15-Deoxy-Delta 12,14-prostaglandin J2 facilitates thyroglobulin production by cultured human thyrocytes

Kikuo Kasai1, Nobuyuki Banba1, Akira Hishinuma2, Michiko Matsumura1, Hirofumi Kakishita, Mihoko Matsumura1, Satoshi Motohashi1, Noriyuki Sato1, and Yoshiyuki Hattori1

1 Department of Endocrinology and Metabolism and 2 Department of Clinical Pathology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A cyclopentenone-type prostaglandin, 15-deoxy-Delta 12,14-prostaglandin J2 (15-d-PGJ2), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma . We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ2-treated cells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production was decreased in untreated cells according to duration of culture. PGD2 and PGJ2, which are precursors to 15-d-PGJ2, exhibited an effect similar to 15-d-PGJ2. However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-gamma , and WY-14643, a specific PPAR-alpha ligand, lacked this effect. 15-d-PGJ2 and its precursors, but not the thiazolidinediones, induced gene expression for heme oxygenase-1 (HO-1), a stress-related protein, and strongly inhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production. Cyclopentenone-type PGs have been recently shown to inhibit nuclear factor-kappa B (NF-kappa B) activation via a direct and PPAR-independent inhibition of inhibitor-kappa B kinase, suggesting that, in human thyrocytes, such PGs may inhibit IL-1-induced NO production, possibly via an inhibition of NF-kappa B activation. On the other hand, sodium arsenite, a known activator of the stress response pathway, induced HO-1 mRNA expression but lacked a promoting effect on TG production. Thus 15-d-PGJ2 and its precursors appear to facilitate TG production via a PPAR-independent mechanism and through a different pathway from the cellular stress response that is available to cyclopentenone-type PGs. Our findings reveal a novel role of these PGs associated with thyrocyte differentiation.

thyrotropin; adenosine 3',5'-cyclic monophosphate; heme oxygenase-1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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EICOSANOIDS, which are oxygenated metabolites of arachidonic acid, modulate cellular function during a variety of physiological and pathological processes (30). Eicosanoids are divided into two groups, according to their mechanism of action: the conventional eicosanoids such as PGE2 and PGI2 and the cyclopentenone-type prostaglandins (PGs). The conventional eicosanoids act on cell surface receptors to exert their actions; the molecular structure of their receptors has recently been revealed (32). Cyclopentenone PGs such as Delta 12-PGJ2, 15-deoxy-Delta 12,14-PGJ2 (15-d-PGJ2), and PGA2 lack cell surface receptors but are actively transported into cells where they exert a wide variety of biological actions, including the cessation of cell growth, antiviral activity, and osteogenesis (9, 31). The actions of the cyclopentenone PGs are attributed to the synthesis of the various proteins. They induce, like the family of cytosolic heat shock proteins (37, 44), gamma -glutamylcysteine synthetase (38), collagen (51), gadd 153 (4), heme oxygenase (HO; see Ref. 25), immunoglobulin-binding protein (Bip/GRP-78; see Ref. 36), p21WAP1/CiP (16), p53 (26), and c-fos (14). Although their intracellular receptor had not been described, 15-d-PGJ2 was recently shown to be a naturally occurring ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma (8, 23) and is a weak activator of PPAR-alpha (8, 23, 24). The PPARs belong to the nuclear receptor superfamily of ligand-dependent transcription factors that activate the transcription of several genes. The differential activation of the PPAR-alpha and -gamma subtypes by several naturally occurring compounds such as fatty acids and 15-d-PGJ2 and by such structurally distinct synthetic compounds such as fibrates and thiazolidinediones has been described and may explain their specific biological activities, including roles in lipid and glucose metabolism and adipocyte differentiation (7, 8, 23, 24, 27, 46-48). In addition, an inhibitory role of PPAR-gamma or PPAR-alpha activation has recently been reported in tissue or in a cell-specific fashion by such ligands on the immune system and/or inflammatory reaction (18, 40, 49). Despite the widespread distribution of the receptors in various tissues, the biological role of PPAR-beta /delta activation and that of its ligands remains obscure.

In a functional rat thyroid epithelial cell line, the FRTL-5 cells, either thyrotropin (TSH) or insulin stimulates the release of arachidonic acid, the expression of cyclooxygenase, and the production of PGD2 and PGE2 (50). In general, thyrocytes cultured in the presence of high concentrations of serum gradually lose their differentiated functions (2, 41). TSH stimulates the differentiation of human thyrocytes and of FRTL-5 cells, including the synthesis of thyroglobulin (TG), a precursor of thyroid hormone, via a cAMP-dependent mechanism (5, 43). In the present study, we observed the promoting effect of 15-d-PGJ2, a metabolite of PGD2, on the basal and the TSH-stimulated production of TG in primary culture of human thyrocytes in the presence of 10% fetal bovine serum (FBS). We then examined whether the effect of 15-d-PGJ2 might be attributed to the activation of PPARs or to some other pathway.


    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
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Materials. PGD2, PGJ2, 15-d-PGJ2, and WY-14643 were obtained from Cayman Chemical (Ann Arbor, MI). The antidiabetic thiazolidinediones, troglitazone and pioglitazone, were kindly supplied by Sankyo Pharmaceutical (Tokyo, Japan) and by Takeda Pharmaceutical (Osaka, Japan), respectively. These agents were dissolved in DMSO. The final concentration of DMSO was <= 0.1%. Other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Cell culture. We obtained specimens of thyroid tissues from two patients with Graves disease. Each specimen was digested with collagenase as described elsewhere (20). The cells (open thyroid follicles) were seeded on 48-well plates (0.2 ml/well) or 60-mm dishes (2 ml/dish) in Ham's F-12 medium supplemented with 10% FBS and then were cultured with 5% CO2 in a humidified atmosphere at 37°C. Cells that reached confluence were used in these experiments. Contamination by fibroblasts was found to be below 5%.

Assay of TG, cAMP, and nitrite in culture supernatant. Assays of cAMP and TG in the culture supernatant were performed using cAMP kits and TG kits, respectively, obtained from Yamasa (Chiba, Japan) and Daiichi Radioisotope (Tokyo, Japan). Nitrite accumulation in the culture medium was quantitated colorimetrically with Griess reagent after a 48-h incubation, as reported elsewhere (20).

Cell respiration. Cell respiration, an indicator of cell viability, was assessed by the mitochondrion-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan (29). To evaluate the cytotoxic effect of 15-d-PGJ2 and other agents, the cells were incubated at 37°C with MTT (0.4 mg/ml in fresh medium) for an additional 60 min after the final 48-h incubation. The culture medium was removed by aspiration, and the cells were solubilized in DMSO. The extent of reduction of MTT to formazan within the cells was quantified by the measurement of absorbance at 550 nm. Formazan production was expressed as a percentage of the value obtained from the control cells (basal condition).

Analysis of mRNA levels by RT-PCR. RNA was extracted from thyrocytes in a 60-mM dish using a modified guanidinium isothiocyanate method (Isogen, Takara, Japan). RT-PCR was performed using the standard method. Briefly, the first-strand cDNA was synthesized using random primers and Moloney murine leukemia virus RT (Promega, Madison, WI), followed by PCR amplification using synthetic gene primers specific for human TG (20), HO-1 (52) , inducible nitric oxide synthase (iNOS; see Ref. 20), PPAR-alpha (39), PPAR-beta /delta (39), PPAR-gamma (11), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as reported previously (20). The following primers used were: TG forward 21-mer, 5'-TGCCCTGGCAATGGAGACAAA-3'; TG reverse 21-mer, 5'-ACACGGGCTGACCTTTCTTAC-3'; HO-1 forward 21-mer, 5'-CAGGCAGAGAATGCTGAGTTC-3'; HO-1 reverse 18-mer, 5'-GCTTCACATAGCGCT GCA-3'; PPAR-alpha forward 21-mer, 5'-AGAACTTCAACATGAACAAGGTCA-3'; PPAR-alpha reverse 24-mer, 5'-GCCAGGACGATCTCCACAGCAAAT-3'; PPAR-beta /delta forward 21-mer, 5'-AGCAGCCTCTTCCTCAACGACCAG-3'; PPAR-beta /delta reverse 21-mer, 5'-GGTCTCGGTTTCGGTCTTCTTGAT-3'; PPAR-gamma forward 21-mer, 5'-GTTCATGCTTGTGAAGGATGC-3'; PPAR-gamma reverse 20-mer, 5'-ACTCTGGATTCAGCTGGTCG-3'; iNOS forward 21-mer, 5'-TGCCCTGGC AATGGAGAGAAA-3'; iNOS reverse 21-mer, 5'-GAGCTGATGGAGTAGAAC CTG-3'; GAPDH forward 26-mer, 5'-TGAAGGTCGGAGTCAACGGATTTG GT-3'; GAPDH reverse 24-mer, 5'-CATGTGGGCCATGAGGTCCACCAC-3'. PCR amplification was performed for 30 cycles using a DNA PCR kit (Perkin-Elmer, Norwalk, CT) as reported previously (20). PCR products were electrophoresed on a 1.5% agarose gel that contained ethidium bromide and were visualized by ultraviolet-induced fluorescence. All three subtypes of PPAR were constitutively expressed in the primary culture of human thyrocytes (Fig. 1). The size of the amplified fragments was consistent with those predicted, being 524 bp for PPAR-alpha , 471 bp for PPAR-beta /delta , 250 bp for PPAR-gamma (Fig. 1), 613 bp for TG, 350 bp for HO-1, and 944 bp for iNOS (data not shown).


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Fig. 1.   Expression of peroxisome proliferator-activated receptor (PPAR) mRNAs in primary culture of human thyrocytes. Expression of mRNAs for PPAR-alpha , PPAR-beta /delta , and PPAR-gamma was evaluated by RT-PCR in thyrocytes cultured under basal conditions [Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS)]. Size markers are on left. The sizes of the amplified fragments were consistent with those predicted (524 bp for PPAR-alpha , 471 bp for PPAR-beta /delta , 250 bp for PPAR-gamma ).

Statistical analysis. Values are expressed as means ± SD of three individual wells or a mean of two individual dishes.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Changes in secretion of TG and cAMP and the gene expression for TG and HO-1 in human thyrocytes over time. We first studied the effects of 15-d-PGJ2, TSH, and their combination on the changes in levels of TG and cAMP in the culture medium of human thyrocytes over time (Fig. 2, A and B). Compared with basal conditions, the accumulation of TG was only slightly higher at 24 h and was increased over twofold at 48 h in cells treated with 15-d-PGJ2 (10 µM) with no apparent increase in the cAMP level. TSH (20 mU/ml) clearly stimulated the accumulation of TG by more than twofold at 24 h and by more than fivefold at 48 h, associated with a rapid, progressive increment in the cAMP level. With their combined administration, the increase in TG level was more prominent and appeared to be additive, or even slightly greater, at 24 h and again at 48 h of incubation. The accumulation of cAMP in the medium was not increased in the early phase for up to 8 h but was increased in the late phase of incubation (24-48 h) compared with that in the case of TSH alone. As shown in Fig. 2C, either actinomycin D (Act D, 2.5 µg/ml) or cycloheximide (CHX, 10 µg/ml) completely inhibited 15-d-PGJ2-, TSH-, 15-d-PGJ2-, and TSH-stimulated production of TG by the cells. We investigated by RT-PCR the effects of 15-d-PGJ2, TSH, and their combination on the gene expression for TG and for HO-1, a stress-related protein (Fig. 3A). Under basal conditions, the level of TG mRNA declined according to the length of incubation from 4 to 48 h. In the presence of 15-d-PGJ2, however, the level of TG mRNA appeared to be slightly higher at 4 h than at baseline and essentially remained at these levels throughout the experiment. TSH markedly increased the level of TG mRNA. Their combined administration also markedly increased such levels. 15-d-PGJ2 clearly increased the level of HO-1 mRNA as early as 4 h; such levels were essentially maintained over 48 h. However, TSH clearly did not induce the gene expression but rather suppressed its basal expression. Even in the presence of TSH, 15-d-PGJ2 markedly increased the level of HO-1 mRNA at 4 h, which remained essentially unchanged at 48 h (Fig. 3A). The effects of Act D and CHX on the levels of mRNA for TG and HO-1 at 24 h of incubation were studied further (Fig. 3B). The level of TG mRNA was almost completely suppressed to the basal level by Act D and was also markedly suppressed by CHX added to cells incubated with 15-d-PGJ2, TSH, or TSH plus 15-d-PGJ2. Act D completely inhibited the 15-d-PGJ2-induced expression of HO-1 mRNA, whereas CHX greatly induced its expression.


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Fig. 2.   Changes in levels of thyroglobulin (TG; A) and cAMP (B) over time in the culture medium of thyrocytes and effects of actinomycin D (Act D) and cycloheximide (CHX) on TG levels (C). Human thyrocytes cultured in 60-mm dishes were incubated in a medium containing 10% FBS alone (basal) or in a medium supplemented with 15-deoxy-Delta 12,14-prostaglandin J2 (15-d-PGJ2, 10 µM), thyrotropin (TSH, 20 mU/ml), or 15-d-PGJ2 + TSH (10 µM + 20 mU/ml) in the presence or absence of Act D (2.5 µg/ml) or CHX (10 µg/ml). At the indicated times, the levels of TG and cAMP were measured in the supernatant. Data represented are means of 2 dishes.



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Fig. 3.   Changes in mRNA levels for TG and heme oxygenase-1 (HO-1) in thyrocytes. A: time course changes in the levels of TG mRNA and HO-1 mRNA in basal and 15-d-PGJ2 (10 µM)-, TSH (20 mU/ml)-, or 15-d-PGJ2 + TSH (10 µM + 20 mU/ml)-stimulated conditions. B: expression of TG and HO-1 mRNAs in thyrocytes 24 h after incubation with 15-d-PGJ2 (10 µM), TSH (20 mU/ml), or 15-d-PGJ2 + TSH (10 µM + 20 mU/ml) in the presence or absence of Act D (2.5 µg/ml) and CHX (10 µg/ml). Levels of mRNA for TG and HO-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference were evaluated by RT-PCR.

Effects of various doses of 15-d-PGJ2 on basal and TSH- or dibutyryl cAMP-stimulated levels of TG. The amount of TG secreted by the cells during an incubation for 48 h was slightly higher in the presence of 1 µM 15-d-PGJ2 and was over three times higher with 10 µM 15-d-PGJ2 than in its absence. However, the level of TG tendered to decrease in the presence of 30 µM 15-d-PGJ2 (Fig. 4A). This dose was cytotoxic as assessed by the MTT assay (Fig. 4C). The amount of cAMP that accumulated under these conditions tended to decrease with the addition of 10 µM or more 15-d-PGJ2 (Fig. 4B). TSH (20 mU/ml) used alone strongly stimulated TG accumulation by about fivefold and was associated with a profound increase in the level of cAMP. This TSH-stimulated accumulation of TG was further promoted and was associated with an increase in cAMP production by 15-d-PGJ2 administered at doses ranging from 1 to 10 µM. Dibutyryl cAMP (0.5 mM) also stimulated TG accumulation. Such stimulation appeared to be slightly increased by the coadministration of 1-10 µM 15-d-PGJ2.


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Fig. 4.   Effects of various doses of 15-d-PGJ2 on basal, dibutyryl cAMP (DBC), and TSH-induced levels of TG (A) and cAMP (B) in the culture medium of thyrocytes. Human thyrocytes cultured in 48-well plates were incubated for 48 h with graded doses of 15-d-PGJ2 in the presence or absence of DBC (0.5 mM) or TSH (20 mU/ml). C: after 48 h of incubation, cell respiration was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Data represented are means ± SD of 3 wells.

Comparable levels of TG and cAMP under basal and TSH-stimulated conditions in untreated thyrocytes vs. those treated with 15-d-PGJ2. We next compared the levels of TG and cAMP under basal and TSH-stimulated conditions during the final 48 h of incubation in thyrocytes treated with or without 5 µM 15-d-PGJ2 for 48-144 h. As shown in Fig. 5, A and B, both the basal and TSH (20 mU/ml)-stimulated levels of TG gradually decreased according to duration of culture in the untreated (control) thyrocytes. The TSH-stimulated accumulation of cAMP decreased in a similar manner in such cells. Both the basal TG and TSH-stimulated TG and cAMP levels were clearly higher in the cells treated with 15-d-PGJ2 compared with untreated cells. However, the basal accumulation of cAMP showed no significant change in the cells untreated or treated with 15-d-PGJ2 throughout the experiment; all of the values were below 1 pmol/ml.


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Fig. 5.   Comparable levels of TG (A) and cAMP (B) and the levels of mRNA for TG and HO-1 (C) under basal and TSH-stimulated conditions in untreated (control) vs. treated thyrocytes with 15-d-PGJ2 for various durations. Human thyrocytes cultured in 60-mm dishes were cultured in the absence and presence of 5 µM 15-d-PGJ2 for various periods of time (48, 96, or 144 h), including a final 48 h of incubation. The basal and TSH (20 mU/ml)-stimulated levels of TG and cAMP during the final 48 h of incubation were measured. Data represent means of 2 dishes. The levels of mRNAs for TG and HO-1 and GAPDH as reference were evaluated by RT-PCR at the indicated times.

The basal level of TG mRNA showed a gradual decrease in untreated cells in accordance with the duration of culture, whereas the response to TSH regarding the expression of TG mRNA remained relatively unchanged throughout the experiment (Fig. 5C). In the 15-d-PGJ2-treated cells, both the expression of TG mRNA in response to TSH and the basal level of TG mRNA were maintained.

Effects of pretreatment, treatment, and continuous treatment with 15-d-PGJ2 on the accumulation of TG and cAMP under basal and TSH-stimulated conditions. We evaluated the effects of the pretreatment, treatment, and continuous treatment (pretreatment and treatment) of the cells with 5 µM 15-d-PGJ2 on the basal and the TSH-stimulated TG and cAMP accumulation during the final 48 h of incubation (Fig. 6, A and B). The administration of 5 µM 15-d-PGJ2 during the final 48 h of incubation promoted both the basal and TSH (0.02-20 mU/ml)-induced production of TG compared with the control condition. Cells pretreated for 48 h with 5 µM 15-d-PGJ2 produced greater amounts of TG in the basal and in the TSH-stimulated conditions during the final 48 h of incubation compared with the untreated cells or cells treated only during the final incubation. Continuous treatment with 5 µM 15-d-PGJ2 showed the highest level of both the basal and TSH-induced production of TG (Fig. 6A). The basal accumulation of cAMP was zero and was not influenced by pretreatment, treatment, or continuous treatment with 5 µM 15-d-PGJ2. However, the TSH (0.02-20 mU/ml)-stimulated accumulation of cAMP was clearly enhanced by treatment, pretreatment, and continuous treatment, in that order (Fig. 6B).


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Fig. 6.   Effects of pretreatment, treatment, and continuous (pretreatment and treatment) treatment with 15-d-PGJ2 on basal and TSH-induced levels of TG (A) and cAMP (B) in the final incubation medium of the thyrocytes. Human thyrocytes in 48-well plates were first preincubated in the presence or absence of 5 µM 15-d-PGJ2 for 48 h. The medium was then aspirated and washed one time with the fresh medium and was finally incubated with various doses of TSH (0-20 mU/ml) in the presence or absence of 5 µM 15-d-PGJ2 for 48 h. The amounts of TG and cAMP in the final incubation medium were measured. Data represented are means ± SD of 3 wells.

Effects of treatment or pretreatment of thyrocytes with PGs and synthetic ligands of PPARs on TG or cAMP accumulation and expression of HO-1 mRNA. The effects of administering various doses of such agents as 15-d-PGJ2, its precursor PGs (PGD2 or PGJ2), the synthetic PPAR-gamma ligands troglitazone and pioglitazone, and PPAR-alpha ligand (WY-14643) on the basal and TSH-stimulated accumulation of TG were examined during 48 h of incubation. Basal TG production was increased by 10 µM 15-d-PGJ2, PGD2, or PGJ2 compared with that by untreated cells. The TSH (20 mU/ml)-induced accumulation of TG was also clearly increased by the cells treated with such PGs dose dependently, from 1 to 10 µM (Fig. 7A). However, neither one of the antidiabetic thiazolidinediones (0.1-10 µM) nor WY-14643 (0.1-50 µM) showed such an effect on the basal or TSH-induced production of TG (Fig. 7B).


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Fig. 7.   Effects of various PGs and ligands for PPAR-gamma /alpha on basal and TSH-induced TG levels during incubation of thyrocytes for 48 h. Human thyrocytes in 48-well plates were incubated for 48 h with various doses of PGD2, PGJ2, or 15-d-PGJ2 (0.1-10 µM; A) and with specific ligands for PPAR-gamma (troglitazone or pioglitazone: 0.1-10 µM) or for PPAR-alpha (WY-14643: 0.1-50 µM; B) in the presence or absence of TSH (20 mU/ml). The amounts of TG accumulated in the medium were measured. Data represented are a means ± SD of 3 wells.

We next examined the effects of 48 h of pretreatment with such agents on the basal or TSH-stimulated accumulation of TG and cAMP during the next 48 h of incubation (Fig. 8, A and B). During preincubation, the accumulation of TG by the cells was higher in the presence of PGD2, PGJ2, or 15-d-PGJ2 (each 5 µM) than in the untreated cells. The accumulation of basal TG clearly declined in the untreated cells during the final incubation period compared with preincubation. In contrast, the basal level of TG in the final incubation by cells pretreated with each PG was similar to that observed during preincubation with no apparent change in cAMP accumulation. The TSH-stimulated accumulation of TG and cAMP during the final 48 h of incubation was clearly higher in the cells pretreated with PGD2, PGJ2, or 15-d-PGJ2 than in the untreated cells (Fig. 8, A and B). Neither troglitazone nor pioglitazone (each 5 µM) or WY-14643 (50 µM) exhibited any effect on either parameter (data not shown).


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Fig. 8.   Effects of pretreatment of thyrocytes with PGs on basal and TSH-induced TG (A) and cAMP (B) levels during final incubation of thyrocytes. Human thyrocytes in 48-well plates were preincubated for 48 h with PGD2, PGJ2, or 15-d-PGJ2 (each 5 µM). The medium was then aspirated and replaced with fresh medium that contained various doses of TSH (0-20 mU/ml) and was further incubated for 48 h. The respective amounts of TG and cAMP in the preincubation or final incubation medium were measured. Data represent means ± SD of 3 wells.

In the absence of TSH, the induction of gene expression of HO-1 in the cells treated with 10 µM 15-d-PGJ2, PGD2, or PGJ2 was observed at 4 and 24 h after their addition, whereas neither troglitazone nor pioglitazone (10 µM) or WY-I4632 (50 µM) showed such an effect (Fig. 9).


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Fig. 9.   Effects of various PGs and ligands for PPAR-gamma /alpha on the expression of HO-1. Human thyrocytes in 60-mm dishes were incubated for 4 or 24 h with 15-d-PGJ2, PGJ2, or PGD2 (each 10 µM) and with specific ligands for PPAR-gamma [troglitazone (Tro) or pioglitazone (Pio): each 10 µM] or for PPAR-alpha [WY-14643 (Wy): 50 µM] in the absence of TSH. Levels of mRNA for HO-1 and GAPDH as a reference were evaluated by RT-PCR.

Effects of 15-d-PGJ2 and thiazolidinediones on inducible nitric oxide production. The effects of administering 15-d-PGJ2, PGD2, and the synthetic PPAR-gamma ligands troglitazone and pioglitazone on IL-1-induced accumulation of nitric oxide (NO) measured as nitrite were examined during 48 h of incubation. IL-1-induced NO production was dose dependently inhibited by 15-d-PGJ2 and PGD2, with the doses ranging from 5 to 20 µM. Pioglitazone did not affect but troglitazone inversely stimulated NO production with the doses from 5 to 20 µM (Fig. 10, A and B). The IL-1-induced expression of iNOS mRNA was suppressed in 15-d-PGJ2 (10 µM)-treated cells but was inversely increased in troglitazone (10 µM)-treated cells at 24 h. In the pioglitazone-treated cells, its level seemed to be unaffected to any major extent (Fig. 10C).


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Fig. 10.   Changes in nitric oxide (NO) production (A and B) and in the levels of inducible nitric oxide synthase (iNOS) mRNA (C) in interleukin (IL)-1-activated thyrocytes treated with and without 15-d-PGJ2, PGD2, troglitazone, or pioglitazone. Human thyrocytes cultured in 48-well plate were incubated for 48 h with graded doses of IL-1 (0-10 ng/ml) in the presence or absence of 10 µM of each agent and with 10 ng/ml IL-1 in the presence or absence of graded doses of each agent (0-20 µM). Nitrite accumulation in the culture medium was measured. Data represent means ± SD of 3 wells. The cells cultured in 60-mm dishes were activated with IL-1 (10 ng/ml) in the presence or absence of 10 µM of each agent. The levels of iNOS mRNA and GAPDH as reference were evaluated by RT-PCR at the indicated times.

Effects of sodium arsenite on basal and TSH-stimulated TG production and HO-1 and TG mRNA expression. After pretreatment of the cells with or without 50 µM sodium arsenite for 1 h, basal and TSH-stimulated TG accumulation in the fresh culture medium was compared with that in control and 15-d-PGJ2 (10 µM)-treated cells during the next 48 h of incubation. Sodium arsenite pretreatment did not increase TG production, but 15-d-PGJ2 treatment facilitated its production (Fig. 11A). Pretreatment with sodium arsenite strongly induced HO-1 mRNA expression in the cells over 4-48 h, whereas TG mRNA expression in response to TSH was similar to that in control cells (Fig. 11B).


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Fig. 11.   Effect of pretreatment with sodium arsenite on basal and TSH-stimulated TG production (A) and the expression of mRNA for TG and HO-1 over time (B). Human thyrocytes were pretreated with or without 50 µM sodium arsenite for 1 h. Basal and TSH (0.1-10 mU/ml)-stimulated TG production for the next 48 h in the fresh medium by arsenite-pretreated thyrocytes was compared with that by the cells incubated in the absence (control) or presence of 10 µM 15-d-PGJ2. Data represent means ± SD of 3 wells. The cells cultured in 60-mm dishes were pretreated with 50 µM sodium arsenite for 1 h. The medium was replaced with the fresh medium, and the cells were cultured further for 48 h in the presence of TSH (10 mU/ml). The levels of TG and HO-1 mRNAs and GAPDH as reference were evaluated by RT-PCR at the indicated times.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

When human thyrocytes were cultured with 10% FBS, both the basal production of TG and the response to TSH, including the production of TG and cAMP production, showed a gradual decrease in accordance with duration of culture. These results are compatible with previous reports showing that thyrocytes cultured in the presence of high concentrations of serum gradually lose the differentiation function (2, 41). Although 15-d-PGJ2 alone failed to stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated in the thyrocytes treated with 15-d-PGJ2. PGD2 and PGJ2 (precursor PGs for 15-d-PGJ2) had an effect similar to 15-d-PGJ2. Because PGD2 rapidly undergoes dehydration in aqueous media to form PGJ2 and is converted in the presence of serum or albumin to Delta 12-PGJ2 and 15-d-PGJ2 (1, 15, 22, 28), the effects of PGD2 and PGJ2 appear to be expressed via this cascade of the PGJ2 series. Furthermore, the promoting effect of these PGs on both the basal production of TG and the response to TSH, including the production of TG and cAMP, was more clearly observed in the continuously treated (pretreated and treated) and pretreated cells than in those treated only in the final incubation (Figs. 6 and 8). Thus the promoting effect of 15-d-PGJ2 or its precursor PGs on the thyrocytes may be attributed to the suppression of dedifferentiation and/or to the facilitation of differentiation via cAMP-independent and TSH- and cAMP-dependent mechanisms.

Various chemicals and conditions can trigger a protective homeostatic mechanism (stress response) in eukaryotic cells. Because it has been reported that the cyclopentenone PGs also activate the stress response pathway, resulting in the induction of various stress-related proteins, including HO-1 and members of molecular chaperones in the cytosol and endoplasmic reticulum (4, 25, 36-38, 44, 51), we investigated whether 15-d-PGJ2, PGJ2, and PGD2 might induce the stress response in human thyrocytes by assessing the gene expression of HO-1 (25). Both 15-d-PGJ2 and PGJ2 markedly and PGD2 less potently induced gene expression for HO-1, indicating that these PGs may in fact activate the stress response pathway in human thyrocytes. 15-d-PGJ2 was recently shown to be a ligand for PPAR-gamma and was also shown to be a weak activator of PPAR-alpha (8, 23, 24). To clarify whether the activation of PPARs by 15-d-PGJ2 may contribute to the promotion of basal TG production and the response to TSH, we further investigated the effects on such parameters of activators of PPAR-gamma and -alpha . We first confirmed that the mRNAs for PPAR subtypes (alpha , beta /delta , and gamma ) were all expressed in human thyrocytes. In contrast to 15-d-PGJ2 and its precursor PGs, the synthetic activators of PPAR-gamma , troglitazone, and pioglitazone, and a synthetic activator of PPAR-alpha , WY-14643, failed to promote the production of TG. 15-d-PGJ2 and its precursors have initially been shown to inhibit the production of proinflammatory cytokines and inducible NO in activated monocytes by a mechanism suggested to PPAR-gamma activation that interferes with nuclear factor (NF)-kappa B transcriptional activity (18, 40). However, it has recently been shown that a cyclopentenone type of PGs inhibits NF-kappa B activation via direct and PPAR-gamma -independent inhibition of inhibitor-kappa B kinase (42). Thus we next examined and clarified that interleukin-1 (IL-1)-induced NO production was strongly inhibited by 15-d-PGJ2 and PGD2 but not by the thiazolidinediones. Troglitazone, but not pioglitazone, inversely stimulated IL-1-induced NO production; these results are compatible with our previous report in rat vascular smooth muscle cells (12). Although IL-1 was shown to inhibit TG mRNA and protein production in human thyrocytes (54), we have observed in a preliminary study that IL-1-induced inhibition of TG production can be restored partially by the coadministration of 15-d-PGJ2 but not by the thiazolidinediones in the cells. These results clearly indicated that 15-d-PGJ2 and its precursor PGs have PPAR-independent effects on thyrocyte functions. We thus conclude in human thyrocytes that the promoting effect of 15-d-PGJ2 and of its precursor PGs on basal TG production and the response to TSH is independent of PPAR activation. It is compatible with a recent report showing that 15-d-PGJ2 and PGJ2 promote the differentiation (i.e., neurite outgrowth) by nerve growth factor in PC-12 cells via a mechanism that is independent of PPAR-gamma activation (45). Accordingly, it is suggested that the promoting effect by such PGs on TG production in human thyrocytes might be associated with an inhibition of NF-kappa B activation, an activation of the stress response pathway, or an unknown mechanism. We finally studied the effect of sodium arsenite, a prototype activator of the stress response pathway (1, 3, 17), on HO-1 mRNA expression and TG production in human thyrocytes. Pretreatment of the cells with sodium arsenite strongly induced HO-1 mRNA expression but lacked a promoting effect on basal and TSH-stimulated TG production. Accordingly, it seems unlikely that an activation of the stress response pathway may be directly related with such promoting effect on TG production.

Arachidonic acid metabolism is highly increased in several pathological conditions such as infection and inflammation (13, 33), and local PG concentrations in the micromolar range have been detected at sites of acute inflammation (35). Elevated cyclopentenone-type PGs have been detected in the late phase of inflammation and are associated with resolution of inflammation (10, 18, 40). Subacute thyroiditis possibly caused by certain viral infections and silent thyroiditis caused by an acute exacerbation of the autoimmune process related with autoimmune thyroiditis are called destructive thyroiditis, which generally exhibits reversible thyroid dysfunction associated with destruction and subsequent regeneration of thyroid follicles (34). In both destructive thyroiditis and chronic autoimmune thyroiditis, monocytes/macrophages, which are known to produce PGD2 (10, 18, 40) and lymphocytes, are prominent in the infiltrates of the thyroid (34, 53). The local expression in autoimmune thyroiditis of various cytokines, including IL-1 and tumor necrosis factor-alpha , has been reported, and these cytokines are produced by infiltrated inflammatory cells and/or thyrocytes themselves (53). Although it has not yet been reported whether human thyrocytes can produce PGD2, TSH and insulin have been shown to stimulate PGD2 and PGE2 production in FRTL-5 cells (50). Furthermore, we already reported that IL-1 stimulates PGE2 production by porcine thyroid cells (21) and monocyte chemoattractant protein-1 by human thyrocytes (19). Thus PGD2 produced locally in the thyroid might have a role on the resolution of inflammation and the functional restoration of human thyrocytes through the cascade of PGJ2 series.

The present findings indicate that a cyclopentenone-type PG, 15-d-PGJ2, and its precursor PGs may stimulate TG production by themselves with no increase in cAMP production and further potentiate the TSH-stimulated production of TG accompanied by an increase in cAMP production in human thyrocytes that were cultured in a high serum concentration. In conclusion, such promoting effect by 15-d-PGJ2 and its precursor PGs in human thyrocytes is independent of the activation of PPAR-gamma or -alpha and also is probably independent of an activation of the stress response pathway. Because the mechanism of dedifferentiation of thyrocytes cultured in the presence of high serum concentrations is unknown at present, the exact mechanism of the promoting effect by 15-d-PGJ2 and its precursor PGs on TG production remains to be elucidated.


    ACKNOWLEDGEMENTS

This work was supported in part by a grant from the Japan Private School Promotion Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Kasai, Dept. of Endocrinology and Metabolism, Dokkyo Univ. School of Medicine, Mibu, Tochigi 321-0293, Japan (E-mail: kkasai{at}dokkyomed.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 September 1999; accepted in final form 26 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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