Metformin ameliorates IL-6-induced hepatic insulin resistance via induction of orphan nuclear receptor small heterodimer partner (SHP) in mouse models.

AIMS/HYPOTHESIS: IL-6 is a proinflammatory cytokine associated with the pathogenesis of hepatic diseases. Metformin is an anti-diabetic drug used for the treatment of type 2 diabetes, and orphan nuclear receptor small heterodimer partner (SHP, also known as NR0B2), a transcriptional co-repressor, plays an important role in maintaining metabolic homeostasis. Here, we demonstrate that metformin-mediated activation of AMP-activated protein kinase (AMPK) increases SHP protein production and regulates IL-6-induced hepatic insulin resistance. METHODS: We investigated metformin-mediated SHP production improved insulin resistance through the regulation of an IL-6-dependent pathway (involving signal transducer and activator of transcription 3 [STAT3] and suppressor of cytokine signalling 3 [SOCS3]) in both Shp knockdown and Shp null mice. RESULTS: IL-6-induced STAT3 transactivation and SOCS3 production were significantly repressed by metformin, adenoviral constitutively active AMPK (Ad-CA-AMPK), and adenoviral SHP (Ad-SHP), but not in Shp knockdown, or with the adenoviral dominant negative form of AMPK (Ad-DN-AMPK). Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP) and protein localisation studies showed that SHP inhibits DNA binding of STAT3 on the Socs3 gene promoter via interaction and colocalisation within the nucleus. Upregulation of inflammatory genes and downregulation of hepatic insulin signalling by acute IL-6 treatment were observed in wild-type mice but not in Shp null mice. Finally, chronic IL-6 exposure caused hepatic insulin resistance, leading to impaired insulin tolerance and elevated gluconeogenesis, and these phenomena were aggravated in Shp null mice. CONCLUSIONS/INTERPRETATION: Our results demonstrate that SHP upregulation by metformin may prevent hepatic disorders by regulating the IL-6-dependent pathway, and that this pathway can help to ameliorate the pathogenesis of cytokine-mediated metabolic dysfunction.

p66Shc expression in proliferating thyroid cells is regulated by thyrotropin receptor signaling.

It is almost unanimously accepted that thyrocyte proliferation is synergistically activated by TSH and insulin/IGF-I. Moreover, it was recently suggested that p66Shc, which is an adaptor molecule of the IGF-I receptor, might play a critical role in this synergistic effect. In this study, we undertook to confirm the role and the mechanism underlying the regulation of p66Shc expression via TSH receptor in thyrocytes. We have found that p66Shc expression is elevated in proliferating human thyroid tissues, including adenomatous goiter, adenoma, Graves' disease, and thyroid cancer, but not in normal thyroid. Among growth factors, TSH increased p66Shc expression both in vivo and in vitro; however, IGF-I, epidermal growth factor, or insulin did not. TSH and Graves' Ig increased the p66Shc expression via the TSH receptor-G(s)-cAMP pathway. However, interestingly, IGF-I or epidermal growth factor increased the tyrosine phosphorylations of p66Shc, and this was enhanced by TSH pretreatment. A similar synergism was observed during the DNA synthesis. When we measured the p66Shc levels induced by individual Igs from 130 patients with Graves' disease, TSH receptor stimulating activity and goiter size showed a weak correlation. We conclude that the expression of p66Shc is regulated by signaling through the TSH receptor in proliferating thyroid cells and that p66Shc appears to be an important mediator of the synergistic effect between TSH and IGF-I with respect to thyrocyte proliferation. Moreover, we suggest that TSH potentiates the regulatory effect of IGF-I on thyrocyte growth, at least in part, by increasing the expression of p66Shc.

Iodide suppression of major histocompatibility class I gene expression in thyroid cells involves enhancer A and the transcription factor NF-kappa B.

High concentrations of iodide can induce transient, clinical improvement in patients with autoimmune Graves' disease. Previous work has related this iodide action to the autoregulatory effect of iodide on the growth and function of the thyroid; more recently, we additionally related this to the ability of iodide to suppress major histocompatibility (MHC) class I RNA levels and antigen expression on thyrocytes. In this report, we describe a transcriptional mechanism involved in iodide suppression of class I gene expression, which is potentially relevant to the autoregulatory action of iodide. Transfection experiments in FRTL-5 cells show that iodide decreases class I promoter activity and that this effect can be ascribed to the ability of iodide to modulate the formation of two specific protein/DNA complexes with enhancer A, -180 to -170 bp, of the class 1 5'-flanking region. Thus, iodide decreases the formation of Mod-1, an enhancer A complex involving the p50 subunit of NF-kappa B and a c-fos family member, fra-2, which was previously shown to be important in the suppression of class I levels by hydrocortisone. Unlike hydrocortisone, iodide also increases the formation of a complex with enhancer A, which we show, in antibody shift experiments, is a heterodimer of the p50 and p65 subunits of NF-kappa B. The changes in these complexes are not duplicated by chloride and are related to the action of iodide on class I RNA levels by the following observations. First, FRTL-5 thyroid cells with an aged phenotype coincidentally lose the ability of iodide to decrease MHC class I RNA levels and to induce changes in either complex. Second, the effect of iodide on class I RNA levels and on enhancer A complex formation with Mod-1 and the p50/p65 heterodimer is inhibited by agents that block the inositol phosphate, Ca++, phospholipase A2, arachidonate signal transduction pathway: acetylsalicylate, indomethacin, and 5,8,11,14-eicosatetraynoic acid. Interestingly, iodide can also decrease formation of the Mod-1 complex and increase formation of the complex with the p50/p65 subunits of NF-kappa B when the NF-kappa B enhancer sequence from the Ig kappa light chain, rather than enhancer A, is used as probe; and both actions mimic the action of a phorbol ester. This suggests that iodide may regulate complex formation with NF-kappa B regulatory elements on multiple genes associated with growth and function, providing a potential mechanism relating the autoregulatory action of iodide on thyroid cells and its action on class I gene expression.

Thyrotropin induces SOCS-1 (suppressor of cytokine signaling-1) and SOCS-3 in FRTL-5 thyroid cells.

TSH has multiple physiological roles: it is required for growth, differentiation, and function of the thyroid gland, and it regulates transcription of interferon-gamma (IFN-gamma)-responsive genes in thyrocytes, including genes for the major histocompatibility complex and intercellular adhesion molecule-1. This report demonstrates that TSH induces the expression of suppressor of cytokine signaling (SOCS)-1 and -3 proteins and alters the phosphorylation state of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1 and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were examined after treatment with TSH or IFN-gamma in either TSH-sensitive FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL) cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells. IFN-gamma up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells, as reported previously for nonthyroid cells. Interestingly, TSH also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5 cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in response to IFN-gamma were reduced. Furthermore, overexpression of either SOCS-1 or SOCS-3 significantly inhibited the IFN-gamma-mediated transactivation of the rat ICAM-1 (intercellular adhesion molecule-1) promoter. TSH and IFN-gamma had different effects on STAT1 and STAT3 phosphorylation. The phosphorylation of Y701 in STAT1, which is responsible for homodimer formation, nuclear translocation, and DNA binding, was specifically stimulated by IFN-gamma, but not by TSH or forskolin. However, the phosphorylation of S727 in STAT1 was induced by IFN-gamma, TSH, and forskolin. TSH induced phosphorylation of both Y705 and S727 in STAT3, while IFN-gamma phosphorylated only the Y705. In addition, we found that SOCS-3 was associated with JAK1 and JAK2 and that these associations were stimulated by TSH. These findings demonstrate that TSH induces SOCS in thyroid cells and provides the evidence of signal cross-talk between TSH and cytokines in thyroid cells.

Involvement of JAK/STAT (Janus kinase/signal transducer and activator of transcription) in the thyrotropin signaling pathway.

TSH is an important physiological regulator of growth and function in thyroid gland. The mechanism of action of TSH depends on interaction with its receptor coupled to heterotrimeric G proteins. We show here that TSH induces the phosphorylation of tyrosine in the intracellular kinases Janus kinase 1 (JAK1) and -2 (JAK2) in rat thyroid cells and in Chinese hamster ovary (CHO) cells transfected with human TSH receptor (TSHR). The JAK family substrates STAT3 (signal transducers and activators of transcription) are rapidly tyrosine phosphorylated in response to TSH. We also find that JAK1, JAK2, and STAT3 coprecipitate with the TSHR, indicating that the TSHR may be able to signal through the intracellular phosphorylation pathway used by the JAK-STAT cascade. TSH increases STAT3-mediated promoter activity and also induces endogenous SOCS-1 (suppressor of cytokine signaling-1) gene expression, a known target gene of STAT3. The expression of a dominant negative form of STAT3 completely inhibited TSH-mediated SOCS-1 expression. These findings suggest that the TSHR is able to signal through JAK/STAT3 pathways.

CR6-interacting factor 1 is a key regulator in Aß-induced mitochondrial disruption and pathogenesis of Alzheimer's disease.

Mitochondrial dysfunction, often characterized by massive fission and other morphological abnormalities, is a well-known risk factor for Alzheimer's disease (AD). One causative mechanism underlying AD-associated mitochondrial dysfunction is thought to be amyloid-ß (Aß), yet the pathways between Aß and mitochondrial dysfunction remain elusive. In this study, we report that CR6-interacting factor 1 (Crif1), a mitochondrial inner membrane protein, is a key player in Aß-induced mitochondrial dysfunction. Specifically, we found that Crif1 levels were downregulated in the pathological regions of Tg6799 mice brains, wherein overexpressed Aß undergoes self-aggregation. Downregulation of Crif1 was similarly observed in human AD brains as well as in SH-SY5Y cells treated with Aß. In addition, knockdown of Crif1, using RNA interference, induced mitochondrial dysfunction with phenotypes similar to those observed in Aß-treated cells. Conversely, Crif1 overexpression prevented Aß-induced mitochondrial dysfunction and cell death. Finally, we show that Aß-induced downregulation of Crif1 is mediated by enhanced reactive oxygen species (ROS) and ROS-dependent sumoylation of the transcription factor specificity protein 1 (Sp1). These results identify the ROS-Sp1-Crif1 pathway to be a new mechanism underlying Aß-induced mitochondrial dysfunction and suggest that ROS-mediated downregulation of Crif1 is a crucial event in AD pathology. We propose that Crif1 may serve as a novel therapeutic target in the treatment of AD.

SIRT2 regulates tumour hypoxia response by promoting HIF-1α hydroxylation.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that has a central role in the regulation of tumour metabolism under hypoxic conditions. HIF-1α stimulates glycolytic energy production and promotes tumour growth. Sirtuins are NAD(+)-dependent protein deacetylases that regulate cellular metabolism in response to stress; however, their involvement in the hypoxic response remains unclear. In this study, it is shown that SIRT2-mediated deacetylation of HIF-1α regulates its stability in tumour cells. SIRT2 overexpression destabilized HIF-1α under hypoxic conditions, whereas HIF-1α protein levels were high in SIRT2-deficient cells. SIRT2 directly interacted with HIF-1α and deacetylated Lys709 of HIF-1α. Deacetylation of HIF-1α by SIRT2 resulted in increased binding affinity for prolyl hydroxylase 2, a key regulator of HIF-1α stability, and increased HIF-1α hydroxylation and ubiquitination. Moreover, a pharmacological agent that increased the intracellular NAD(+)/NADH ratio led to the degradation of HIF-1α by increasing SIRT2-mediated deacetylation and subsequent hydroxylation. These findings suggest that SIRT2-mediated HIF-1α deacetylation is critical for the destablization of HIF-1α and the hypoxic response of tumour cells.

Thyrotropin modulates interferon-gamma-mediated intercellular adhesion molecule-1 gene expression by inhibiting Janus kinase-1 and signal transducer and activator of transcription-1 activation in thyroid cells.

TSH is known as an important hormone that plays the major role not only in the maintenance of normal physiology but also in the regulation of immunomodulatory gene expression in thyrocytes. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) was identified as one of the proteins that are abnormally expressed in the thyroid gland during autoimmune thyroid diseases. In this study we found that TSH inhibits interferon-gamma (IFNgamma)-mediated expression of the ICAM-1 gene, and we investigated the involved mechanisms in rat FRTL-5 thyroid cells. After exposure to IFNgamma, ICAM-1 expression is positively regulated at the level of transcription. This effect occurs via the IFNgamma-activated site (GAS) element in the ICAM-1 promoter as a consequence of the activation of STAT1 (signal transducer and activator of transcription-1), but not of STAT3. On the other hand, after exposure to TSH plus IFNgamma, ICAM-1 transcription is negatively modulated. We found that this inhibitory effect of TSH also occurs via the GAS element. Electrophoretic mobility shift assays confirmed that the IFNgamma-induced DNA-binding activities of STAT1 were reduced by TSH. Furthermore, our results showed that the inhibitory effect of TSH on IFNgamma signaling is caused by inhibition of tyrosine phosphorylation on STAT1, Janus kinase-1 (Jak1), and IFNgamma receptor a, but not Jak2. In conclusion, we have identified a novel mechanism in which TSH modulates the IFNgamma-mediated Jak/STAT signaling pathway through the inhibition of Jak1 and STAT1.

Major histocompatibility class II HLA-DR alpha gene expression in thyrocytes: counter regulation by the class II transactivator and the thyroid Y box protein.

Aberrant expression of major histocompatibility complex (MHC) class II proteins on thyrocytes, which is associated with autoimmune thyroid disease, is mimicked by gamma-interferon (gamma-IFN). To define elements and factors that regulate class II gene expression in thyrocytes and that might be involved in aberrant expression, we have studied gamma-IFN-induced HLA-DR alpha gene expression in rat FRTL-5 thyroid cells. The present report shows that class II expression in FRTL-5 thyrocytes is positively regulated by the class II transactivator (CIITA), and that CIITA mimics the action of gamma-IFN. Thus, as is the case for gamma-IFN, several distinct and highly conserved elements on the 5'-flanking region of the HLA-DR alpha gene, the S, X1, X2, and Y boxes between -137 to -65 bp, are required for class II gene expression induced by pCIITA transfection in FRTL-5 thyroid cells. CIITA and gamma-IFN do not cause additive increases in HLA-DR alpha gene expression in FRTL-5 cells, consistent with the possibility that CIITA is an intermediate factor in the gamma-IFN pathway to increased class II gene expression. Additionally, gamma-IFN treatment of FRTL-5 cells induces an endogenous CIITA transcript; pCIITA transfection mimics the ability of gamma-IFN treatment of FRTL-5 thyroid cells to increase the formation of a specific and novel protein/DNA complex containing CBP, a coactivator of CRE binding proteins important for cAMP-induced gene expression; and the action of both gamma-IFN and CIITA to increase class II gene expression and increase complex formation is reduced by cotransfection of a thyroid Y box protein, which suppresses MHC class I gene expression in FRTL-5 thyroid cells and is a homolog of human YB-1, which suppresses MHC class II expression in human glioma cells. We conclude that CIITA and TSH receptor suppressor element binding protein-1 are components of the gamma-IFN-regulated transduction system which, respectively, increase or decrease class II gene expression in thyrocytes and may, therefore, be involved in aberrant class II expression associated with autoimmune thyroid disease.

Regulation of major histocompatibility class II gene expression in FRTL-5 thyrocytes: opposite effects of interferon and methimazole.

Aberrant expression of major histocompatibility complex (MHC) class II antigens is associated with autoimmune thyroid disease; aberrant expression duplicating the autoimmune state can be induced by interferon-gamma (IFNgamma). We have studied IFNgamma-induced human leukocyte antigen (HLA)-DR alpha gene expression in rat FRTL-5 thyroid cells to identify the elements and factors important for aberrant expression. Using an HLA-DR alpha 5'-flanking region construct from -176 to +45 bp coupled to the chloramphenicol acetyltransferase reporter gene, we show that there is no basal class II gene expression in FRTL-5 thyroid cells, that IFNgamma can induce expression, and, as is the case for antigen-presenting cells from the immune system, that IFNgamma-induced expression requires several highly conserved elements on the 5'-flanking region, which, from 5' to 3', are the S, X1, X2, and Y boxes. Methimazole (MMI), a drug used to treat patients with Graves' disease and experimental thyroiditis in rats or mice, can suppress the IFNgamma-induced increase in HLA-DR alpha gene expression as a function of time and concentration; MMI simultaneously decreases IFNgamma-induced endogenous antigen presentation by the cell. Using gel shift assays and the HLA-DR alpha 5'-flanking region from -176 or -137 to +45 bp as radiolabeled probes, we observed the formation of a major protein-DNA complex with extracts from FRTL-5 cells untreated with IFNgamma, termed the basal or constitutive complex, and formation of an additional complex with a slightly faster mobility in extracts from cells treated with IFNgamma. MMI treatment of cells prevents IFNgamma from increasing the formation of this faster migrating complex. Formation of both complexes is specific, as evidenced in competition studies with unlabeled fragments between -137 and -38 bp from the start of transcription; nevertheless, they can be distinguished in such studies. Thus, high concentrations of double stranded oligonucleotides containing the sequence of the Y box, but not S, X1, or X2 box sequences, can prevent formation of the IFNgamma-increased faster migrating complex, but not the basal complex. Both complexes involve multiple proteins and can be distinguished by differences in their protein composition. Thus, using specific antisera, we show that two cAMP response element-binding proteins, activating transcription factor-1 and/or -2, are dominant proteins in the upper or basal complex. The upper or basal complex also includes c-Fos, Fra-2, Ets-2, and Oct-1. A dominant protein that distinguishes the IFNgamma-increased lower complex is CREB-binding protein (CBP), a coactivator of cAMP response element-binding proteins. We, therefore, show that aberrant expression of MHC class II in thyrocytes, induced by IFNgamma, is associated with the induction or increased formation of a novel protein-DNA complex and that its formation as well as aberrant class II expression are suppressed by MMI, a drug used to treat human and experimental autoimmune thyroid disease. Its component proteins differ from those in a major, basal, or constitutive protein-DNA complex formed with the class II 5'-flanking region in cells that are not treated with IFNgamma and that do not express the class II gene.

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.

TSH regulates a gene expression encoding ERp29, an endoplasmic reticulum stress protein, in the thyrocytes of FRTL-5 cells.

This experiment was performed to evaluate the effect of thyroid-stimulating hormone (TSH) on the endoplasmic reticulum resident 29 kDa protein (ERp29) gene expression in the thyrocytes of FRTL-5 cells. Although ERp29 mRNA was constantly expressed, its expression began to increase remarkably from 10(-9) M TSH. On the other hand, the effect of TSH on the abundance of ERp29 mRNA started within 6 h and peaked at 8 h. Actinomycin D strongly blocked this effect while cycloheximide did not. The half-life of ERp29 mRNA was about 4-4.5 h in the presence or absence of TSH that was not affected by the stability of ERp29 mRNA. The effect of TSH on the ERp29 gene expression was specific, while other growth factors (transferrin, insulin and hydrocortisone) did not alter its expression. Our data indicate for the first time that the expression of ERp29 is regulated transcriptionally by TSH in thyrocytes.

Regulation of phosphatidylinositol-phosphate kinase IIgamma gene transcription by thyroid-stimulating hormone in thyroid cells.

This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.

Involvement of the protein kinase C pathway in thyrotropin-induced STAT3 activation in FRTL-5 thyroid cells.

The binding of thyrotropin (TSH) to the TSH receptor (TSHR) activates two signaling pathways: the cAMP-protein kinase A (PKA) and the protein kinase C (PKC) systems. We have recently demonstrated that TSH activates the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway via TSHR. This study aimed to investigate whether the cAMP/PKA or the PKC system is involved in STAT3 activation in response to TSH. Treatment with TSH activated STAT3 phosphorylation in FRTL-5 thyrocytes and human TSHR-expressing Chinese hamster ovary cells. TSH-induced STAT3 activation was inhibited by a blocking antibody directed against TSHR that was isolated from patients with primary myxoedema. Increased intracellular cAMP activated STAT3 but inhibition of PKA did not affect STAT3 activation. On the other hand, the PKC stimulant PMA induced STAT3 phosphorylation and the PKC inhibitors inhibited it. Moreover, inhibition of PKC blocked STAT3 activation induced by a stimulator of cAMP. Our data suggest that TSH activates STAT3 via TSHR and cAMP- and PKC-dependent pathways, and provide evidence that PKC may be involved in the pathway downstream from cAMP.

Hormone-dependent regulation of intercellular adhesion molecule-1 gene expression: cloning and analysis of 5'-regulatory region of rat intercellular adhesion molecule-1 gene in FRTL-5 rat thyroid cells.

Intercellular adhesion molecule-1 (ICAM-1) has been suggested to play an important role in the perpetuation of autoimmune thyroid disease. To clarify the regulation of ICAM-1 gene in thyroid cells, we investigated ICAM-1 expression in the FRTL-5 thyroid cell model and defined several elements in the 5'-regulatory region that are important for transcriptional regulation of the rat ICAM-1 gene. Cells maintained in medium with 5% serum but without hydrocortisone, insulin, and thyrotropin (TSH) express the highest levels of ICAM-1 RNA. TSH/forskolin downregulate ICAM-1 RNA levels independent of the presence or absence of hydrocortisone or insulin. Moreover, TSH/forskolin decrease ICAM-1 RNA levels that are maximally induced by two cytokines: 100 ng/mL tumor necrosis factor-alpha (TNF-alpha) or 100 U/ml interferon-gamma (IFN-gamma). The effect of TSH/forskolin, as well as TNF-alpha and IFN-gamma, on ICAM-1 RNA levels is transcriptional. Thus, we cloned a 1.8-kb fragment of the 5'-flanking region of the rat ICAM-1 gene, upstream of the translational start site, and showed that TNF-alpha or IFN-gamma caused a 3.5- and greater than 12-fold increase respectively, in its promoter activity, when linked to a luciferase reporter gene and stably transfected into FRTL-5 cells. TSH or forskolin, in contrast, halved the activity of the full length chimera within 24 hours and significantly suppressed the TNF-alpha and IFN-gamma-induced increase (>50%; p < 0.02). Using 5'-deletion mutants, we located the element important for the TNF-alpha effect between -431 and -175 bp; we additionally show that deletion of a NF-kappaB core element within this region, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-alpha inducibility. The effect of IFN-gamma could be localized between -175 bp and -97 bp from the start of translation. This region contains 2 regulatory elements known to be involved in IFN-gamma action in other eukaryotic cells, an IFN-gamma activated site (GAS), -138 to -128 bp, and Spl site, -112 to -108 bp. Deletion of the 10 bp GAS sequence resulted in the complete loss of IFN-gamma induction of pCAM-175 promoter activity. TSH and forskolin action was also mapped between -175 bp and -97 bp from the start of translation. The mutant construct, pCAM-175delGAS mutl, which has no GAS sequence, exhibited no TSH-mediated suppression of promoter activity. We thus show that TSH/cAMP can downregulate ICAM-1 gene expression and inhibit the activity of cytokines (TNF-alpha and IFN-gamma) to increase ICAM-1 gene expression in FRTL-5 thyroid cells. We also localized elements on the 5'-flanking region of ICAM-1 important for these actions. We propose that this TSH/cyclic adenosine monophosphate (cAMP) action is a component of the mechanism to preserve self-tolerance of the thyroid during hormone-induced growth and function of the gland, and it may attenuate cytokine action during inflammatory reactions.

Identification of genes in a thyroid cell line regulated by thyroid-stimulating hormone (TSH).

Differential display (DD) PCR (Liang and Pardee, 1992) is a recently described technique to identify genes whose expression has changed during a biological process. We used this method to detect genes thyroid stimulating hormone-dependently regulated in a rat thyroid cell line, because thyroid stimulating hormone (TSH) is the most important hormone for cell proliferation and differentiation including prehormonal proteins secretion in thyrocytes (Kim and Arvan, 1991; Kim and Arvan, 1993). Following DD-PCR experimentation, thyroid stimulating hormone-dependently regulated gene fragments of 15 species were obtained. The genes were used as molecular probes in Northern blot analysis and then sequenced. Two of the clones (#123 and #205) were up-regulated and two more (#107 and #111) were down-regulated thyroid stimulating hormone-dependently in the thyroid cells, as demonstrated by Northern blot analysis. Following partial sequencing, each of the clones #107, #111 and #205 were shown to be homologues of the apoptosis-related gene, aldolase A, and a-2 collagen (IV), respectively, while clone #123 showed no homology with known genes. These findings suggest that the four genes mentioned above may have an a important physiological function in the thyrocytes, which is thyroid stimulating hormone-dependently up-/down-regulated.

Expression of an HSP110 family, ischemia-responsive protein (irp94), in the rat brain after transient forebrain ischemia.

The transcriptional expression of an ischemia responsive protein (irp94) in the hippocampus of rats was analyzed by Northern blotting. A transient forebrain ischemia was induced in the rats by temporary occluding of the bilateral common carotid arteries (CCAs) for various periods, and then reperfusion. Among the frontal, parietal, temporal and occipital lobes, and the cerebellum and hippocampus, the maximum mRNA expression of irp94 was at the occipital lobe, and the minimum was at the parietal lobe following ten min of forebrain ischemia. The irp94 mRNA expression reached a maximum fifteen min after the transient ischemia. From twenty min on after the ischemia its expression decreased. After a ten-min ischemia and the following reperfusion, irp94 mRNA expression gradually increased in the first twelve h, and then decreased. The expression pattern was like that of the endoplasmic reticulum chaperone, Erp72, but not that of the cytosol chaperone, hsp72. In addition, when intracellular ATP was depleted with antimycin A the mRNA level of irp94 increased in a thyrocyte cell culture model. The results suggest that irp94, like a molecular chaperone, may play a role in protecting the cell against external stimulation, especially after a transient forebrain ischemia. Although future studies of irp94 will be required to clarify the interactions with other intracellular factors inducing ischemia or showing molecular chaperone activity, what is offered here is an insight into its functional role as a component of stress response in neurons that should be considered as a new therapeutic approach for the treatment of ischemia.

The endoplasmic reticulum chaperone GRP94 is induced in the thyrocytes by cadmium.

We established a relationship between the toxic effects of cadmium on the expression of the endoplasmic reticulum (ER) chaperone GRP94 (glucose regulated protein 94) and cell survival in cultured rat-thyrocytes of FRTL5 cells. There are no data reporting that the enhanced expression of GRP94 by Cd stimulation is detectable in thyrocytes. Western blot analysis revealed higher levels of GRP94 expression in those cells post-treated with low concentrations of Cd, following a step-down treatment method, than in Cd pre-treated cells or cells not treated with any Cd, due to changes in cellular sensitivity after pre-treatment with Cd and the possible induction of GRP94 expression after removal of a low concentration of Cd. Elevated GRP94 expression in thyrocytes post-treated with Cd confers a survival advantage by rendering them resistant to cytotoxic stress, and the existence in the thyrocytes of a Cd-specific pathway regulates the expression of stress proteins by Cd.

RAF kinase inhibitor-independent constitutive activation of Yes-associated protein 1 promotes tumor progression in thyroid cancer.

The transcription coactivator Yes-associated protein 1 (YAP1) is regulated by the Hippo tumor suppressor pathway. However, the role of YAP1 in thyroid cancer, which is frequently associated with the BRAF(V600E) mutation, remains unknown. This study aimed to investigate the role of YAP1 in thyroid cancer. YAP1 was overexpressed in papillary (PTC) and anaplastic thyroid cancer, and nuclear YAP1 was more frequently detected in BRAF(V600E) (+) PTC. In the thyroid cancer cell lines TPC-1 and HTH7, which do not have the BRAF(V600E) mutation, YAP1 was cytosolic and inactive at high cell densities. In contrast, YAP1 was retained in the nucleus and its target genes were expressed in the thyroid cancer cells 8505C and K1, which harbor the BRAF(V600E) mutation, regardless of cell density. Furthermore, the nuclear activation of YAP1 in 8505C was not inhibited by RAF or MEK inhibitor. In vitro experiments, YAP1 silencing or overexpression affected migratory capacities of 8505C and TPC-1 cells. YAP1 knockdown resulted in marked decrease of tumor volume, invasion and distant metastasis in orthotopic tumor xenograft mouse models using the 8505C thyroid cancer cell line. Taken together, YAP1 is involved in the tumor progression of thyroid cancer and YAP1-mediated effects might not be affected by the currently used RAF kinase inhibitors.

Identification of genes in thyrocytes regulated by unfolded protein response by using disulfide bond reducing agent of dithiothreitol.

Disulfide bonds are formed between the sulfhydryl groups in two cysteine residues of a protein. The formation of these bonds is necessary for the proper folding of a protein into its active three-dimensional form. In this study, the genes associated with disulfide bond formation of proteins from the rat thyroid cell line, FRTL-5 cell, were investigated using disulfide bond reducing agent of dithiothreitol (DTT), which prevented disulfide formation of newly synthesized proteins. The expression of six genes, they being the cAMP phosphodiesterase 7A1, neuronal cell death inducible putative kinase (NIPK), cytosolic LIM protein (Ajuba), Eker, early growth response 1 and the ferritin heavy chain, was specifically enhanced under both reductive conditions and various endoplasmic reticulum (ER) stresses inducing drugs such as Brefeldin A (BFA), calcium ionophore A23187 (A23187) and tunicamycin. These results suggest that a suitable redox environment is necessary for the correct disulfide bond conformation in thyrocytes in a complex system.