the downstream regions II-III and ChEL to complete its folding. A combination of nonsense mutations, frameshift mutations, splice site mutations, and missense mutations in Tg occurs spontaneously to cause congenital hypothyroidism and thyroidal ER stress. These Tg mutants are unable to achieve a native conformation within the ER, interfering with the efficiency of Tg maturation and export to the thyroid follicle lumen for iodide storage and hormonogenesis.

Differential response of normal, dedifferentiated and transformed thyroid cell lines to cisplatin treatment.

The effects of cisplatin (cisPt) on the extra cellular signal-regulated kinase (ERK) and the protein kinase B (PKB/Akt), known to play important roles in promoting cell survival and in down regulating apoptosis, were investigated in thyroid cell lines. The cytotoxic effect of cisPt was highest in normal PC-Cl3 cells, intermediate in dedifferentiated PC-E1A and PC-raf cells and lowest in fully transformed and tumorigenic PC-E1Araf cells. CisPt provoked ERK phosphorylation; such phosphorylation was unaltered by Gö6976, a conventional PKC inhibitor, whilst blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC-Cl3 and in PC-E1Araf cells, respectively. In PC-E1Araf, but not in PC-Cl3 cells, the cisPt-provoked ERK phosphorylation was also blocked by a myristoylated PKC-zeta pseudo substrate peptide (PS-zeta). The cytotoxic effects of cisPt increased when cells were pre-incubated with the mitogen-activated protein kinase (MEK) inhibitor PD98059. CisPt provoked the phosphorylation of PKB/Akt and this effect was blocked by LY294002, a PI3K inhibitor. In PC-Cl3 cells pre-incubated with LY294002 the effects of cisPt on ERK phosphorylation and cell mortality resulted unaffected; conversely, LY294002 reduced the ERK phosphorylation and increased cisPt cytotoxity of in PC-E1Araf cells. Furthermore, in PC-E1Araf cells pre-incubated with LY294002 and PS-zeta ERK phosphorylation was abolished and cisPt cytotoxicity was highest. Altogether results highlight a role for PKCs in the upstream regulation of ERK pathway facing the cell response to cisPt treatments. Understanding the mechanisms by which cells process cisPt provides important insights for designing more efficient platinum-based drugs.

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.

Endoplasmic reticulum stress causes thyroglobulin retention in this organelle and triggers activation of nuclear factor-kappa B via tumor necrosis factor receptor-associated factor 2.

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.

Promoter identification of CIKS, a novel NF-kappaB activating gene, and regulation of its expression.

We have recently identified a novel gene, named CIKS (Connection to IKK-complex and SAPK), able to activate the transcription factor NF-kappaB, after interaction with the regulatory subunit NEMO/IKKgamma of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5'-flanking region of CIKS gene was 'TATA-less', but contained other consensus promoter elements including an initiator (Inr), 'GC' and 'CAAT' boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5'-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from -464 to -220 bp of the 5'-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase-PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and transforming growth factor (TGF)-beta, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5'-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.