Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells

A rat thyroid peroxidase cDNA has been isolated from a FRTL-5 thyroid cell library and sequenced. The cDNA is 2776 base pairs long with an open reading frame of 770 amino acids. By comparison to full-length human thyroid peroxidase cDNA and based on its identification of a 3.2 kilobase mRNA in rat thyroid FRTL-5 cell Northern blots, the rat peroxidase cDNA appears to lack 400-500 base pairs at the 5'-end of the mRNA. It exhibits only a 74% nucleotide and 77% amino acid sequence similarity to human thyroid peroxidase cDNA within the total aligned sequences, although the predicted active site regions are highly conserved (greater than 90-100%). The cDNA has been used to map the thyroid peroxidase gene in mice to chromosome 12 and to compare thyroid peroxidase and thyroglobulin gene expression in FRTL-5 rat thyroid cells. Despite the fact TSH action in both cases is duplicated, and presumably mediated, by cAMP, TSH-induced increases in thyroid peroxidase and thyroglobulin mRNA levels differ. Differences exist with respect to hormone concentration and time. The ability of TSH to increase thyroglobulin, but not thyroid peroxidase mRNA levels, requires insulin, 5% serum, or insulin-like growth factor-I. Insulin or insulin-like growth factor-I alone can increase thyroglobulin mRNA levels as well as or better than TSH but have only a small effect on thyroid peroxidase mRNA levels by comparison to TSH. The ability of TSH to increase thyroglobulin gene expression is readily detected in nuclear run-on assays but not the ability of TSH to increase thyroid peroxidase gene expression. Cycloheximide inhibits TSH-increased thyroglobulin but not peroxidase mRNA levels. Finally, methimazole and phorbol 12-myristate 13-acetate show different effects on TSH-induced increases in thyroglobulin and thyroid peroxidase mRNA levels.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

Regulation of geranylgeranyl pyrophosphate synthase in the proliferation of rat FRTL-5 cells: involvement of both cAMP-PKA and PI3-AKT pathways

We have reported that geranylgeranyl pyrophosphate (GGPP), one of the isoprenoids in the mevalonate pathway, plays an essential role for cell growth through the geranylgeranylation of Rho small GTPases, which control the degradation of P27Kip1 at G1/S transition in rat thyroid FRTL-5 cells. Since GGPP is synthesized from isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) by GGPP synthase, we analyzed the regulatory roles of GGPP synthase in the proliferation of FRTL-5 cells stimulated by thyrotropin and insulin in the presence of 5% calf serum (TSH+Ins). We found that: (1) GGPP synthase was activated at G1/S transition with increasing mRNA accumulation followed by protein expression, (2) pravastatin, an inhibitor of HMG-CoA reductase, did not suppress the increasing activity of GGPP synthase with its protein expression although it inhibits proliferation in growth-stimulated FRTL-5 cells, (3) forskolin stimulated proliferation with activation of GGPP synthase in FRTL-5 cells, and (4) LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited proliferation with the decreasing activity of GGPP synthase in growth-stimulated FRTL-5 cells. These data indicated that growth stimulation by TSH+Ins increased the activity of GGPP synthase with its increasing protein expression from G1/S transition, in which both cAMP-PKA and PI3-kinase pathways are involved in the proliferation of FRTL-5 cells.     

Inhibitory effect of IL-1 on the TSH dependent growth of rat thyroid cells (FRTL-5)

Using a functioning rat thyroid cell line (FRTL-5), we examined the effects of some cytokines, particularly interleukin-1 (IL-1) on the growth of thyroid cells. In 5H medium, namely Coon's modified Ham's F-12 medium supplemented with 5% calf serum and a five-hormone preparation consisting of insulin, hydrocortisone, transferrin, glycyl-L-histidyl-L-lysine acetate and somatostatin, IL-1 enhanced the growth of FRTL-5 cells detected by [3H]TdR incorporation. However, in 6H medium (5H medium plus bovine TSH), IL-1 inhibited the growth of FRTL-5 cells. Both effects were neutralized by the addition of anti-IL-1 antibody. Furthermore, IL-1 inhibited the growth of FRTL-5 cells induced by forskolin which is known as an adenylate cyclase activator. FRTL-5 cells have specific IL-1 receptors detected by the binding of 125I-labeled IL-1 alpha. By Scatchard plot analysis, the numbers and the dissociation constants of IL-1 receptors on FRTL-5 cells were shown to be 5225/cell and 8.69 x 10(-10) M. Interleukin-2, interleukin-6 and interferon-gamma (IFN-gamma) had no significant effects on the cell growth in 6H medium, while IFN-gamma and insulin-like growth factor I stimulated cell growth somewhat in 5H medium. These results suggest that IL-1 plays a regulatory role in the growth of thyroid cells through binding to the IL-1 receptors.     

Interaction of secreted insulin-like growth factor-I (IGF-I) with cell surface receptors is the dominant mechanism of IGF-I's autocrine actions.

In a prior report we presented evidence that insulin-like growth factor-I (IGF-I) can act in an autocrine fashion by demonstrating that FRTL-5 cells transfected with hIGF-IA fusion genes express and secrete biologically active IGF-I that renders the stimulation of DNA synthesis in FRTL-5 cells independent of their requirement for exogenous IGFs or insulin. To determine if IGF-I's autocrine actions require secretion or can be mediated by interactions with intracellular receptors, we have created a new line of FRTL-5 cells that express a mutant IGF-IA precursor containing the endoplasmic reticulum retention amino acid sequence, Lys-Asp-Glu-Leu (KDEL), at its carboxyl terminus. The mutant IGF-IA/KDEL precursor expressed by stably transfected FRTL-5 cells was shown to be retained intracellularly and to have biological activity comparable with mature IGF-I, as judged by the activity of partially purified IGF-IA/KDEL in wild type FRTL-5 cells. Expression of IGF-IA/KDEL in FRTL-5 cells, however, neither augmented TSH-stimulated DNA synthesis nor stimulated IGF-binding protein-5 expression, as does IGF-IA expression in transfected FRTL-5 cells and the addition of exogenous IGF-I to wild type FRTL-5 cells. IGF-IA/KDEL expression, however, desensitized FRTL-5 cells to the actions of exogenous IGF-I despite having only minimal effects on cell surface type I receptor number, suggesting that intracellular IGF-I is capable of significant biological actions. The failure of IGF-IA/KDEL to replicate the actions of secreted IGF-I, taken together with the findings that a monoclonal antibody against IGF-I blocked IGF-I's actions in IGF-I-secreting transfected FRTL-5 cells, provides evidence that IGF-I secretion and interaction with cell surface type I IGF receptors is the dominant mechanism of IGF-I's autocrine actions.     

Increases in cytosolic Ca++ down regulate thyrotropin receptor gene expression by a mechanism different from the cAMP signal.

Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation.     

TSH is able to induce cell cycle-related gene expression in rat thyroid cell.

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Changes in the synthesis of histone H1(0) and H1 in rat FRTL-5 thyroid cells exposed to thyrotropin

In absence of thyrotropin (TSH), FRTL-5 rat thyroid cells stop proliferating and lose the functional characteristics of thyroid tissue. FRTL-5 cells regain their differentiated state and their proliferation activity upon addition of TSH. In this study we investigated the synthesis of histone H1 variants and H19(0) in FRTL-5 cells exposed to 10(-8) M TSH, two days after TSH withdrawal. TSH induced the synthesis of some H1 variants and H1. This effect was already evident six hours after TSH addition, thus well before proliferation, DNA or thyroglobulin synthesis was induced. These data indicate that the induction of H1(0) and some H1 variants is an early event after TSH stimulation and may thus be related to the functional differentiation of FRTL-5 cells.     

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity

Lactacystin, an inhibitor of proteasome activity, amplifies prostaglandin I2 production by rat liver cells stimulated by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor-alpha or interleukin-1. Lactacystin also stimulates the cell's release of arachidonic acid (AA) and increases the cyclooxygenase activity in these cells. In serum deprived cells, the enhanced AA release is reduced, cyclooxygenase activity on exogenous AA is increased and endogenous production of prostaglandin I2 is unchanged. These findings suggest that, in vivo, the ratio of dividing to quiescent cells in a tissue may influence eicosanoid production. The increases in prostaglandin I2 production, AA release and cyclooxygenase activity on exogenous AA resulting from the combined lactacystin and 12-O-tetradecanoylphorbol-13-acetate treatment are inhibited by actinomycin or cycloheximide.     

The tissue-specific pathways regulating cell proliferation are inherited independently in somatic hybrid between thyroid and liver cells

Thyroid stimulating hormone (TSH) and insulin-like growth factors type 1 (IGF-I) regulate the proliferation and differentiation of cultured thyroid cells but not of cultured liver cells. We have examined the influence of TSH and IGF-I on the metabolic functions and proliferation of somatic hybrids obtained by fusing rat thyroid cells (FRTL5) with rat liver cells (BRL). While IGF-I is able to stimulate the proliferation of the hybrid cells (TxL) TSH fails to induce their growth. However, the hybrid TxL cells have surface TSH receptors with normal ligand characteristics. The addition of TSH to TxL cells led to typical enhancement of cAMP production and depolymerization of actin filaments. Yet, TSH failed to stimulate iodine uptake in the hybrid cells. Interestingly, iodine inhibited TxL proliferation induced by IGF- I but not by serum. It is concluded that the hybrid TxL cells inherited from the parental thyroid cells several important differentiated traits including mitogenic pathways induced and used by IGF-I, functional TSH receptors, and sensitivity to the inhibitory action of iodine.     

Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations of palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of cellular lipids in C-9 cells prelabelled with [3H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E2 and F2 alpha by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the two effects taken independently.