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.

Acidosis inhibits 1,25-(OH)2D3 but not cAMP production in response to parathyroid hormone in the rat.

Parathyroid hormone (PTH) is a major activator of renal proximal tubule 25-hydroxyvitamin D3-1-hydroxylase (1-OHase). Chronic metabolic acidosis (CMA) inhibits 1-OHase and reduces circulating 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels in rats fed a low-Ca diet (LCD, 0.002% Ca). To examine the cellular mechanism whereby CMA inhibits 1-OHase, PTH-dependent renal 1-OHase activity and cAMP were measured in proximal tubules isolated from rats fed LCD for 14 days and made acidotic by the addition of 1.5% ammonium chloride to the drinking water. Serum 1,25-(OH)2D3 and proximal tubule 1-OHase activity and cAMP content were lower in acidotic rats. hPTH-(1-34) (10(-7) M) in vitro increased cAMP content to equivalent concentrations in tubules from rats with CMA and from nonacidotic controls; however, PTH increased 1-OHase activity only in tubules from nonacidotic animals. Although forskolin increased tubule cAMP content to equivalent levels in tubules from acidotic and nonacidotic rats, 1-OHase activity declined in tubules from nonacidotic rats and remained suppressed in acidotic tubules. The results suggest that chronic metabolic acidosis inhibits the PTH activation of 1-OHase through alteration of one or more steps in a cAMP-independent messenger system. PTH and forskolin can increase cAMP production by acidotic and nonacidotic proximal tubules; however, 1-OHase activity is not restored to normal in acidotic tubules and nonacidotic tubule 1-OHase may be inhibited.

Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D3 production in aging does not involve protein kinase A or C pathways.

Intestinal calcium absorption declines with aging as a result of decreased renal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] biosynthesis. At least part of the decline in 1,25-(OH)2D3 may be due to acquired resistance to parathyroid hormone (PTH) stimulation of renal 25-hydroxyvitamin D1-hydroxylase (1-OHase) activity. To test whether aging rats can increase 1,25-(OH)2D3 production in response to PTH, male rats of the same litter were fed a normal Ca diet and were sacrificed at 175-225 g (young rats) or 3 months later at 350-425 g (aging rats). At sacrifice, basal serum 1,25-(OH)2D3 levels (88 +/- 16 versus 49 +/- 8 pg/ml, P < 0.05) and in vitro renal proximal tubule 1-OHase activity (178 +/- 15 versus 77 +/- 5 pmol/mg protein/5 minutes, n = 6, P < 0.001) were lower in aging animals. rPTH-(1-34) (10(-11) or 10(-7) M) increased in vitro 1,25-(OH)2D3 secretion by perifused renal proximal tubules from young but not aging rats. For young and aging rats, rPTH-(1-34) (10(-7) M) increased proximal tubule cAMP-dependent protein kinase (PKA) activity, and lower concentrations (10(-11) M) stimulated translocation of protein kinase C (PKC) activity from cytosolic to soluble membrane proximal tubule cell fractions. The results of this study show that PTH activation of 1,25-(OH)2D3 production may involve both signaling pathways, with the PKC pathway responsive to lower concentrations of the hormone. The acquired resistance to PTH stimulation of 1,25-(OH)2D3 production in aging appears not to involve the hormonal activation of PKA or PKC.

Evidence that activation of protein kinase-C can stimulate 1,25-dihydroxyvitamin D3 secretion by rat proximal tubules.

PTH stimulates mammalian renal proximal tubule cell synthesis and secretion of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a Ca-dependent process. In the present study regulation of 1,25-(OH)2D3 secretion by PTH, phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the Ca ionophore A23187, and calcitonin was evaluated in perifused rat proximal tubule cells isolated by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low Ca diet secreted 1,25-(OH)2D3 at a rate 2.5 times that of tubule cells from rats fed a normal Ca diet. Perifusion of tubules with human PTH-(1-34) (10(-7) M) induced an immediate and sustained increase in 1,25-(OH)2D3 secretion. Perifusion with either A23187 or 12-O-tetradecanoylphorbol 13-acetate caused transient increases in hormone secretion, while both agents perifused simultaneously resulted in a sustained increase in 1,25-(OH)2D3 secretion. Perifusion of tubule cells with the protein kinase-C (PKC) inhibitor staurosporine blocked the PTH-induced increase in 1,25-(OH)2D3 secretion. Calcitonin had no effect on 1,25-(OH)2D3 secretion rates. The results of the present studies show that an activator of PKC increases 1,25-(OH)2D3 secretion by mammalian proximal tubule cells and suggest that the phospholipase-C/PKC signalling system may mediate PTH stimulation of 1,25-(OH)2D3 secretion.

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.

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.

Taurine intake of Korean breast-fed infants during lactation.

i. Taurine concentrations of human milk of nonvegetarians and lacto-ovovegetarians decreased significantly during the course of lactation. Taurine concentrations in lacto-ovovegetarians after 90 days postpartum were lower than those of nonvegetarians. Taurine concentrations of human milk by 150 days postpartum were: nonvegetarian 248-434 nmol/ml (31.0-54.4 mg/L); lacto-ovovegetarian 153-418 nmol/ml (19.1-52.3 mg/L). ii. Taurine intakes in infants of lacto-ovovegetarians decreased significantly during lactation, unlike from the pattern in infants of nonvegetarians. Taurine intakes in infants of lacto-ovovegetarians at 90, 120 and 150 days postpartum were lower, compared with those in nonvegetarians. Taurine intakes in infants by 150 days postpartum were: Infants of nonvegetarians 169-229 mumol/day (21.1-28.6 mg/day); Infants of lacto-ovovegetarians 106-210 mumol/day (21.1-28.6 mg/day). iii. This study showed that the taurine concentration in human milk and the taurine intake of infants were different during lactation between nonvegetarians and lacto-ovovegetarians, and that the intake per kg body weight decreased during lactation. Results suggest that the differences observed might be due to the effect of consumption of different types of food.

Tc-99m dimercaptosuccinic acid(DMSA) renal scintigraphy in patients with acute pyelonephritis.

OBJECTIVES: Recently, several authors reported that Tc-99m DMSA renal scan frequently showed cortical defects of the involved kidneys even in the patients with acute pyelonephritis who did not show abnormal findings in the ultrasonography and intravenous pyelography (IVP). METHODS: In order to evaluate the utilities of Tc-99m DMSA renal scan and the clinical meaning of cortical defects in the Tc-99m DMSA renal scan of the patients with acute pyelonephritis, ninety two patients with acute pyelonephritis, from March 1991 to February 1994 in Chungnam National University Hospital(CNUH), were included in this study. Patients were subdivided as Group A:Patients showing normal Tc-99m DMSA renal scan (n = 42) and Group B:Patients with definite cortical defects on the Tc-99m DMSA renal scan (n = 50). We compared clinical characteristics such as age and sex, recurrency, duration of fever, bacterial culture study, incidence of renal insufficiency and the results of renal ultrasonography and intravenous pyelography between the two groups. RESULTS: Fifty four percents of 92 patients with acute pyelonephritis showed a significantly longer febrile period after admission, higher positive rates on the urine and blood culture studies and higher incidence of renal insufficiency than those of the Group A patients. Sixty nine percents of Group B patients showed normal results in ultrasonography or IVP study. CONCLUSIONS: Tc-99m DMSA renal scan was a more sensitive imaging test than ultrasonography in kidneys and IVP to detect pyelonephritis lesions and may be useful to predict the patient group with a severe disease course. These patients may need more careful management and further studies to evaluate the possibility of complications.

Etiologic considerations of nonspecific pleuritis.

Twenty-three patients with nonspecific pleuritis were studied to determine clinical outcome. After a mean follow-up period of 6 months (1 to 36 months), a diagnosis was reached in 17 patients, while 6 patients remained unknown. The causes of the nonspecific pleuritis diagnosed on initial pleural biopsy were tuberculosis (11 patients, 48%), neoplasm (2 patients, 8.7%), parapneumonic effusion (1 patient), subphrenic abscess (1 patient), congestive heart failure (1 patients), and nephrotic syndrome (1 patient). The diagnosis was made by therapeutic trials (tuberculosis: 11 patients, parapneumonic effusion: 1 patient, congestive heart failure: 1 patient), by repeat pleural biopsy in 1 hepatoma, by open thoractomy in 1 lung cancer, by exploratory laparotomy in 1 subphrenic abscess, and by kidney biopsy in 1 nephrotic syndrome. The WBC counts (more than 2,000/mm3) and lymphocyte percentage (more than 60%) in the pleural fluid were significantly elevated in the patients with tuberculosis compared to those with malignant pleurisy, and other laboratory data were meaningless. As a result of this investigation, we suggest that tuberculous pleurisy is the most common cause of nonspecific pleuritis in Korea and that therapeutic trial with antituberculous medication for patients with high WBC count and lymphocyte percent in pleural fluid can help to locate the nonspecific pleuritis.

Circulating intercellular adhesion molecule-1(ICAM-1) in sera of patients with Graves' disease and Hashimoto disease.

OBJECTIVES: Intercellular adhesion molecule-1 (ICAM-1), a 80-110 kD glycoprotein, has been found to be a ligand for the lymphocyte function associated antigen-1 (LFA-1) molecule and has important roles in inflammatory and immune mediated mechanisms. ICAM-1 is expressed on thyroid follicular cells of patients with Hashimoto disease and cultured thyroid monolayer cells derived from the thyroid surgical specimen. In addition to the expression of ICAM-1 on the surface of cells, soluble variants of several adhesion molecules have been reported. METHODS: We evaluated the circulating ICAM-1 in sera of representative autoimmune thyroid disease, Hashimoto and Graves' disease, and analyzed correlations between circulating ICAM-1 and thyroid-directed autoantibodies. Sera were collected from 58 patients with autoimmune thyroid disease, 28 patients with Graves' disease and 30 patients with Hashimoto disease. Serum concentrations for circulating ICAM-1 were determined with sandwitch enzyme immunoassay. RESULTS: Compared with normal individuals, mean serum concentrations for circulating ICAM-1 were significantly elevated in patients with Hashimoto disease and antithyroperoxidase-positive Graves' disease. Patients with antithyroperoxidase-positive Graves' disease revealed significantly higher serum circulating ICAM-1 concentrations than antithyroperoxidase-negative Graves' disease. Circulating ICAM-1 showed significant positive correlation with serum titers of antithyroglobulin and antithyroperoxidase antibody (r = 0.44, n = 28, p = 0.009, and r = 0.55, n = 28, p = 0.001 respectively). There was a significant positive correlation between circulating ICAM-1 levels and serum antithyroperoxidase level in the group of autoimmune thyroid disease and also circulating ICAM-1 levels were significantly correlated with serum antithyroperoxidase antibody levels in antithyroperoxidase antibody-positive Graves' disease(r = 0.55, n = 28, p = 0.001) and in Hashimoto disease (r = 0.5, n = 30, p = 0.002). The thyrotropin binding inhibiting immunoglobulins(TBII) showed no significant correlation with circulating ICAM-1 levels. CONCLUSIONS: In the present study, high serum levels of ICAM-1 were associated with autoimmune thyroid disease. Graves' disease and Hashimoto disease and positively correlates with levels of antithyroperoxidase antibody.

The cytokines, interleukin-1 beta, interleukin-6 and interferon-gamma upregulate the expression of intercellular adhesion molecule-1(ICAM-1) in rat thyroid cell line, FRTL-5.

OBJECTIVES: Recently, the role of adhesion molecules in the immune system has been recognized. ICAM-1 plays an important role in a variety of inflammatory and immune mediated mechanisms, including recruitment and targeting of lymphocytes. We observed the effects of cytokines on expression of rat homologue of human intercellular adhesion molecule-1 in rat thyroid cell line, FRTL-5. METHODS: We have examined expression of rat intercellular adhesion molecule-1 (ICAM-1, CD54), a homologue of human intercellular adhesion molecule-1, by immunocytochemistry (immunoperoxidase staining) in the continuously growing rat thyroid cell line, FRTL-5. RESULTS: Low level of ICAM-1 expression was noted at basal condition and this basal expression was not influenced by thyrotropin. Expression in rat homologue of ICAM-1 is increased by interferon-gamma, interleukin-1 beta and interleukin-6 with a dose dependent manner. CONCLUSION: These results show that a pure line of rat thyroid cells can express an ICAM-1 homologue and this is directly enhanced by cytokines such as rat interferon-gamma, human interleukin-1 beta and interleukin-6. Expression of this homologue is partially responsible for lymphocyte adhesion to thyroid cells, which is likely to be a major event in T cell recognition of thyroid antigens in autoimmune thyroiditis.

Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells.

Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H(2)O(2))-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H(2)O(2) produced in response to thyrotropin (TSH). Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H(2)O(2). In addition, methimazole induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhances the elimination of H(2)O(2) produced by TSH in FRTL-5 cells. Treatment with 500 micrometer H(2)O(2) causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e. terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling, BAX expression, and poly(ADP-ribose) polymerase cleavage. Overexpression of Prx I and Prx II reduces the amount of H(2)O(2)-induced apoptosis measured by these assays. These results suggest that Prx I and Prx II are involved in the removal of H(2)O(2) in thyroid cells and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H(2)O(2) in thyroid cells.

Methimazole as an antioxidant and immunomodulator in thyroid cells: mechanisms involving interferon-gamma signaling and H(2)O(2) scavenging.

The antithyroid drug, methimazole (MMI) is used to treat patients with Graves' hyperthyroidism. The major action of MMI is to inhibit synthesis of thyroid hormone in the thyroid gland. However, MMI also has antioxidant and immunomodulatory effects on thyrocytes and/or immune cells. This study identifies novel antioxidant and immunomodulatory effects of MMI involving the interferon-gamma (IFN-gamma) signaling pathway in thyroid cells. MMI inhibits transcription of the intercellular adhesion molecule-1 (ICAM-1) gene by modulating the function of transcription factor STAT1 (signal transducer and activator of transcription 1), which binds to the IFN-gamma activated site of the ICAM-1 promoter. Furthermore, MMI rapidly eliminates H(2)O(2) produced by IFN-gamma treatment in thyroid cells and thus inhibits the H(2)O(2)-mediated phosphorylation of tyrosine 701 in STAT1. MMI also eliminates H(2)O(2) in vitro. MMI facilitates electron transfer from NADPH to H(2)O(2) using thioredoxin or glutathione, fulfilling a role similar to peroxiredoxin or glutathione peroxidase, respectively. MMI prevents the IFN-gamma and H(2)O(2)-mediated reversible inactivation of phosphatases. These effects inhibit full activation of the IFN-gamma-induced Janus kinase(JAK)/STAT signaling pathway in FRTL-5 thyroid cells. These results may in part explain the antioxidant and immunomodulatory effects of MMI in thyroid cells of Graves' disease patients.