Protein synthesis-dependent potentiation by thyroxine of antiviral activity of interferon-gamma

Hung-Yun Lin, Paul M. Yen, Faith B. Davis, and Paul J. Davis

Department of Medicine, Albany Medical College and Stratton Veterans Affairs Medical Center, Albany, New York 12208; and Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have studied the prenuclear signal transduction pathway by which thyroid hormone potentiates the antiviral activity of human interferon-gamma (IFN-gamma ) in HeLa cells, which are deficient in thyroid hormone receptor (TR). The action of thyroid hormone was compared with that of milrinone, which has structural homologies with thyroid hormone. L-Thyroxine (T4), 3,5,3'-L-triiodothyronine (T3), and milrinone enhanced the antiviral activity of IFN-gamma up to 100-fold, a potentiation blocked by cycloheximide. The 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil did not block the T4 effect. 3,3',5,5'-Tetraiodothyroacetic acid prevented the effect of T4 but not of milrinone. The effects of T4 and milrinone were blocked by inhibitors of protein kinases C (PKC) and A (PKA) and restored by PKC and PKA agonists; only the effect of T4 was blocked by genistein, a tyrosine kinase inhibitor. In separate models, milrinone was shown not to interact with nuclear TR-beta . T4 potentiation of the antiviral activity of IFN-gamma requires PKC, PKA, and tyrosine kinase activities but not traditional TR.

thyroid hormone action; protein kinase C; protein kinase A; tyrosine kinase

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

WE HAVE DEMONSTRATED that iodothyronines potentiate the antiviral activity of recombinant human interferon-gamma (IFN-gamma ) by up to 140-fold in HeLa cells and human fibroblasts (13). This thyroid hormone potentiation occurs by two mechanisms, one protein synthesis dependent and the other postnuclear or nongenomic and independent of protein synthesis (11). The nongenomic, or protein synthesis-independent effect of thyroid hormone has been shown to involve activities of both protein kinase C (PKC) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) (12). The possible contributions of these kinases to the protein synthesis-dependent actions of thyroid hormone have not been explored. In early studies of this latter effect, a physiological concentration of L-thyroxine (T4) (13) has been shown to be more effective than a physiological concentration of 3,5,3'-L-triiodothyronine (T3), and 3,3',5'-L-triiodothyronine [reverse T3 (rT3)] has also been found to be active (11). This action of T4 is blocked by 3,3',5,5'-tetraiodothyroacetic acid (TETRAC), whereas the nongenomic effect of T4 is not blocked by TETRAC (11).

Milrinone is a bipyridine with cardiac inotropic properties (1) that also stimulates bone turnover and calcium release from bone (10). Its mechanisms of action include inhibition of cyclic nucleotide phosphodiesterase activity (7) and stimulation of tissue-specific membrane calcium- and magnesium-dependent adenosinetriphosphatase (Ca2+-ATPase) activity (14, 23). We have reported that milrinone has structural homologies with thyroid hormone (14) and bioactivities that overlap those of thyroid hormone, including stimulation of myocardial membrane (14) and skeletal muscle sarcoplasmic reticulum (23) Ca2+-ATPase activities and binding to the thyroid hormone binding protein, transthyretin (5).

Because of structural similarities between milrinone and thyroid hormone, we have used milrinone as a tool, along with selected PKC and PKA agonists and PKC, PKA, and tyrosine kinase antagonists, to identify possible signal transduction pathways that may contribute to the enhancement of IFN-gamma 's antiviral activity by both thyroid hormone and milrinone. We have also compared the effects of milrinone with those of T3 in traditional thyroid hormone receptor-beta (TR-beta ) binding and cotransfection studies to further contrast the bipyridine's effects with those of thyroid hormone.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents. Recombinant human IFN-gamma (>1 × 107 U/mg) was obtained from BioSource International (Camarillo, CA) and was used for all studies. T4, T3, 3,3',5-triiodothyroacetic acid (TRIAC), 6-n-propyl-2-thiouracil (PTU), cycloheximide, milrinone, amrinone, 3-isobutyl-1-methylxanthine (IBMX), phorbol 12-myristate 13-acetate (PMA), and 8-bromo-cAMP (8-BrcAMP) were obtained from Sigma Chemical (St. Louis, MO). TETRAC was obtained in pure form except for minimal salt content from Henning Berlin (G. Weickgenannt, personal communication); there is no thyroid hormone or TRIAC in this product. KT-5720, a PKA inhibitor with an inhibitor constant of 6 × 10-8 M (9), was obtained from Kamiya Biomedical (Thousand Oaks, CA), and CGP-41251, a PKC inhibitor (3), was kindly provided by Ciba-Geigy (Basel, Switzerland). The tyrosine kinase inhibitor genistein (17) was obtained from ICN Biochemicals (Costa Mesa, CA).

Cell cultures. HeLa cell cultures were grown and maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), as described previously (13). HeLa cells are deficient in TR (19). The serum was depleted of thyroid hormone by treatment with an anion exchange resin (AG1-X8, Bio-Rad Laboratories, Richmond, CA), according to the method of Weinstein et al. (24). As a result, the free and total T4 concentrations in hormone-depleted serum-supplemented medium (SSM), measured by analog radioimmunoassay and chemiluminescent assay, were 0.4 × 10-12 and 0.9 × 10-9 M, respectively, and the total T3 concentration was <10-10 M (13). There was no free T3 measurable in hormone-depleted serum by a commercial assay (Laboratory Corporation of America). Murine fibroblasts (L-929) were grown and maintained in FBS-supplemented minimal essential medium (MEM); these cells were used for assay of antiviral activity (see below).

Virus culture and quantitation of antiviral state. Confluent monolayers of HeLa cells (105 cells/well) were grown in 24-well trays at 37°C and refed with fresh medium containing thyroid hormone-depleted FBS (10%) for 24 h before addition of IFN-gamma . Cultures then received 1.0 IU/ml IFN-gamma or diluent in medium (control) for an additional 24 h at 37°C, followed by viral challenge. A plaque-purified preparation of vesicular stomatitis virus (VSV, Indiana serotype) was maintained for determination of antiviral state and IFN-gamma titration. Antiviral state was measured by infecting control and IFN-gamma -treated cells with VSV at an input multiplicity of infection of 10 plaque-forming units (pfu) per cell; virus yield (pfu/ml) was quantitated by plaque assay in L-929 cells according to methods previously described (13).

Antiviral activity of IFN-gamma and effect of thyroid hormone and bipyridines. Stock solutions (10-4 M) of T4 and analogs in 4% propylene glycol-0.04 N KOH and 10-3 M preparations of milrinone and amrinone in dimethyl sulfoxide (DMSO) were diluted 1:100 or more in SSM as needed. IBMX was dissolved in 100% ethanol in a stock concentration of 5 × 10-2 M and applied to cells in a final concentration of 5 × 10-4 M. PTU was dissolved directly in SSM in a concentration of 10-3 M. Control incubations contained diluent in appropriate concentrations. Diluents used had no antiviral effect when applied alone in SSM to cells and did not alter antiviral response to IFN-gamma . HeLa cells were incubated with T4 or bipyridine for either a 24-h preincubation before 24-h treatment with IFN-gamma (1.0 IU/ml) or the last 4 h of a 24-h IFN-gamma exposure. Antiviral assays were then performed. Results are shown as means ± SE of two or more experiments. One-way analysis of variance (ANOVA) was used to determine statistical significance of hormone potentiation and inhibition of that potentiation.

Effect of cycloheximide and protein kinase agonists and antagonists on potentiation of the antiviral activity of IFN-gamma . In selected studies, cycloheximide (CHX, 5 µg/ml) was added to cultured cells with or without T4 or milrinone for 24 h before IFN-gamma treatment in the preincubation paradigm described above. The medium containing CHX and hormone or bipyridine was then removed and replaced with fresh medium containing 1.0 IU/ml IFN-gamma for 24 h, after which the antiviral state was determined. Under these conditions, protein synthesis, measured by [35S]methionine incorporation into proteins and subsequent counting of trichloroacetic acid precipitates, was inhibited by 63% in the cells exposed to CHX. Because CHX was removed before addition of medium containing IFN-gamma , there was no inhibition of the IFN's antiviral effect during the subsequent 24-h incubation.

The effect of kinase agonists and antagonists was similarly tested by adding those reagents to the culture medium during the 24-h preincubation with thyroid hormone or milrinone. 8-BrcAMP was dissolved in distilled water. The agonist PMA and kinase antagonists were dissolved in DMSO so that a final DMSO concentration of 0.1% or less was attained. As in the studies with CHX, the media were changed before the addition of IFN-gamma to the cells, so that agonists and/or inhibitors were not present during IFN-gamma treatment.

T3 binding studies using nuclear TR-beta . A previously described expression vector (16) containing human TR-beta cDNA was subcloned into the Nde I and BamH I sites of pET16b vector containing sequences encoding histidine residues in the leader sequence (Novagen; gift of Dr. Kevin Petty, University of Texas Southwestern). TR was then expressed in BL21(DE3) pLys S Escherichia coli cells, and the expressed protein was purified using nickel-bound resin (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The molecular weight and purity of isolated protein were verified by electrophoresis and Coomassie blue staining on a 10% polyacrylamide gel. T3 binding studies were performed as previously described (21).

Cotransfection studies. Previously described cDNA clones of rat TR-alpha in pSG5 (20) or TR-beta in pCDNA (27) were used in the cotransfection experiments. A previously described reporter plasmid containing the chicken lysozyme thyroid hormone response element (TRE) F2 and luciferase cDNA was also used (26). CV-1 cells were grown in DMEM containing 10% fetal calf serum. The serum for these studies was depleted of T3 and steroid hormones by charcoal stripping for 12 h at 4°C and constant mixing with 5% (wt/vol) AG1-X8 resin twice for 12 h at 4°C before ultrafiltration. The cells were transfected with expression (0.1 µg) and reporter (1.7 µg) plasmids as well as respiratory syncytial virus (RSV) beta -galactosidase control plasmid (1 µg) in 3.5-cm plates using the calcium phosphate precipitation method (26). Cells were grown for 48 h in the absence or presence of various concentrations of the T3 or milrinone and harvested. Cell extracts were then analyzed for both luciferase and beta -galactosidase activity (26). Luciferase activity was normalized to beta -galactosidase activity, and the magnitude of induction relative to basal transcription was calculated, with onefold basal equaling transcription activity with empty expression vector alone in the absence of ligand.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of thyroid hormone and bipyridines on antiviral activity of IFN-gamma in HeLa cells. The potentiating effects of pretreatment with graded concentrations of T4 and T3 on the antiviral action of IFN-gamma subsequently applied to HeLa cells are shown in Fig. 1. There is a 10-fold, or one log, decrease in virus yield in cells treated with IFN-gamma (1.0 IU/ml) compared with untreated (control) cells. Although 10-11 to 10-10 M concentrations of both hormones were ineffective, a progressive increase in T4 and T3 concentration to 10-7 M produced 54- and 42-fold increases, respectively, in antiviral activity (P < 0.01), shown as decreases in virus yield compared with the yield with IFN-gamma alone. The hormone-depleted SSM contained <10-9 M total T4 and <10-10 M total T3. We regard 10-7 M total T4 and 10-9 M total T3 as physiological concentrations. Thus our studies show that, at physiological concentrations, T4 is more potent than T3 in this intact HeLa cell antiviral model. To document that the conversion of T4 to T3 is not contributing to the T4 effect, PTU was preincubated with T4. PTU neither altered the antiviral response to IFN-gamma nor had any effect on T4 potentiation as shown in Fig. 2.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration-dependent potentiation by L-thyroxine (T4) and 3,5,3'-L-triiodothyronine (T3) of antiviral effect of interferon-gamma (IFN-gamma ). In this and subsequent antiviral figures, open bar represents control virus yield and filled bar represents antiviral effect of IFN-gamma (1.0 IU/ml). Horizontal arrow below graph also indicates samples that received IFN-gamma . T4 or T3 (10-11 to 10-7 M) was added to cells for a 24-h preincubation before 24-h IFN-gamma treatment as described in MATERIALS AND METHODS. In all studies, there was a complete change of medium between preincubation and IFN-gamma treatment periods. Statistical significance of potentiation by T4 and T3 at each concentration was measured by 1-way analysis of variance (ANOVA), comparing virus yield with that in samples treated with IFN-gamma alone. pfu, Plaque-forming units. Levels of significance: * P < 0.05; ** P < 0.01.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Lack of effect of 6-n-propyl-2-thiouracil (PTU) on T4 potentiation of IFN-gamma 's antiviral action. PTU did not inhibit antiviral action of IFN-gamma (1.0 IU/ml) and did not inhibit T4 potentiation of antiviral effect. ** P < 0.01, significant effect of T4 pretreatment on virus yield in IFN-gamma -treated cells.

The effect of T4, milrinone, and amrinone on the antiviral action of IFN-gamma is shown in Fig. 3. Cells preincubated with 10-7 M T4 for 24 h before IFN-gamma exposure showed a 71-fold reduction in virus yield, compared with the yield in cells treated with IFN-gamma alone. A similar effect was seen when T4 was added to cells during only the last 4 h of the 24-h IFN-gamma incubation (4-h coincubation). Milrinone (10-7 to 10-5 M) enhanced the antiviral effect of IFN-gamma from 7- to 41-fold, respectively, when added to HeLa cells in the 24-h pretreatment paradigm (Fig. 3) but had no effect during a 4-h coincubation with IFN-gamma . Neither T4 nor milrinone had antiviral activity in the absence of IFN-gamma (results not shown). Amrinone (10-7 M) did not enhance the antiviral effect of IFN-gamma (Fig. 3) and 10-5 M amrinone was also ineffective (not shown). Similarly, IBMX (5 × 10-4 M), like milrinone and amrinone a phosphodiesterase inhibitor, had no effect on virus yield either in the presence or absence of IFN-gamma (results not shown).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of T4, milrinone and amrinone on antiviral action of IFN-gamma in HeLa cells. T4 or bipyridine was added to cells either for a 24-h preincubation before a 24-h treatment with IFN-gamma (1.0 IU/ml) or coincubated for last 4 h of a 24-h IFN-gamma treatment. T4 potentiated IFN-gamma action almost 100-fold in both experimental models. Milrinone potentiated antiviral effect of IFN-gamma but only when applied to cells in preincubation model. Amrinone was without effect. Significance of T4 or milrinone potentiation: * P < 0.05; ** P < 0.01.

We have previously demonstrated that thyroid hormone potentiation of IFN-gamma 's antiviral effect in the preincubation experimental model is inhibited by CHX (11). We show this effect in Fig. 4, along with additional results demonstrating that the addition of CHX to the cell medium during treatment with milrinone (10-6 or 10-5 M) inhibited the bipyridine's enhancement of IFN's antiviral effect. Thus milrinone's action requires protein synthesis in a manner similar to the action of T4 and T3 in the 24-h preincubation model (11). The addition of milrinone to a maximally effective concentration of T4 did not further enhance IFN-gamma potentiation (results not shown).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibition by cycloheximide (CHX) of potentiation by T4 and milrinone of IFN-gamma antiviral effect. T4 and milrinone were applied to cells during 24-h preincubation with or without CHX (25 µg/ml). Medium was then removed and replaced with medium containing IFN-gamma (1.0 IU/ml) for 24 h before antiviral assay. T4 and milrinone each potentiated antiviral action of IFN-gamma (* P < 0.05); these effects were completely blocked by CHX.

Interaction of TETRAC with effects of T4 and milrinone. We have reported that TETRAC and TRIAC (10-7 M) do not potentiate the antiviral action of IFN-gamma but do, however, block the protein synthesis-dependent potentiation by T4 of IFN-gamma 's action (11). We therefore tested whether TETRAC also blocked milrinone's potentiation in the preincubation experimental model. Results in Fig. 5 indicate that although TETRAC blocked the antiviral potentiation by T4, there was no effect of TETRAC on the potentiating action of milrinone. Parallel studies using TRIAC and milrinone yielded similar results (not shown). To further demonstrate the disparate responses to TETRAC of T4- and milrinone-induced antiviral potentiation, cells were pretreated for 24 h with T4, milrinone, and TETRAC before IFN-gamma treatment. Results in Fig. 5 show that milrinone restored potentiation of IFN-gamma 's antiviral activity in the presence of T4 and TETRAC. These experiments indicate that the actions of T4 and milrinone in potentiating the effect of IFN-gamma do not depend on the identical mechanism.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Interaction of T4, milrinone, and tetraiodothyroacetic acid (TETRAC) in potentiation of antiviral effect of IFN-gamma . Cells were pretreated with T4, milrinone (Mil) or both, with or without TETRAC, before 24-h IFN-gamma exposure, and effect on virus yield was then determined. TETRAC blocked potentiation by T4 but not potentiation by milrinone. Addition of milrinone to TETRAC and T4 caused potentiation to be expressed. *** P < 0.001, significant potentiation by both T4 and milrinone.

Effect of protein kinase agonists and antagonists. The effects of PKC agonist, PMA, and PKC inhibitor, CGP-41251, on potentiation of IFN-gamma 's antiviral effect by T4 and milrinone are seen in Fig. 6. CGP-41251 has a concentration inhibiting 50% of the maximal response of 5 × 10-8 M for PKC (3); at a concentration of 5 × 10-10 M, the inhibitor partially blocked the T4 effect and completely blocked the milrinone effect. It was not possible to use higher concentrations of CGP-41251 because of their toxic effect on subsequent incubation of cells with IFN-gamma , even though the medium with inhibitor was replaced with fresh medium before the IFN-gamma treatment. When used alone before IFN-gamma but without hormone or bipyridine, neither CGP-41251 nor PKC agonist PMA had any effect on virus yield (not shown). In cells treated with either T4 or milrinone before IFN-gamma , PMA partially blocked the antiviral potentiation, presumably reflecting partial depletion of PKC in cells. In the presence of T4 or milrinone and CGP-41251, PMA reversed the CGP-41251 effect completely in the T4-treated cells and partially in the milrinone-treated cells. It is presumed that PMA and CGP-41251 are mutually inhibitory (22) but permit endogenous diacylglycerol, generated during exposure of cells to thyroid hormone, to activate PKC, since the affinity of PKC for diacylglycerol is 250-fold that for phorbol ester (15).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of CGP-41251 and phorbol 12-myristate 13-acetate (PMA) on T4 and milrinone potentiation of antiviral effect of IFN-gamma . CGP-41251 (CGP, 5 × 10-10 M) and PMA (10-7 M) were added individually or together during T4 or milrinone preincubations. Medium was then removed and replaced with fresh medium for IFN-gamma incubation. CGP impaired potentiating effect of both T4 and milrinone (*** P < 0.001), as did PMA (not significant with T4; * P < 0.05 with milrinone). When added together, effects of CGP and PMA were partially or completely neutralized. Neither CGP nor PMA altered antiviral effect of IFN-gamma alone (not shown).

A similar pattern was seen in studies utilizing the PKA inhibitor KT-5720 and agonist 8-BrcAMP (Fig. 7). Partial or complete inhibition of the potentiation by T4 and milrinone was seen when cells were also pretreated with KT-5720 (5 × 10-7 M). Preincubation with 8-BrcAMP did not affect potentiation of antiviral effect by T4 or milrinone, but 8-BrcAMP did reverse the action of KT-5720 in the presence of T4 and partially reversed the KT-5720 effect on milrinone potentiation. Neither KT-5720 nor 8-BrcAMP, in the absence of T4, had any effect on the subsequent antiviral effect of IFN-gamma (results not shown).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of KT-5720 and 8-bromo-cAMP (8-BrcAMP) on T4 and milrinone potentiation of antiviral action of IFN-gamma . KT-5720 (KT, 5 × 10-7 M) and 8-BrcAMP (8-Br, 10-3 M) were added individually or together during T4 or milrinone preincubations. Both T4 and milrinone enhanced antiviral action of IFN-gamma significantly (*** P < 0.001). KT blocked potentiating effects of T4 and milrinone (* P < 0.05; ** P < 0.01), whereas 8-Br had no effect. 8-Br did, however, partially or completely reverse effect of KT. Neither KT nor 8-Br had any effect on antiviral action of IFN-gamma alone (not shown).

We also tested whether the two kinase agonists, PMA and 8-BrcAMP, if added together to cells for 24 h before IFN-gamma treatment could simulate the effects of T4 and milrinone. Although each agonist alone did not potentiate the IFN-gamma effect, the two together did enhance the antiviral action (results not shown), suggesting that T4 and milrinone exert their effects by stimulating activities of both PKC and PKA and that both kinase activities are necessary for potentiation.

The effects of the tyrosine kinase inhibitor, genistein (17), on T4 and milrinone action are shown in Fig. 8. Genistein was an effective concentration-dependent inhibitor of T4 potentiation when both were added during the 24-h preincubation. On the other hand, genistein had no effect on the potentiation of the antiviral action of IFN-gamma by milrinone. Genistein did not inhibit the antiviral effect of IFN-gamma alone (not shown), since the inhibitor was washed out before the IFN-gamma treatment. Thus tyrosine phosphorylation also plays a role in potentiation by thyroid hormone, but milrinone's action does not involve that signaling mechanism.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 8.   Inhibition by genistein of potentiation by T4 but not milrinone of antiviral action of IFN-gamma . Increasing concentrations of genistein caused progressive loss of T4 potentiation but no loss of milrinone potentiation. Significant effect of T4 or milrinone or loss of potentiation due to genistein: ** P < 0.01; *** P < 0.001.

Comparison of ligand binding and transcriptional effects of T3 and milrinone. To examine whether milrinone interacts with nuclear TRs, we compared the ligand binding and transcriptional effects of milrinone with those of T3. E. coli-expressed TR-beta was incubated with 10-9 M [125I]T3 and increasing concentrations of T3 or milrinone were added (Fig. 9). A three-fold excess of unlabeled T3 decreased [125I]T3 binding by 50%, whereas a 300-fold excess of milrinone had no significant effect on labeled T3 binding.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 9.   Comparison of ligand binding effects of T3 and milrinone. Tracer [125I]T3 concentration was 10-9 M. T3 binding to thyroid hormone receptor-beta (TR-beta ) was decreased 50% by a 3-fold excess of unlabeled T3, whereas a 300-fold excess of milrinone had no significant effect on hormone binding.

CV-1 cells, which lack TR (27), were cotransfected with TR-beta expression plasmid and a TRE-containing reporter plasmid F2, and the effects of T3 and milrinone were examined (Fig. 10). In the absence of T3, unliganded TR-beta repressed basal transcription as previously reported (2, 26, 28). T3 in concentrations of 10-6 and 10-8 M stimulated transcription four- and twofold above basal levels, respectively. Milrinone (10-6 M) had no effect on basal repression and did not stimulate transcription. Additionally, 10-6 M milrinone did not block transcriptional activation by 10-8 M T3. Similar results were also observed for these ligands when CV-1 cells were cotransfected with TR-alpha expression plasmid and F2 reporter (data not shown).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 10.   Comparison of effects of T3 and milrinone on transcription of TR-beta . Both 10-6 and 10-8 M T3 stimulated transcription. Milrinone (10-6 M) had no effect on transcription and did not block effect of T3, 10-8 M. Data are shown as means ± SD.

Additional studies were performed to examine the effect of TETRAC on transcriptional activation of TR-beta . TETRAC allowed derepression of basal transcriptional activity and generated weak agonist activity (results not shown), whereas, in additional studies, we have demonstrated that TRIAC is equipotent with T3 in functional studies with TR-beta . Our findings suggest that TETRAC can bind to nuclear TR and function as a weak agonist in contrast to milrinone, which does not bind to TR. However, it should be noted that, in the potentiation of the antiviral action of IFN-gamma , neither TETRAC nor TRIAC is an agonist.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present studies, we examined the signal transduction pathways of a recently recognized action of thyroid hormone in human cells, namely, the potentiation by T4 and T3 of the antiviral activity of homologous IFN-gamma (11, 13). This is a complex model of hormone action, in that T4 and T3 can potentiate the action of IFN-gamma by two mechanisms, one that requires protein synthesis and a second, postnuclear pathway that is independent of protein synthesis (11). These pathways are depicted in Fig. 11. The first pathway is susceptible to stimulation by T4, T3, and rT3 and is blocked by TETRAC, whereas the postnuclear pathway is unresponsive to rT3 and is unaffected by TETRAC (11). Both pathways are more responsive to T4 than T3 in physiological concentrations, and the effect of T4 is not altered by coincubation of cells with PTU. We have demonstrated this potentiation by thyroid hormone in HeLa and CV-1 cells (H.-Y. Lin, unpublished observations); HeLa cells, like CV-1 cells, are deficient in TR (19, 27).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 11.   Proposed protein synthesis-dependent mechanism by which thyroid hormone potentiates antiviral action of IFN-gamma . In absence of thyroid hormone, IFN-gamma , via a cell surface receptor, induces phosphorylation of the signal transduction and activation of transcription protein STAT1alpha by tyrosine kinase activity of Janus kinases (JAK1 and JAK2) (4, 8, 18). Dimerized STAT1alpha translocates to cell nucleus and binds to an IFN-gamma -activated sequence element in promoter region of an IFN-gamma -responsive gene, leading to induction of antiviral proteins (ER, endoplasmic reticulum). Maximal activity of STAT1alpha is achieved when both tyrosine (Tyr) and serine (Ser) residues are phosphorylated (25), but tyrosine phosphorylation is sufficient to induce antiviral activity. In present studies, potentiation by thyroid hormone of IFN-gamma action is shown to require PKC and PKA (serine kinase) activities as well as tyrosine kinase and to be inhibited by TETRAC. It is possible that T4 induces tyrosine phosphorylation of STAT1alpha , but hormone alone is not antiviral. We postulate that T4-induced PKC and PKA activities relate to serine phosphorylation of STAT1alpha . Inhibition by genistein of hormone action but not IFN-gamma action implicates tyrosine phosphorylation in activation of serine kinase(s) relevant to STAT1alpha rather than direct stimulation of tyrosine phosphorylation of STAT1alpha . Milrinone is shown here to require serine kinase but not tyrosine kinase activity and to effect potentiated antiviral state by a pathway that is not sensitive to TETRAC. A second pathway, postnuclear and CHX insensitive, by which T4 enhances antiviral action of IFN-gamma has previously been described (12) and is also shown here.

The use of milrinone, which shares structural homologies with thyroid hormone (14), allowed us to further characterize the interaction of iodothyronines with the IFN-gamma signal transduction pathway. The experiments described here showed that milrinone, at concentrations that are achieved clinically, also enhanced the antiviral action of IFN-gamma by a protein synthesis-dependent mechanism. This action mimicked that of T4, T3, and rT3. Like thyroid hormone, milrinone had no antiviral activity in the absence of IFN-gamma . Milrinone was inactive in the protein synthesis-independent pathway, and amrinone was wholly inactive in both protein synthesis-dependent and -independent experimental paradigms. The difference in activities of the two bipyridines was not surprising, since X-ray crystallographic analysis has shown that the ring structure of milrinone, but not amrinone, resembles that of iodothyronines (14).

Our observations show that activities of PKC and PKA together are necessary components of the protein synthesis-dependent potentiation of IFN-gamma 's action by thyroid hormone and milrinone. PMA and 8-BrcAMP, when added concurrently but not separately, partially reproduced the IFN potentiation achieved with thyroid hormone and the bipyridine. That the roles of PKC and PKA in the protein synthesis-dependent and -independent pathways are different is shown by the fact that milrinone, though requiring PKC and PKA in the protein synthesis-dependent pathway, is inactive in the postnuclear pathway.

Although milrinone and thyroid hormone have structural homologies and both depend for their action on PKC and PKA in these studies, they do not enhance the action of IFN-gamma in the protein synthesis-dependent pathway by a wholly identical mechanism. That this is the case was shown by the ability of two deaminated analogues of thyroid hormone, TETRAC and TRIAC, to block the action of T4 but not that of milrinone. This is likely to represent inhibition by TETRAC and TRIAC of binding of T4 at an extranuclear site that is linked to kinase activation. We have previously shown that TETRAC blocks T4 binding to erythrocyte membranes and also inhibits T4 activation of membrane Ca2+-ATPase but does not itself increase Ca2+-ATPase activity (6). Furthermore, genistein, an inhibitor of tyrosine kinases (17), inhibited thyroid hormone action but did not affect the potentiating activity of milrinone. The sites in a signal transduction pathway at which TETRAC and genistein effect their actions must be proximal to PKC and PKA activation, or both TETRAC and genistein would also inhibit milrinone's action. Additional support for this conclusion was provided by experiments in which a full milrinone response was achieved in the presence of T4 with a T4-inhibiting concentration of TETRAC.

The apparent dependence of thyroid hormone action on a mechanism that involves tyrosine kinase activity as well as serine-threonine kinases (PKC and PKA) suggests that thyroid hormone action is mediated via the signal transduction pathway that includes activities of the Janus kinase (JAK) family of tyrosine kinases and proteins involved in signal transduction and activation of transcription (STAT proteins). This pathway has been established as a major mechanism for intracellular signaling initiated by many cytokines including the IFNs (4, 8, 18). Exposure of the cellular IFN-gamma receptor to IFN-gamma results in receptor tyrosine phosphorylation and activation of JAK1 and JAK2 by tyrosine transphosphorylation. Subsequently, the 91-kDa protein STAT1alpha is also activated by tyrosine phosphorylation, leading to homodimerization, nuclear translocation of the dimer, and ultimately binding to the IFN-gamma response elements on target DNA called IFN-gamma activation sites. Results of the present studies are consistent with the hypothesis that one of the actions of thyroid hormone in the HeLa cell model is stimulation of tyrosine kinase activity in the JAK-STAT pathway.

We also postulate that the PKC- and PKA-requiring pathway which we have demonstrated to be shared by the actions of milrinone and thyroid hormone, distal to steps influenced by deaminated iodothyronines and by genistein, involves further phosphorylation of STAT1 on serine residues, resulting in heightened binding affinity of the STAT1-STAT1 dimer for promoter regions on IFN-gamma -responsive genes. Wen et al. (25) have shown that both tyrosine and serine phosphorylation are necessary for maximal activation of transcription by STAT1. We do not believe that T4 acts via a traditional nuclear TR in this IFN-gamma -HeLa cell model but, rather, that IFN-gamma potentiation is achieved by a thyroid hormone-directed protein kinase cascade, which is summarized in Fig. 11.

Although these studies were carried out in a cell line deficient in TR, we nonetheless pursued the possibility that milrinone and TETRAC were capable of interacting with TR. The lack of interaction of TETRAC with TR-beta is consistent with the action of TETRAC in the antiviral studies at an extranuclear binding site for thyroid hormone. We studied the possibility that milrinone could bind to nuclear receptors for thyroid hormone and that such receptors were associated with potentiation of the cellular antiviral response directed by IFN-gamma . Milrinone, however, was found not to compete with T3 for binding sites on bacterially expressed TR-beta . In addition, milrinone did not transactivate or block T3-mediated transcriptional activity measured via the TRE F2 that was cotransfected with TR-alpha or TR-beta . These results indicate that milrinone 1) does not have sufficient structural homology with thyroid hormone to permit binding of the bipyridine to a principal nuclear receptor for T3 and 2) does not potentiate the antiviral activity of IFN-gamma by means of a mechanism dependent on TR-alpha or TR-beta .

    ACKNOWLEDGEMENTS

We thank Jeannie Whang and Dr. Ying Liu for valuable technical assistance in ligand binding and cotransfection studies.

    FOOTNOTES

This work was supported in part by the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P. J. Davis), and by a grant from the Candace King Weir Foundation.

Address for reprint requests: P. J. Davis, Dept. of Medicine A-57, Albany Medical College, Albany, NY 12208.

Received 11 March 1997; accepted in final form 21 May 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Baim, D. S., A. V. McDowell, J. Cherniles, E. S. Monrad, J. A. Parker, J. Edelson, E. Braunwald, and W. Grossman. Evaluation of a new bipyridine inotropic agent---milrinone---in patients with severe congestive heart failure. N. Engl. J. Med. 309: 748-756, 1983[Abstract].

2.   Brent, G. A., M. K. Dunn, J. W. Harney, T. Gulick, P. R. Larsen, and D. D. Moore. Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor. New Biologist 1: 329-336, 1989[Medline].

3.   Caravatti, G., T. Meyer, A. Fredenhagen, U. Trinks, H. Mett, and D. Fabbro. Inhibitory activity and selectivity of staurosporine derivatives towards protein kinase C. Bioorg. Med. Chem. Lett. 4: 399-404, 1994.

4.   Darnell, J. E., Jr., I. M. Kerr, and G. R. Stark. Jak-STAT pathway and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415-1421, 1994[Medline].

5.   Davis, P. J., V. Cody, F. B. Davis, P. R. Warnick, M. Schoenl, and L. Edwards. Competition of milrinone, a non-iodinated cardiac inotropic agent, with thyroid hormone for binding sites on human serum prealbumin (TBPA). Biochem. Pharmacol. 36: 3635-3640, 1987[Medline].

6.   Davis, P. J., F. B. Davis, and S. D. Blas. Studies on the mechanism of thyroid hormone stimulation in vitro of human red cell Ca2+-ATPase activity. Life Sci. 30: 675-682, 1982[Medline].

7.   Honerjager, P. Pharmacology of bipyridine phosphodiesterase III inhibitors. Am. Heart J. 121: 1939-1944, 1991[Medline].

8.   Ihle, J. N., B. A. Witthuhn, F. W. Quelle, K. Yamamoto, W. E. Thierfelder, B. Kreider, and O. Silvennoinen. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem. Sci. 19: 222-227, 1994[Medline].

9.   Kase, H., K. Iwahashi, S. Nakanishi, Y. Matsuda, K. Yamada, M. Takahashi, C. Murakata, A. Sato, and M. Kaneko. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem. Biophys. Res. Commun. 142: 436-440, 1987[Medline].

10.   Krieger, N. S., T. S. Stappenbeck, and P. H. Stern. Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture. J. Clin. Invest. 79: 444-448, 1987[Medline].

11.   Lin, H.-Y., H. R. Thacore, F. B. Davis, and P. J. Davis. Thyroid hormone analogues potentiate the antiviral action of interferon-gamma by two mechanisms. J. Cell. Physiol. 167: 269-276, 1996[Medline].

12.   Lin, H.-Y., H. R. Thacore, F. B. Davis, and P. J. Davis. Potentiation by thyroxine of interferon-gamma -induced antiviral state requires PKA and PKC activities. Am. J. Physiol. 271 (Cell Physiol. 40): C1256-C1261, 1996[Abstract/Free Full Text].

13.   Lin, H.-Y., H. R. Thacore, P. J. Davis, and F. B. Davis. Thyroid hormone potentiates the antiviral action of interferon-gamma in cultured human cells. J. Clin. Endocrinol. Metab. 79: 62-65, 1994[Abstract].

14.   Mylotte, K. M., V. Cody, P. J. Davis, F. B. Davis, S. D. Blas, and M. Schoenl. Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies. Proc. Natl. Acad. Sci. USA 82: 7974-7978, 1985[Abstract].

15.   Newton, A. C. Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270: 28495-28498, 1995[Free Full Text].

16.   Petty, K. J. Tissue- and cell-specific distribution of proteins that interact with the human thyroid hormone receptor-beta . Mol. Cell. Endocrinol. 108: 131-142, 1995[Medline].

17.   Ryu, K., Y. Koide, Y. Yamashita, and T. O. Yoshida. Inhibition of tyrosine phosphorylation prevents IFN-gamma -induced HLA-DR molecule expression. J. Immunol. 150: 1253-1262, 1993[Abstract/Free Full Text].

18.   Schindler, C., and J. E. Darnell, Jr. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64: 621-651, 1995[Medline].

19.   Selmi, S., and H. H. Samuels. Thyroid hormone receptor and v-erb A. A single amino acid difference in the C-terminal region influences dominant negative activity and receptor dimer formation. J. Biol. Chem. 266: 11589-11593, 1991[Abstract/Free Full Text].

20.   Spanjaard, R. A., D. S. Darling, and W. W. Chin. Ligand-binding and heterodimerization activities of a conserved region in the ligand-binding domain of the thyroid hormone receptor. Proc. Natl. Acad. Sci. USA 88: 8587-8591, 1991[Abstract].

21.   Suen, C.-S., P. M. Yen, and W. W. Chin. In vitro transcriptional studies of the roles of the thyroid hormone (T3) response elements and the minimal promoters in T3-stimulated gene transcription. J. Biol. Chem. 269: 1314-1322, 1994[Abstract/Free Full Text].

22.   Van der Bruggen, T., P. T. M. Kok, M. Blom, A. J. Verhoeven, J. A. M. Raaijmakers, J.-W. J. Lammers, and L. Koenderman. Transient exposure of human eosinophils to the protein kinase C inhibitors CGP39-360, CGP41-251 and CGP44-800 leads to priming of the respiratory burst induced by opsonized particles. J. Leukoc. Biol. 54: 552-557, 1993[Abstract].

23.   Warnick, P. R., P. J. Davis, F. B. Davis, V. Cody, J. Galindo, Jr., and S. D. Blas. Rabbit skeletal muscle sarcoplasmic reticulum Ca2+-ATPase activity: stimulation in vitro by thyroid hormone analogues and bipyridines. Biochim. Biophys. Acta 1153: 184-190, 1993[Medline].

24.   Weinstein, S. P., J. P. Watts, P. N. Graves, and R. S. Haber. Stimulation of glucose transport by thyroid hormone in ARL 15 cells: increased abundance of glucose transporter protein and messenger ribonucleic acid. Endocrinology 126: 1421-1429, 1990[Abstract].

25.   Wen, Z., Z. Zhong, and J. E. Darnell, Jr. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82: 241-250, 1995[Medline].

26.   Yen, P. M., M. Ikeda, J. H. Brubaker, M. Forgione, A. Sugawara, and W. W. Chin. Roles of v-erb A homodimers and heterodimers in mediating dominant negative activity by v-erbA. J. Biol. Chem. 269: 903-909, 1994[Abstract/Free Full Text].

27.   Yen, P. M., E. C. Wilcox, Y. Hayashi, S. Refetoff, and W. W. Chin. Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants. Endocrinology 136: 2845-2851, 1995[Abstract].

28.   Zhang, X. K., K. N. Wills, G. Graupner, M. Tzukerman, T. Hermann, and M. Pfahl. Ligand-binding domain of thyroid hormone receptors modulates DNA binding and determines their bifunctional roles. New Biologist 3: 169-181, 1991[Medline].


AJP Cell Physiol 273(4):C1225-C1232