Effects of interleukin 1 on growth and adenosine 3',5'-monophosphate generation of the rat thyroid cell line, FRTL-5 cells.

We investigated the effects of IL-1, IL-2, IL-6, interferon-gamma and tumour necrosis factor-alpha on growth and cAMP generation of FRTL-5 cells. IL-1 produced a significant stimulation of [3H]thymidine incorporation into FRTL-5 cells without TSH, whereas IL-1 caused significant reductions in [3H]thymidine incorporation induced by TSH or forskolin, which is known as an adenylate cyclase activator. Intracellular cAMP generation of FRTL-5 cells was stimulated by IL-1, whereas TSH-stimulated cAMP generation was inhibited by IL-1. These effects of IL-1 was neutralized by addition of anti-IL-1 antibody. The studies suggested that IL-1 blocks the effects of TSH on proliferation and cAMP generation of FRTL-5 cells on a post-receptor site of TSH.     

Transforming growth factor-beta blocks protein kinase-A-mediated iodide transport and protein kinase-C-mediated DNA synthesis in FRTL-5 rat thyroid cells.

Recent studies have shown that transforming growth factor-beta (TGF beta) alters DNA synthesis and iodide metabolism in human, porcine, and rat thyroid cells. In the present work we studied the mechanism of TGF beta action in FRTL-5 rat thyroid cells. The cells were treated with TGF beta in the presence of TSH, growth factors, and cellular modulators for various periods of time; then, [3H]thymidine incorporation and DNA content were measured as indicators of DNA synthesis, and [125I]iodide uptake was measured to assess cell function. TGF beta (10 ng/ml) inhibited TSH-induced DNA synthesis and iodide uptake. TGF beta also inhibited DNA synthesis induced by insulin-like growth factor-I, fibroblast growth factor, and endothelial cell growth factor. The protein kinase-A (PKA) activator 8-bromo-cAMP increased both iodide uptake and DNA synthesis; TGF beta inhibited 8-bromo-cAMP-induced [125I]iodide uptake, but not [3H]thymidine incorporation. The protein kinase-C (PKC) activator phorbol 12-myristate 13-acetate increased [3H]thymidine incorporation, and TGF beta inhibited this action of phorbol 12-myristate 13-acetate. The results show that activation of PKA or PKC increases DNA synthesis. TGF beta inhibited PKC-mediated, but not PKA-mediated, DNA synthesis in these cells. The results also show that TGF beta selectively inhibits PKA-mediated iodide uptake, but not PKA-mediated DNA synthesis. These findings suggest that TGF beta is a strong inhibitor of the proliferation and function of thyroid cells.     

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.     

Influence of cytokines on growth and differentiated function of FRTL5 cells

A functioning rat thyroid cell line (FRTL5) was used to study interactions of TSH with interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha). We examined effects on growth and differentiated function. Growth was assessed by DNA, incorporation of [3H]thymidine ([3H]Tdr) into DNA, and cell number; uptake of 125I (I- uptake) and the concentration of cAMP were measured simultaneously with growth assessment. IFN gamma stimulated the 30-min I- uptake and enhanced the effect of TSH. TNF alpha had minimal effects on growth indices (slight increase in [3H]Tdr incorporation) and had no influence on I- uptake; it inhibited TSH stimulation of both growth and I- uptake. When combined, IFN gamma and TNF alpha synergized in inhibiting TSH-stimulated growth. By itself TNF alpha inhibited stimulation of I- uptake by TSH, but augmented the enhancement seen with IFN gamma. The influence of calf serum (CS) was to increase the rate of incorporation of [3H]Tdr, but a similar qualitative pattern for the actions of the cytokines remained. A reverse profile (stimulation by IFN gamma, inhibition by TNF alpha, and stimulation by the combination) was seen for I- uptake, with CS increasingly diminishing all values. TSH stimulation of growth was progressively effective with increments of CS in the medium, but consistently there was inhibition that was greater with IFN gamma than with TNF alpha and was almost total with the combined cytokines. Stimulation of I- uptake by TSH was enhanced by IFN gamma, reduced by TNF alpha, and, when serum was present, increased to a degree that was greater than additive by the combined cytokines. Growth stimulation by insulin or insulin-like growth factor-I was inhibited partially by the individual cytokines and completely by the combination. Both insulin and insulin-like growth factor-I inhibited TSH stimulation of I- uptake, but similar stimulation by the cytokines was not affected. Simultaneous with inhibition of TSH-stimulated growth, both IFN gamma and TNF alpha enhanced cAMP accumulation. The mechanism of these multiple effects of IFN gamma and TNF alpha, especially on the actions of TSH, may not currently be fully explained, but they almost certainly reflect differing modes of action. The relevance to thyroid function in man is conjectural. Especially in Graves' disease, where thyroid infiltration with cells that secrete these cytokines is common, it seems probable that both IFN gamma and TNF alpha will have significant influences on both growth and differentiated cell function.     

Characterization of tumor necrosis factor-alpha receptors in human and rat thyroid cells and regulation of the receptors by thyrotropin

Administration of recombinant human tumor necrosis factor-alpha (TNF) to rats and mice produces a model of nonthyroid illness in which there is impairment of hypothalamic-pituitary thyroid function, including reduced serum concentrations of T4 and T3, reduced thyroid radioiodine uptake, and reduced response to TSH. In this study, we tested the binding and effects of TNF on FRTL-5 cells and on four human thyroid carcinoma cell lines. The TSH-stimulated [125I]iodide uptake by FRTL-5 cells was inhibited by TNF in a dose-dependent manner. The four human thyroid carcinoma cell lines (NPA, MRO, ARO, WRO) have TSH receptors but did not respond to TSH in regard to iodide uptake and thymidine incorporation. Both human thyroid carcinoma cells and FRTL-5 cells contain specific receptors for TNF. Scatchard analysis showed that the receptor numbers and dissociation constants in human thyroid carcinoma cells and FRTL-5 cells were, respectively; 2.4 x 10(4), 5.4 nM (WRO); 8 x 10(3), 3.4 nM (MRO); 4 x 10(3), 1 nM (ARO); 7 x 10(3), 1 nM (NPA); 3 x 10(3), 1 nM (FRTL-5), and 9 x 10(3), 1 nM (FRTL-5 cells treated with TSH). The results indicate that TNF affects thyroid cell function through binding to the TNF receptor and that the number of TNF receptors is regulated by TSH.     

Mitogen-activated protein kinase activation in hepatocyte growth factor-stimulated rat hepatocytes: Involvement of protein tyrosine kinase and protein kinase C

Hepatocyte growth factor (HGF) stimulated mitogen-activated protein (MAP) kinases and MAP kinase kinase in primary cultured rat hepatocytes. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced MAP kinase activity. A PKC activator, phorbol myristate acetate (PMA), induced MAP kinase activation in a concentration-dependent manner. Protein tyrosine kinase (PTK) inhibitors, genistein, and ST638 also inhibited HGF-induced MAP kinase activation. Furthermore, HGF increased formation of Ras guanosine triphosphate (GTP) complex, indicating Ras activation. Genistein inhibited HGF-induced Ras activation, but Ro31-8425 was without effect. On the other hand, Ro31-8425 decreased HGF-induced [³H]arachidonic acid (AA) release and [³H]thymidine incorporation. Genistein also prevented [³H]AA release and [³H]-thymidine incorporation. Moreover, a commonly used phospholipase A2 (PLA2) inhibitor, quinacrine, decreased HGF- induced [³H]AA release and [³H]thymidine incorporation. The inhibitory profile of [³H]AA release was well correlated with that of [³H]thymidine incorporation in Ro31-8425-, genistein-, and quinacrine- treated cells. A cyclooxygenase inhibitor, indomethacin, which suppressed HGF-induced DNA synthesis, had minimal effect on MAP kinase activation. In contrast, prostaglandin (PG) E1, E2, or F2 alpha, which stimulate [³H]thymidine incorporation to the same level as that caused by HGF in hepatocytes, caused very weak activation of MAP kinases. These results suggest that PTK, Ras, and PKC play roles in MAP kinase activation induced by HGF and that MAP kinase activation resulting in AA release is involved in DNA synthesis in rat hepatocytes. (Hepatology 1996 May;23(5):1244-53)     

Effects of tumor necrosis factor on bone formation in vitro

Tumor necrosis factor (TNF) was studied for its effects on bone formation in cultured rat calvariae. TNF alpha at 100-100,000 U/ml stimulated [3H]thymidine incorporation into DNA, an effect that appeared after 24 h of treatment and lasted 96 h. Transient (24-h) treatment with TNF alpha increased [3H]proline incorporation into type I collagen 24-72 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. Transient treatment with TNF alpha also increased alkaline phosphatase activity. In contrast, continuous treatment with TNF alpha for 48-96 h caused a marked inhibition on [3H]proline incorporation into type I collagen and alkaline phosphatase activity. TNF alpha caused a small increase in collagen degradation. Lymphotoxin had similar effects to those of TNF alpha. In conclusion, TNF alpha stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but TNF alpha has a direct inhibitory effect on osteoblastic function.     

Antibody-dependent cell-mediated growth inhibition of rat thyroid cells, FRTL-5, in patients with autoimmune thyroid diseases

We studied antibody-dependent mononuclear cell-mediated growth inhibition of thyroid cells in 18 untreated patients with Graves' disease, 18 patients with chronic thyroiditis, and 15 normal subjects by measuring the ability of their sera to inhibit [3H]thymidine incorporation into DNA in a rat thyroid cell line, FRTL-5, in the presence of normal mononuclear cells. [3H]thymidine incorporation was significantly inhibited in the presence of sera from patients with Graves' disease and chronic thyroiditis (P less than 0.001), whereas it was not affected in normal subjects. A significant correlation was observed between the inhibition of [3H]thymidine incorporation and the titre of anti-microsomal antibodies (P less than 0.05). The inhibitory effect on [3H]thymidine incorporation was significantly abolished when serum pre-absorbed with human thyroid membranes was used (P less than 0.005). These inhibitory effects on [3H]thymidine incorporation significantly correlated with those obtained by using IgG fractions (P less than 0.01). These data indicate that antibody-dependent mononuclear cell-mediated growth inhibition may play a role in thyroid cell growth regulation in patients with autoimmune thyroid disease.     

Tumor necrosis factor-alpha increases release of arachidonate and prolactin from rat anterior pituitary cells

We investigated the effect of tumor necrosis factor-alpha (TNF alpha), a product of activated macrophages, on the release of arachidonate from dispersed anterior pituitary cells. Primary cultures of anterior pituitary cells from rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents, and PRL release into the medium and [3H]arachidonate cleaved from phospholipid were measured. TNF alpha significantly increased the release of both PRL and [3H] arachidonate release in a time- and dose-dependent manner. Other cytokines, such as interleukin-1 alpha, interleukin-1 beta, and gamma-interferon, had no effect on [3H]arachidonate release. To define the role of calcium in TNF alpha-induced arachidonate release, dispersed pituitary cells were incubated with low calcium medium, which decreased arachidonate release in response to TNF alpha. TNF alpha potentiated the release of [3H]arachidonate and PRL promoted by phospholipase-A2 and melittin, and markedly shifted the dose-response curve to the left. Inhibitors of phospholipase-A2, such as p-bromophenacyl bromide and quinacrine, had no effect on TNF alpha-induced [3H]arachidonate and PRL release. BW755C, an inhibitor of the conversion of arachidonate to its metabolites, decreased TNF alpha-induced PRL release, while indomethacin, a prostaglandin synthesis inhibitor, had no effect on TNF alpha-induced PRL release. These data indicate that arachidonate metabolites may be involved in the process of TNF alpha-induced PRL release.     

Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL5 thyroid follicular cells.

Specific thyroid hormone (T3) receptors are present in thyroid follicular cells, including the rat FRTL5 clonal line, but little is known about the effects of T3 on the growth and differentiated function of the thyroid. Unlike primary cultures of animal or human thyroid cells, FRTL5 do not secrete appreciable amounts of thyroid hormones. We now have studied the effects of T3 by itself and in combination with TSH and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation into DNA, iodide uptake, and cAMP production in FRTL5. We also have investigated the expression of different c-erbA mRNAs in these cells. Specific binding of T3 to FRTL5 cell nuclei in intact cells occurred with a binding capacity of 0.1-0.15 ng T3/mg DNA and an apparent Kd of 0.4 nM. Using an RNase protection assay on total cellular FRTL5 RNA and specific cRNA probes, we demonstrated the presence of c-erbA alpha and -beta mRNAs, both encoding T3 receptors. Biological effects were assessed in serum-free medium or buffer containing 0.1% BSA after maintaining quiescent culture of cells for at least 5 days in hormone-free medium containing 5% calf serum. T3 alone stimulated a dose-dependent increase in [3H]thymidine incorporation that reached a plateau at 188% of the control value at 10 nM T3. At 10(-11) M TSH, T3 potentiated TSH-stimulated [3H]thymidine incorporation (2.2-fold), but at TSH concentrations greater than 5 x 10(-11) M, T3 had no effect or reduced the response to TSH. T3 potentiated the [3H]thymidine response to 2 and 10 ng/ml IGF-I by 1.5- to 1.7-fold. T3 alone had no effect on iodide uptake, but attenuated iodide uptake stimulated by TSH. T3 was more potent in inhibiting TSH-stimulated iodide uptake than in enhancing TSH-stimulated DNA synthesis. T3 did not affect either basal or TSH-stimulated cAMP accumulation. Thus, in FRTL5 thyroid follicular cells 1) T3 receptors are expressed, as measured by direct binding assays and by the expression of c-erbA mRNAs; and 2) T3 acts as a growth factor and weak antidifferentiation factor. We suggest that T3 may modulate the actions of TSH and growth factors in thyroid epithelium.     

Evidence for thyrotropin (TSH)-blocking activity in goitrous Hashimoto's thyroiditis with assays measuring inhibition of TSH receptor binding and TSH-stimulated thyroid adenosine 3',5'-monophosphate responses/cell growth by immunoglobulins

There are two forms of autoimmune thyroiditis that may cause hypothyroidism: autoimmune atrophic thyroiditis (primary idiopathic hypothyroidism or primary myxedema) and autoimmune goitrous thyroiditis (Hashimoto's disease). Patients with the former have impalpable thyroid glands, and those with the latter have goiters. We studied TSH binding inhibitory immunoglobulins (TBII), TSH-stimulated cAMP response inhibitory immunoglobulins (TSII), and TSH-stimulated cell growth inhibitory immunoglobulins (TGII) in 42 patients with the former (group 1) and 115 patients with the latter (group 2). Porcine thyroid cells in primary culture and rat thyroid cells in continuous culture (FRTL-5 cells) were used to study TSII and TGII activities, respectively; TSII was expressed as percent inhibition of 0.1 mU/ml TSH-stimulated cAMP response by the patient's immunoglobulin (IgG; 1 mg/ml) during 2-h incubation, and TGII was expressed as percent inhibition of 10 mU/ml TSH-stimulated [14C]thymidine incorporation by the patient's IgG (1 mg/ml) during 24-h incubation. The new findings in this report are: some patients in both groups had TBII, TSII, and/or TGII; the frequency of the presence of TBII, TSII, and TGII in the patients with autoimmune atrophic thyroiditis was higher than that in the patients with autoimmune goitrous thyroiditis, and TSII and TGII were significantly associated with autoimmune atrophic thyroiditis; no correlation was found between goiter size and TBII, TSII, or TGII activity; and there were good correlations between TBII, TSII, and TGII activities. We also found that TSH-stimulated thymidine incorporation was through cAMP production and that the inhibitory IgGs inhibited TSH-stimulated thymidine incorporation by decreasing cAMP production in FRTL-5 cells, but not in porcine or human thyroid cells.     

Independent and interactive effects of tetradecanoyl phorbol acetate on growth and differentiated functions of FRTL5 cells

In studies of regulation of the growth and differentiated function of the thyroid follicular cell, we have employed the FRTL5 cell line to evaluate both the effects of agents that activate protein kinase-C (PKC) and their interaction with other agents that influence the growth and/or function of the FRTL5 cell. The PKC activator tetradecanoyl-phorbol acetate (TPA) alone induced a time- and concentration-dependent stimulation of the incorporation of [3H]thymidine into the DNA of quiescent FRTL5 cells, an effect anteceded by an increase in the levels of the mRNAs of the proto-oncogene c-myc and associated with a stimulation of cell replication. TPA also produced a dose-dependent inhibition of the low levels of radioiodine uptake in quiescent FRTL5 cells. These effects of TPA were unaccompanied by any change in the cellular cAMP concentration. TPA also modified a variety of responses to TSH, attenuating the TSH-induced stimulation of [3H]thymidine incorporation into DNA, cell replication, cAMP generation, and iodine uptake. Inhibition of TSH-stimulated growth and iodine uptake by TPA could not be ascribed solely to a decrease in cAMP generation, since TPA also inhibited the increase in [3H]thymidine incorporation and iodide uptake induced by the cAMP analog (Bu)2cAMP. In contrast, the independent stimulatory effects of TPA and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation and cell replication were at least additive when the two stimulators were added together. We have previously reported that both TSH and (Bu)2cAMP amplify the enhancement of DNA synthesis and cell replication in FRTL5 cells induced by IGF-I, and that the response of DNA synthesis to IGF-I is also enhanced if cells are preincubated with either TSH or (Bu)2cAMP. Both the former amplification of mitogenesis and the latter priming effect were decreased by exposing cells to TPA concomitant with their exposure to TSH or (Bu)2cAMP. The effects of TPA were mimicked by other activators of PKC, but not by a phorbol ester that fails to activate this enzyme. In general, we conclude that in the FRTL5 cell, regulation of cell growth is extremely complex; there are at least three mitogenic pathways that are separate from but interact with one another. The first is the well known cAMP-dependent pathway, which is activated by TSH. The second is activated by IGF-I and is cAMP independent. These two pathways interact to produce a marked amplification of their individual mitogenic effects. The third pathway is that stimulated by TPA and involves activation of PKC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine has divergent effects on deoxyribonucleic acid synthesis in FRTL5 cells: inhibition of thyrotropin-stimulated and potentiation of insulin-like growth factor-I-stimulated thymidine incorporation

Adenosine inhibits TSH-stimulated [3H]thymidine incorporation into DNA in FRTL5 thyroid follicular cells by both inhibiting cAMP generation and acting at a locus beyond adenylate cyclase. On the other hand, adenosine markedly potentiates DNA synthesis in FRTL5 stimulated by insulin-like growth factor-I (IGF-I). The mechanisms of this latter effect are unknown, but require the coincubation of adenosine and IGF-I and not mediated by an increase in intracellular cAMP concentration. Adenosine increases the maximal response of FRTL5 to [3H]thymidine incorporation stimulated by IGF-I and increases the sensitivity of FRTL5 to IGF-I. These effects of adenosine are reflected by an increase in nuclear labeling as well as by an increase in [3H]thymidine incorporation into DNA. Adenosine also plays a role as an autocrine growth factor in FRTL5, since adenosine deaminase increases the response of these cells to TSH. The effects of adenosine on both TSH- and IGF-I-stimulated DNA synthesis are shared by guanosine and inosine, although with different potencies for the various guanine nucleosides. Inosine potentiates IGF-I-stimulated DNA synthesis, but inhibits TSH-stimulated DNA synthesis only weakly. Adenosine interacts with multiple receptors and with multiple postreceptor pathways in FRTL5 to produce divergent effects on the control of cell replication by two growth factors (TSH and IGF-I) that act through different postreceptor pathways.     

Ethanol-inhibited [3H]thymidine incorporation via protein kinase C-p44/42 mitogen-activated protein kinase/phospholipase A2 signal pathway in renal proximal tubule cells

BACKGROUND: Ethanol exposure leads to changes of cell proliferation in a variety of cell types. However, how ethanol affects the proliferation of renal proximal tubule cells is not known. METHODS: To examine the effect of ethanol on cell proliferation and its related signaling pathway, [H]thymidine incorporation, release of [H]arachidonic acid (AA), and Western blotting of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) were performed in primary cultured rabbit renal proximal tubule cells. RESULTS: Ethanol inhibited [H]thymidine incorporation in a time- and dose-dependent manner. An inhibitory effect of ethanol on [H]thymidine incorporation was predominantly observed after 12 hr of treatment with 100 mM ethanol. Ethanol increased AA release and prostaglandin E2 production. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by phospholipase A2 inhibitors and was significantly blocked by PKC inhibitors. Indeed, ethanol induced a PKC translocation from the cytosolic to the membrane fraction. In addition, ethanol-induced inhibition of [H]thymidine incorporation was blocked by PD 98059 (a p44/42 MAPK inhibitor), but not by SB 203580 (a p38 MAPK inhibitor), and ethanol increased the phosphorylation of p44/42 MAPK. Results of phosphorylated p44/42 MAPK by ethanol were consistent with those of [H]thymidine incorporation and [H]AA-release experiments. CONCLUSIONS: Ethanol inhibited [H]thymidine incorporation via PKC, p44/42 MAPK, and phospholipase A2 signaling pathways in primary cultured renal proximal tubule cells.     

Aging alters somatomedin-C-dexamethasone synergism in the stimulation of deoxyribonucleic acid synthesis and replication of cultured human fibroblasts

The effects of dexamethasone on somatomedin-C (SM-C) stimulation of [3H]thymidine incorporation and cell replication were studied in early passage fibroblasts from normal donors, aged 7-24 yr (young) and 85-96 yr (old), and one patient with progeria. Preincubation of cells from young donors with dexamethasone dramatically enhanced SM-C stimulation of [3H]thymidine incorporation [e.g. 19- vs. 3-fold in serum-free medium; 66- vs. 14-fold in 0.25% human hypopituitary serum (HHS)], with no alteration in the timing of peak thymidine incorporation. In contrast, preincubation of cells from old and progeric donors with dexamethasone resulted in a 6- to 12-hr lengthening of the prereplicative period and, generally, little or no synergism with SM-C. Cells from old and progeric donors had a normal replicative response to SM-C with or without 0.25% HHS. In cells from young donors, dexamethasone enhanced the SM-C-stimulated increase in cell number 32-49% in serum-free medium and 70-189% in 0.25% HHS. In comparison, dexamethasone had no potentiating effect on SM-C stimulation of multiplication of cells from old and progeric donors. These data indicate that dexamethasone and SM-C are synergistic in stimulating DNA synthesis and replication of fibroblasts from young donors, but that this synergism is impaired in cells from aged and progeric donors.     

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts.

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.     

Somatomedin C-binding and action in fibroblasts from aged and progeric subjects

Aging is associated with diminished cell growth, which has been ascribed in part to decreased cellular responsiveness to serum mitogens. To investigate whether there is an age-related loss of responsiveness to somatomedin-C (SM-C), we studied SM-C binding and action in early passage fibroblasts from normal donors, aged 7-96 yr, and one progeric subject. SM-C stimulated [3H]thymidine incorporation 4- to 16-fold in young cells, 4- to 17-fold in aged cells, and 4- to 11-fold in progeric cells. SM-C was synergistic with 0.25% human hypopituitary serum in stimulating [3H]thymidine incorporation in all cell lines. Dose-response curves for SM-C stimulation of thymidine incorporation were not significantly altered in aged or progeric cells. Half-maximal responses occurred at 5-15 ng/ml SM-C for all cell lines. [3H]Thymidine incorporation results were supported by cell replication studies. In addition, binding of [125I] SM-C was virtually identical in all cell lines, with 50% displacement at 2-5 ng/ml SM-C. Thus, in vivo aging does not appear to be associated with either an alteration in SM-C receptors or a diminished cellular responsiveness to SM-C's mitogenic effects.     

Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.     

Inhibition of thyrotropin-stimulated adenylate cyclase activation and growth of rat thyroid cells, FRTL-5, by immunoglobulin G from patients with primary myxedema: comparison with activities of thyrotropin-binding inhibitor immunoglobulins

We studied the blocking type TSH receptor antibodies in 28 patients with primary myxedema and 21 patients with goitrous Hashimoto's thyroiditis by measuring the ability of their IgGs to inhibit TSH binding to its receptor, and to inhibit TSH-stimulated cAMP increase and [3H]thymidine incorporation in a rat thyroid cell line, FRTL-5. The incidences of TSH binding inhibitor immunoglobulin, thyroid stimulation inhibiting immunoglobulin and thyroid growth inhibiting immunoglobulin in patients with primary myxedema were 54.6, 75 and 65.2%, respectively, against 14.3, 0 and 17.7%, respectively, in goitrous Hashimoto's thyroiditis. The antibodies inhibited dose-dependently not only TSH stimulated but also Graves' IgG-stimulated cAMP increase and [3H]thymidine incorporation. The TSH binding inhibitor immunoglobulin activities in patients with primary myxedema were significantly correlated with both the thyroid stimulation inhibiting immunoglobulin (r = 0.665; P less than 0.01) and the thyroid growth inhibiting immunoglobulin (r = 0.618; P less than 0.01) activity. Thirteen patients whose TSH binding inhibitor immunoglobulin activities were more than 50% had both strong thyroid stimulation inhibiting immunoglobulin (75.1-100%) and thyroid growth inhibiting immunoglobulin (57.4-100%) activities. These data suggest that the vast majority of patients with primary myxedema have potent blocking type TSH receptor antibodies. These might play a role in primary myxedema causing hypothyroidism and thyroid atrophy through inhibiting TSH-stimulated cAMP generation.     

The mechanism of action of tumour necrosis factor-alpha and interleukin 1 on FRTL-5 rat thyroid cells

Previous work showed that treatment of rats with tumour necrosis factor-alpha produced a model of nonthyroid illness in which there was reduction of circulating thyroid hormones and TSH, reduced thyroid response to TSH, and reduced thyroid iodide uptake. In vitro studies showed that tumour necrosis factor-alpha binds to a specific receptor on FRTL-5 rat thyroid cells, that TSH increases the number of tumour necrosis factor-alpha receptors, and that tumour necrosis factor-alpha inhibits iodide uptake by these cells. In the present study, we obtained additional data on the effects of tumour necrosis factor-alpha on FRTL-5 cells and studied the mechanism of action of tumour necrosis factor-alpha in these cells. Tumour necrosis factor-alpha inhibited both basal and TSH-stimulated [125I]iodide uptake: tumour necrosis factor-alpha slowed the recovery of [125I]iodide trapping after the cells were exposed to TSH and augmented the loss of the [125I]iodide trapping function after the cells were deprived of TSH: tumour necrosis factor-alpha inhibited [125I]iodide trapping in a noncompetitive manner; tumour necrosis factor-alpha did not affect cell growth of FRTL-5 cells. Interleukin-1 (IL-1) also inhibited basal and TSH-stimulated [125I]iodide uptake, but it stimulated cell growth. Tumour necrosis factor-alpha and IL-1 did not affect the generation of cAMP in the presence or absence of TSH; these cytokines blocked the cAMP-induced stimulation of [125I]iodide uptake. Tumour necrosis factor-alpha did not affect [3H]arachidonic acid uptake or release by FRTL-5 cells. The inhibitors of the phospholipase A2-arachidonic acid pathway did not affect the action of tumour necrosis factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)