Biochimica et Biophysica Acta, 1093(1991) 223-228 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100204W
223
BBAMCR 12959
Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils Kunihiko Aoyagi, Koichiro Takeshige and Shigeki Minakami Department of Biochemistry, Kyushu Unit'ersitySchool of Medicine, Fukuoka (Japan) (Received 2 November 1990)
Key words: N-Formylmethionylleucylphenylalanine;Chemotactic peptide receptor; lodothyronine; Superoxide; (Human neutrophil)
We studied the action of iodinated thyronines on the superoxide (02-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucyiphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced Oz" production with ICso values of about 10-6 M and 7 • 10-6 M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the Oz._ production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O ~ production stimulated with either the fifth component of'the complement (CSa) or phorbol 12-myristate 13-acetate. L-Thyroxine and L.triiodothyronine were found to block [3H]FMLP binding to its own receptor with ICso values similar to those for the inhibition of the Oz" production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O Z production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.
Introduction
Neutrophils exposed to a chemotactic peptide Nformylmethionylleueylphenylalanine (FMLP) elicit superoxide (O~) production [1] as well as chemotaxis and the release of granule enzymes [2]. These cellular responses are mediated by binding of FMLP to specific receptors on the plasma membrane of the ceils [3]. The signal transduction induced by FMLP involves an increase of the intracellular Ca 2+ concentration [4,5] and the activation of protein kinase C [6], which is considered to induce the activation of the NADPH-O~- generating oxidase [7,8]. Additionally, other pathways for the activation are also reported [9,10].
Abbreviations: FMLP, N.formylmethionylleucylphenylalanine;C5a, the fifth component of complement; PMA, phorbol 12-myristate 13-acetate; O~-, superoxide; IC~, the concentration causing 50% inhibition; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Correspondence: Kunihiko Aoyagi, Department of Biochemistry, Kyushu University School of Medicine, Maidashi 3-1-1, Fukuoka 812, Japan.
It is generally assumed that most of the biological effects of iodothyronines are mediated through the selective binding of 3,5,3'-triiodothyronine to nuclear receptors and subsequent protein synthesis as a hormonal action, but several investigators have demonstrated that iodothyronines can also initiate cellular responses through an interaction with extranuclear components in the plasma membrane [11,12] and mitochondria [13]. The compounds are shown in vitro to inhibit aggregation and serotonin release of human platelets induced by collagen [14] and to stimulate human erythrocyte Ca2+-ATPase activity [15]. Thyroxine is more effective on these blood cells than triiodothyronine. The action of iodothyronines on neutrophils, however, has not been fully studied. The present study was undertaken to investigate the effects of iodothyronines on human neutrophils stimulated with FMLP in vitro. Thyroxine and triiodothyronine inhibited dose dependently the O~ production induced by the peptide, but did not inhibit the production induced by the fifth component of the complement (C5a), which also acts through binding to its receptors, or by phorbol 12-myristate 13-acetate, an activator of protein kinase C. They also decreased the binding of
224 the peptide to its receptor with IC50 values similar to those for the inhibition of the O~ production. These results suggest that thyroxine and triiodothyronine modulate the O~ production of neutrophils by changing the binding of the peptide to the receptor, and iodothyronines might be useful for characterizing the metabolic changes of human neutrophils stimulated by FMLP. Materials and Methods
Reagents L-Thyroxine, D-thyroxine, 3,5,3'-triiodo-L-thyronine, 3,5,3'-triiodo-D-thyronine, DL-thyronine, 3,5-diiodo-Ltyrosine, D,L-tyrosine and 5,5'-dithiobis(2-nitrobenzoic acid) were purchased from Nacalai tesque, inc., Japan, and 3,5-diiodo-L-thyronine and cytochrome c (type III) was from Sigma Chemical Co., St. Louis, MO, U.S.A. Glutathione reductase (yeast) was from Boehringer Mannheim, F.R.G., and N-forrnylmethionylleucylphenylalanine (FMLP), the synthetic tripeptide, was from Peptide Institute, Inc., Japan. Human CSa was a generous gift from Dr. T.E. Hugli (Research Institute of Scripps Clinic, CA, U.S.A.) through Dr. M. Abe (Research Institute for Disease of the Chest, Faculty of Medicine, Kyushu University, Japan). Dibutyl phthalate and dinonyl phthalate were purchased from Wako Pure Chemical, Osaka, Japan, Bio-Gel P-2 was from Bio-Rad, and [3H]FMLP (53.6 Ci/mmol) was from New England Nuclear, Boston, MA, U.S.A. Other reagents were of analytical grade. Thyroxine, triiodothyronine, thyronine and tyrosine were dissolved in a solvent containing dimethyl sulfoxide (10%, v/v) and 50 mM NaOH. Diiodothyronine was dissolved in a solvent containing 15 mM HCI instead of NaOH. Diiodotyrosine was dissolved in 50 mM NaOH. All stock solutions were stored at - 20 ° C. The solvents used had no significant effect on the FMLP-indueed O i production.
Cell preparation Human neutrophils were isolated from healthy volunteers by dextran sedimentation, hypotonic lysis and the Conray-FicoU density gradient centrifugation as described previously [16]. The population of cell types were more than 95% neutrophils and less than 5% lymphocytes, monocytes or eosinophils.
Assay of the superoxide production The activity to release superoxide was measured by the reduction of eytochrome c at 550-540 nm using a
dual-wavelength spectrophotometer (Hitachi 557) as described previously [17]. The cells were suspended in 1 ml of a Hepes-buffered saline containing 135 mM
NaC1, 5 mM KCI, 5 mM glucose and 20 mM Hepes (pH 7.4). Before the addition of a stimulant, the mixture was preineubated with 1 mM CaCI z and 50/zM cytochrome c for 10 min at 37 ° C.
Reversibility of the inhibition 1 ml of neutrophil suspension (2.106 cells) was incubated with or without L-thyroxine (10 /zM) for 2 rain at 37 ° C, and then washed by the addition of 9 ml of the ice-cold Hepes-buffered saline containing 1 mM CaCI2. The suspension was centrifuged at 500 x g for 30 s at 4 ° C and 9 ml of the supernatant was aspirated. The washing procedure was repeated as indicated, and the cells were then resuspended in the assay system of the O~ production.
[ 3H]FMLP binding assay Binding of [3H]FMLP to neutrophils was measured by centrifuging the ceils through an immiscible layer [18,19]. A 200 /zl aliquot of neutrophil suspension (5.106 ceUs/ml) containing 1 mM CaCi 2 was incubated for 2 min at 4°C with L-thyroxine followed by the addition of [3H]FMLP and the incubation for 60 min at 4 *C with occasional shaking. The cell suspension was transferred to a microtube containing 6 M urea (10 /zl, bottom layer) and a mixture of dibutyl phthalate and dinonyl phthalate (10:3, v/v) (200/zl, upper layer), and centrifuged for 5 rain at 10000 x g. The cells at the bottom of each tube were cut off and placed in a liquid scintillation vial for radioactivity determination. Nonspecific binding of [3H]FMLP was determined in the presence of 10 -5 M unlabelled FMLP for each concentration of [3H]FMLP.
Molecular sieve chromatography Chromatographic analyses of FMLP were performed essentially as described by Stenson et al. [20] using a Bio-Gel P-2 column. A mixture of [3H]FMLP (40 nM) with or without L-thyroxine (10 /.~M) in the Hepes-buffered saline (pH 7.4) was mixed with unlabelled FMLP to achieve the FMLP concentration at 1 /zM. A 200/zl aliquot of the mixture was applied to a Bio-Gel P-2 column (1 × 18 cm) and eluted with the same buffer. The flow rate was 4.5 m l / h and V0 was 5.4 ml. Fractions of 0.9 ml were collected and the radioactivity was measured in a liquid scintillation counter. Molecular weight standards are oxidized glutathione (612), reduced glutathione (307), and sodium azide (65). They were determined as follows: oxidized and reduced glutathione were detected using 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence and absence of glutathione reductase, respectively [21] and sodium azide was detected by measuring electroconductivity.
225 Results
The effect of thyroxine on the stimulation of 0 2 production Pretreatment of human neutrophils with L-thyroxine abolished the FMLP-induced 0 2 production and the ongoing O 2 production induced by FMLP was also rapidly inhibited by the addition of 5 p M L-thyroxine (Fig. 1A). L-Thyroxine, however, had no significant effect on C5a-stimulated 0 2 production at concentrations lower than 30/~M (Fig. 1B). L-Thyroxine did not act at a site following protein kinase C, because it did not affect 0 2 production stimulated with phorbol 12myristate 13-acetate (PMA), which directly activates protein kinase C (Fig. 1C). These findings indicate that the effects of the compound on FMLP-induced 0 2 production are not toxic. This is supported by the results that iodothyronines at the concentrations used did not alter cell viability, which was determined by Trypan blue dye exclusion. Furthermore, the leakage of lactate dehydrogenase was measured and was less than 5% of the total cell content in the presence of the compounds. The inhibition of FMLP-induced 0 2 production by L-thyroxine was dose dependent and the concentration of L-thyroxine required for the IC50 of 0 2 production stimulated by 1/.~M FMLP was approx. 10 -6 M (Fig. 2). L-Triiodothyronine also prevented FMLP-induced 0 2 production in a dose-dependent manner with an IC50 value of 7 . 1 0 - 6 M. Fig. 3 shows that the effects of various concentrations of the peptide on O 2 production were inhibited by L-thyroxine.
(A)
(B)
(C)
T
9
100
0 0
80+
~
60,
G
A
C
.o ~
"0
40,
P
20.
b
0
T -4
-5
Iodothyronines (IogM)
Fig. 2. Dose-dependent inhibition of the FMLP-induced O~ production by iodothyronines. Neutrophils (2.106 eeils/ml) were treated with L-thyroxine (o) or L-triiodothyronine (e) at various concentrations for 2 rain at 37 ° C, and then 1 ~,M FMLP was added. Each point represents the mean of five experiments. The results are presented as percent of the control value (7.02+0.55 nmol/min per 2. l0 n cells).
Inhibition of the FMLP-induced 0~- production by carious iodothyronines and their structural analogues The effects of various iodothyronines and their structural analogues on the FMLP-induced 0 2 production were examined (Table I). O,L-Thyroxine, at a concentration of 10 #M, inhibited 0 2 production completely and the same concentration of O,L-triiodothyronine inhibited the production by 60-70%. The related compounds including L-diiodothyronine, thyronine, L-diiodotyrosine and O,L-tyrosine, however, had no inhibitory effects on the O~- production. There was no difference between the abilities of the o and L forms to inhibit the O~ producticm, but there was a marked difference between triiodc~thyronine and diiodothyronine, suggesting that the inhibitory potencies depend mainly on the number of iodines. Reversibility of the inhibitory effect When neutrophils were incubated with thyroxine and then washed with the Hepes-buffered saline to
0,02 A
i
A
~ f . - - ' ' "
"6 4. U
;:
1 pM FMLP
::
2 rain
t
20 ng/ml CSa
I
10 ng/rnl PMA
Fig. 1. Effect of L-thyroxine on the O~- production by human neutrophils stimulated with FMLP, C5a or PMA. Neutrophils, as indicated cell counts, were preincubated with 1 mM CaCI: for 10 rain at 37 ° C. The cells were treated with or without L-thyroxine for 2 rain at 37 ° C and then stimulated. (A) 1/zM FMLP was added to the cell suspension (106/ml) (curve l), 1 /~M FMLP was added 1 min prior to 5/xM L-thyroxine (arrow T) (curve 2), 5/~M L-thyroxine was added 2 rain prior to 1 /zM FMLP (curve 3). (B) Either 20 ng/ml C5a alone (. . . . . . ) or 5 p M L-thyroxine 2 rain prior to 20 ng/ml CSa ( ) was added to the cell suspension (4.106/ml). (C) Either 10 ng/ml PMA alone (. . . . . . ) or 5/zM L-thyroxine 2 rain prior to 10 n g / m l PMA ( ) was added to the cell suspension (106/ml).
=% 0 'ID "
0 •8
-7
-6
-5
FMLP (logM)
Fig. 3. Effect of L.thyroxine on O~ production by human neutrophils exposed to different concentrations of FMLP. Neutrophils (2.10 +' cells/ml) were treated with the indicated concentrations of L-thyroxine: none (O), 1 /~M (o), 2.5 p.M (t:3) and 5 p M (Ill), and 2 rain later different doses of FMLP were added. Each point represents the mean of duplicate determinations.
226 TABLE I
o lOO-
Inhibition of FMLP-induced 0 2 production by iodothyronines and their structural analogues Neutrophils (2-106 cells/ml) were incubated with iodothyronines or their analogues at a concentration of 10 /~M for 2 rain at 37°C followed by the addition of 1 tzM FMLP. Each value represents the mean+S.D, of four experiments. The results are presented as a percent of the control value (5.43+_0.43 nmol/min per 2.106 cells). lnhibitors
% of control
L-Thyroxine D-Thyroxine t.-Triiodothyronine D-Triiodothyronine t.-Diiodothyronine Thyronine L-Diiodotyrosine t.-Tyrosine D.Tyrosine
0.6+ 0.4 0.7+ 0.3 36.9+ 5.4 32.5 + 7.5 106.5 :l: 4.5 93,1 + 6.2 100.6 + 6.2 96.3 + 6.7 93.8 ± 10.4
remove L-thyroxine, inhibition of 0~" production revealed restoration (Fig. 4). By washing three times, the rate of the O~ production recovered to approximately 80% of the control.
[ 3H]FMLP binding studies To determine the site at which L-thyroxine and L-triiodothyronine interrupt the signal transduction induced by FMLP, we examined the effects of the iodinated compounds on the specific binding of [3H]FMLP to the neutrophils. As shown in Fig. 5, the compounds ir/,i;ited the binding of [aH]FMLP to the cells. [aH]FMLP binding was reduced by 50% at about 10 -6 M for L-thyroxine and at about 10 -5 M for L-triiodothyronine. L-Thyroxine reduced [3H]FMLP binding with a similar dose response at 37 °C as that at 4 °C (results not shown). A representative result in the Scatchard plots [22] showed the affinity of the receptor
+ ~
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40 -
.~_
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z,o-
.~
o
.,+
.;
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,
+5
.4
Iodothyronlnes (IogM) Fig. 5. Effects of various concentrations of iodothyronines on the binding of ['~H]FMLP. Neutrophils (10~ cells/200/zl) were preincubated with various concentrations of L-thyroxine (O) or L-triiodothyronine (e) for 2 rain at 4°C followed by the addition of [3H]FMLP (40 riM). The cells were incubated for 60 rain at 4°C, and then separated from unbound [3H]FMLP, Specific [aH]FMLP binding in the presence of various concentrations of the iodothyronines was expressed as a percent of the binding observed in the absence of the iodothyronines. Each point represents the mean of triplicate determinations.
to the ligand changed by L-thyroxine (Fig. 6). Binding affinities and the numbers of surface FMLP receptors per cell in the control and L-thyroxine-pretreated cells were 36.2 nM and 69.3 nM, and 22700 and 22200, respectively. Thus, the inhibition of [aH]FMLP binding to neutrophils by L-thyroxine seems mainly to be due to a decrease in the affinity of the receptor to the ligand.
Molecular sieve chromatography The possibility remains that L-thyroxine directly interacts with the peptide leading to a decrease in the formation of the ligand-receptor complex. This possibility was ruled out by the analyses using gel filtration
s
lOO(
~ 0
o
.
~
~.
i 0
,
0
Number o f wash
Fig. 4, Inhibition by L-thyroxine of FMLP-induced O~ production was reversed by washing, Neutrophiis (2. ]06 cells/m]) were incubated with (e) or without (o) 10 #M L-thyroxine for 2 rain at 37 ° C. The cells were washed once, twice or three times with ice-cold Hepes-buffered saline to remove L-thyroxine, and then the O~ production by FMLP (1 p,M) was determined. Each point represents the mean of three experiments. The results are presented as a percent of the control value (5.70+0.25 nmol/min per 2.106 cells).
.
,
1000
2000
Bound (CPM)
Fig. 6. Scatchard analysis of the specific binding of [3H]FMLP to human neutrophils. Neutrophils (106 cells/200 p,I) were pteincubated with (e) or without (o) L-thyroxine (1 p,M) for 2 rain at 4°C followed by the addition of various concentrations of [3H]FMLP. The cells were incubated for 60 min at 4 ° C and then separated from unbound [3H]FMLP. Each point represents the mean of triplicate determinations. The data are representative of three separate experiments.
227 chromatography (results not shown). When [3H]FMLP alone was applied to a Bio-Gel P-2 column, the radioactivity eluted at the No. 11 fraction as a single peak. When L-thyroxine alone was applied and monitored at 325 nm, no peak was detected - probably due to the hydrophobic interaction of the compound with the gel. If L-thyroxine binds with [3H]FMLP, the elution profile of the radioactivity would be expected to change compared to that of [3H]FMLP alone: the peak of the radioactivity may be retarded because the complex interacts with the gel or may be accelerated because the complex possesses a larger molecular weight than [ 3H]FMLP alone. The same count of radioactivity eluted as a single peak at the No. 11 fraction when the mixture of labelled FMLP and L-thyroxine was applied to the column, indicating that FMLP does not form a complex with L-thyroxine. This is supported by the finding that non-specific binding of [3H]FMLP to neutrophils was not affected by L-thyroxine. Discussion
In the present work we demonstrated that thyroxine and triiodothyronine inhibited the respiratory burst of human neutrophils stimulated with FMLP, but not with C5a or PMA. L-Thyroxine also prevented the FMLP-induced Increase in intracellular free Ca 2+ concentrations using the fluorescence probe fura-2 [23] (results not shown). Additionally 10 /~M L-thyroxine reduced the lysozyme release in response to 1 p.M FMLP by 85.1 _+3.6% (mean + S.D.; n -- 6). FMLP-induced O~- production ceased within 1 min after the addition of L-thyroxine (Fig. 1A), and the observation is consistent with the findings [24] that continuous new interactions between FMLP and receptors are required for maintaining the NADPH-O z generating oxidase in the activated state. C5a, like FMLP, has been shown to elicit various biological responses, such as ehemotaxis and 0 2 production, by acting through specific cellular receptors on the plasma membrane of neutrophils [2527]. Of two similar receptor-mediated responses, only that induced by FMLP was prevented by L-thyroxine. The order of the inhibitory potency of the iodothyronines was thyroxine > triiodothyronine, which is different from that of the binding activity to the nuclear thyroid hormone receptor (triiodothyronine > thyroxine), and there was no difference between the L- and the/)-forms. It is unlikely that protein synthesis mediated through nuclear receptors is involved in the inhibitory effects, because the inhibitions occurred within I min after the addition of FMLP and were restored by washing. A similar order of potency of iodothyronines has been reported in the regulation of certain enz'yme~. Hagiwara et al. [28] observed that O,L-thyroxine and O,L-triiodothyronine inhibit myosin light chain kinase of chicken gizzard with IC5o values of about 8/zM and
about 20/~M, respectively, with a lack of stereospecificity. It has also been demonstrated that L-thyroxine is more active than L-triiodothyronine in the stimulation of human erythrocyte Ca2+-ATPase [15] and the inhibition of rat cerebrocortical type II iodothyronine 5'-deiodinase [29]. These and our findings suggest that thyroxine acts as a more potcnt modulator of some cellular functions compared with triiodothyronine and that their stereospecifieities are not necessarily required. The evidences obtained show that the receptor for the chemotactic peptide is modulated by iodothyronines. Possible mechanisms by which the compounds could alter receptor affinity are as follows. The first possibility is that the iodinated compounds may bind the FMLP receptor near the binding site. The second is that they modulate the ligand-receptor complex by binding to membrane components other than the receptor. The third is that they may bind with the specific thyroid hormone receptor on the plasma membrane and alter the FMLP receptor function. The fourth is that they may uncouple the receptor by some direct or indirect effect from the transducing proteins such as GTP-binding proteins. Inhibition of FMLP binding to neutrophils by corticosteroids [30,31], drugs such as phenylbutazone [32,33], indomethacin [34], ibuprofen [35], sulfasalazine [20] and dielofenac [36], and ammonia [37] has, so far, been reported, although the precise mechanisms of their inhibitory effects have not been clarified. The FMLP receptor has been partially purified, characterized and shown to be an integral membrane glycoprotein [38-42]. Recently, the FMLP receptor has been cloned and sequenced [43] and its expression in Xenopus oocytes has been documented [44,45]. The molecular nature of the FMLP receptor, however, has as yet been poorly characterized. Becker et al. [46,47] have postulated that this receptor has three hydrophobic regions which interact with the methionyl, the leucyi and the aromatic ring side chain of FMLP. In our study, several structural analogues including diiodothyronine, thyronine, diiodotyrosine and tyrosine had no significant inhibitory effect in contrast to the potency of thyroxine and triiodothyronine. Thus, the number of iodines is critical for the inhibitory effects and the attachment of more than three iodines to thyronine is required for an inhibitory property. Because iodine is bulky and lipophilic, the additional attachment of iodine may alter the structural and hydrophobic character of the compounds, thereby leading to interaction with the FMLP receptor or another component responsible for the receptor affinity. The inhibitory iodothyronines are assumed to be of less physiological significance because the thyroxine concentration required for inhibition (10 -7 to 10 -5 M) was higher than that of total thyroxine (10 -s to 10 -6 M) or that of free thyroxine (10 - ~ to 10 -1° M) in the
228 serum. However, the fact that the number of iodines of the iodothyronine is related to the inhibitory property might be useful for studying the characteristics of FMLP receptors and the regulation of 0 2 production induced by FMLP. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science and Culture, of Japan. References 1 Beckcr, E,L,, Sigman, M. and Oliver, J.M, (1979) Am. J. Pathol. 95, 81-98. 2 Showell, H J., Freer, gJ., Zigmond, S,H., Sehiffmann, E., Aswanikumar, S,, Corcoran, B. and Beeker, E.L. (1976) J. Exp. Med. 143, 1154-1169, 3 Williams, L.T., Snyderman, R., Pike, M.C, and Lefkowitz, RJ. (1977) Prec. Natl. Aead. Sei. USA 74, 1204-1208. 4 Nakagawara, M., Takeshige, K., Sumimoto, H., Yoshitake, J. and Minakami, S, (1984) Biochim. Biophys. Aeta 805, 97-103. 5 Krause, K,H., Schlegel, W., Wollheim, C.B., Andersson, T., Waldvogel, F.A. and Lew, P.D. (1985) J. Clin. Invest. 76, 13481354. 6 Snyderman, R., Smith, C.D. and Verghese, M.W. (1986) J. Leuk. Biol. 40, 785-800. 7 Fujita, !., Irita, K, Takeshige, K. and Minakami, S. (1984) Biochem. Biophys. Res. Commun. 120, 318-324. 8 Fujita, !., Takeshige, K. and Minakami, S. (1986) Biochem. Pharmacol. 35, 4555-4562. 9 Billah, M.M., Eckel, S., Mullmann, T.J., Egan, R.W. and Siegel, M.I. (1989) J. Biol. Chem. 264, 17069-17077. 10 Koenderman, L., Tool, A., Roos, D. and Verhoeven, A.J. (1989) FEllS Lett. 243, 399-403. 11 Segal, J. and lngbar, S,H. (1982)J. Clin. Invest. 70, 919-926. 12 Angel, R.C., Botta, J.A. and Farias, R.N. (1989) J. Biol. Chem. 264, 19143-19146. 13 Sterling, K., Campbell, G.A. and Brenner, M.A. (1984) Acta Endocrinol. 105, 391-397. 14 Mamiya, S., Hagiwara, M., lnoue, S. and Hidaka, H. (1989) J. Biol. Chem. 264, 8575-8579, 15 Davis, P J. and Bias, S,D. (1981) Biochem. Biophys, Res. Cornmen, 99, 1073-1080. 16 Umei, T,, Takeshige, K. and Minakami, S. (1987) Bioehem. J. 243, 467-472. 17 Sumimoto, H., Takeshige, K. and Minakami, S. (V'~4) Biochim. Biophys. Acta 803, 271-277. 18 Yuo, A., Kitagawa, S., Ohsaka, A., Ohta, M., Miyazono, K., Okabe, T., Urabe, A., Saito, M. and Takaku, F. (1989) Blood 74: 2144-2149.
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223
BBAMCR 12959
Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils Kunihiko Aoyagi, Koichiro Takeshige and Shigeki Minakami Department of Biochemistry, Kyushu Unit'ersitySchool of Medicine, Fukuoka (Japan) (Received 2 November 1990)
Key words: N-Formylmethionylleucylphenylalanine;Chemotactic peptide receptor; lodothyronine; Superoxide; (Human neutrophil)
We studied the action of iodinated thyronines on the superoxide (02-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucyiphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced Oz" production with ICso values of about 10-6 M and 7 • 10-6 M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the Oz._ production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O ~ production stimulated with either the fifth component of'the complement (CSa) or phorbol 12-myristate 13-acetate. L-Thyroxine and L.triiodothyronine were found to block [3H]FMLP binding to its own receptor with ICso values similar to those for the inhibition of the Oz" production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O Z production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.
Introduction
Neutrophils exposed to a chemotactic peptide Nformylmethionylleueylphenylalanine (FMLP) elicit superoxide (O~) production [1] as well as chemotaxis and the release of granule enzymes [2]. These cellular responses are mediated by binding of FMLP to specific receptors on the plasma membrane of the ceils [3]. The signal transduction induced by FMLP involves an increase of the intracellular Ca 2+ concentration [4,5] and the activation of protein kinase C [6], which is considered to induce the activation of the NADPH-O~- generating oxidase [7,8]. Additionally, other pathways for the activation are also reported [9,10].
Abbreviations: FMLP, N.formylmethionylleucylphenylalanine;C5a, the fifth component of complement; PMA, phorbol 12-myristate 13-acetate; O~-, superoxide; IC~, the concentration causing 50% inhibition; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Correspondence: Kunihiko Aoyagi, Department of Biochemistry, Kyushu University School of Medicine, Maidashi 3-1-1, Fukuoka 812, Japan.
It is generally assumed that most of the biological effects of iodothyronines are mediated through the selective binding of 3,5,3'-triiodothyronine to nuclear receptors and subsequent protein synthesis as a hormonal action, but several investigators have demonstrated that iodothyronines can also initiate cellular responses through an interaction with extranuclear components in the plasma membrane [11,12] and mitochondria [13]. The compounds are shown in vitro to inhibit aggregation and serotonin release of human platelets induced by collagen [14] and to stimulate human erythrocyte Ca2+-ATPase activity [15]. Thyroxine is more effective on these blood cells than triiodothyronine. The action of iodothyronines on neutrophils, however, has not been fully studied. The present study was undertaken to investigate the effects of iodothyronines on human neutrophils stimulated with FMLP in vitro. Thyroxine and triiodothyronine inhibited dose dependently the O~ production induced by the peptide, but did not inhibit the production induced by the fifth component of the complement (C5a), which also acts through binding to its receptors, or by phorbol 12-myristate 13-acetate, an activator of protein kinase C. They also decreased the binding of
224 the peptide to its receptor with IC50 values similar to those for the inhibition of the O~ production. These results suggest that thyroxine and triiodothyronine modulate the O~ production of neutrophils by changing the binding of the peptide to the receptor, and iodothyronines might be useful for characterizing the metabolic changes of human neutrophils stimulated by FMLP. Materials and Methods
Reagents L-Thyroxine, D-thyroxine, 3,5,3'-triiodo-L-thyronine, 3,5,3'-triiodo-D-thyronine, DL-thyronine, 3,5-diiodo-Ltyrosine, D,L-tyrosine and 5,5'-dithiobis(2-nitrobenzoic acid) were purchased from Nacalai tesque, inc., Japan, and 3,5-diiodo-L-thyronine and cytochrome c (type III) was from Sigma Chemical Co., St. Louis, MO, U.S.A. Glutathione reductase (yeast) was from Boehringer Mannheim, F.R.G., and N-forrnylmethionylleucylphenylalanine (FMLP), the synthetic tripeptide, was from Peptide Institute, Inc., Japan. Human CSa was a generous gift from Dr. T.E. Hugli (Research Institute of Scripps Clinic, CA, U.S.A.) through Dr. M. Abe (Research Institute for Disease of the Chest, Faculty of Medicine, Kyushu University, Japan). Dibutyl phthalate and dinonyl phthalate were purchased from Wako Pure Chemical, Osaka, Japan, Bio-Gel P-2 was from Bio-Rad, and [3H]FMLP (53.6 Ci/mmol) was from New England Nuclear, Boston, MA, U.S.A. Other reagents were of analytical grade. Thyroxine, triiodothyronine, thyronine and tyrosine were dissolved in a solvent containing dimethyl sulfoxide (10%, v/v) and 50 mM NaOH. Diiodothyronine was dissolved in a solvent containing 15 mM HCI instead of NaOH. Diiodotyrosine was dissolved in 50 mM NaOH. All stock solutions were stored at - 20 ° C. The solvents used had no significant effect on the FMLP-indueed O i production.
Cell preparation Human neutrophils were isolated from healthy volunteers by dextran sedimentation, hypotonic lysis and the Conray-FicoU density gradient centrifugation as described previously [16]. The population of cell types were more than 95% neutrophils and less than 5% lymphocytes, monocytes or eosinophils.
Assay of the superoxide production The activity to release superoxide was measured by the reduction of eytochrome c at 550-540 nm using a
dual-wavelength spectrophotometer (Hitachi 557) as described previously [17]. The cells were suspended in 1 ml of a Hepes-buffered saline containing 135 mM
NaC1, 5 mM KCI, 5 mM glucose and 20 mM Hepes (pH 7.4). Before the addition of a stimulant, the mixture was preineubated with 1 mM CaCI z and 50/zM cytochrome c for 10 min at 37 ° C.
Reversibility of the inhibition 1 ml of neutrophil suspension (2.106 cells) was incubated with or without L-thyroxine (10 /zM) for 2 rain at 37 ° C, and then washed by the addition of 9 ml of the ice-cold Hepes-buffered saline containing 1 mM CaCI2. The suspension was centrifuged at 500 x g for 30 s at 4 ° C and 9 ml of the supernatant was aspirated. The washing procedure was repeated as indicated, and the cells were then resuspended in the assay system of the O~ production.
[ 3H]FMLP binding assay Binding of [3H]FMLP to neutrophils was measured by centrifuging the ceils through an immiscible layer [18,19]. A 200 /zl aliquot of neutrophil suspension (5.106 ceUs/ml) containing 1 mM CaCi 2 was incubated for 2 min at 4°C with L-thyroxine followed by the addition of [3H]FMLP and the incubation for 60 min at 4 *C with occasional shaking. The cell suspension was transferred to a microtube containing 6 M urea (10 /zl, bottom layer) and a mixture of dibutyl phthalate and dinonyl phthalate (10:3, v/v) (200/zl, upper layer), and centrifuged for 5 rain at 10000 x g. The cells at the bottom of each tube were cut off and placed in a liquid scintillation vial for radioactivity determination. Nonspecific binding of [3H]FMLP was determined in the presence of 10 -5 M unlabelled FMLP for each concentration of [3H]FMLP.
Molecular sieve chromatography Chromatographic analyses of FMLP were performed essentially as described by Stenson et al. [20] using a Bio-Gel P-2 column. A mixture of [3H]FMLP (40 nM) with or without L-thyroxine (10 /.~M) in the Hepes-buffered saline (pH 7.4) was mixed with unlabelled FMLP to achieve the FMLP concentration at 1 /zM. A 200/zl aliquot of the mixture was applied to a Bio-Gel P-2 column (1 × 18 cm) and eluted with the same buffer. The flow rate was 4.5 m l / h and V0 was 5.4 ml. Fractions of 0.9 ml were collected and the radioactivity was measured in a liquid scintillation counter. Molecular weight standards are oxidized glutathione (612), reduced glutathione (307), and sodium azide (65). They were determined as follows: oxidized and reduced glutathione were detected using 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence and absence of glutathione reductase, respectively [21] and sodium azide was detected by measuring electroconductivity.
225 Results
The effect of thyroxine on the stimulation of 0 2 production Pretreatment of human neutrophils with L-thyroxine abolished the FMLP-induced 0 2 production and the ongoing O 2 production induced by FMLP was also rapidly inhibited by the addition of 5 p M L-thyroxine (Fig. 1A). L-Thyroxine, however, had no significant effect on C5a-stimulated 0 2 production at concentrations lower than 30/~M (Fig. 1B). L-Thyroxine did not act at a site following protein kinase C, because it did not affect 0 2 production stimulated with phorbol 12myristate 13-acetate (PMA), which directly activates protein kinase C (Fig. 1C). These findings indicate that the effects of the compound on FMLP-induced 0 2 production are not toxic. This is supported by the results that iodothyronines at the concentrations used did not alter cell viability, which was determined by Trypan blue dye exclusion. Furthermore, the leakage of lactate dehydrogenase was measured and was less than 5% of the total cell content in the presence of the compounds. The inhibition of FMLP-induced 0 2 production by L-thyroxine was dose dependent and the concentration of L-thyroxine required for the IC50 of 0 2 production stimulated by 1/.~M FMLP was approx. 10 -6 M (Fig. 2). L-Triiodothyronine also prevented FMLP-induced 0 2 production in a dose-dependent manner with an IC50 value of 7 . 1 0 - 6 M. Fig. 3 shows that the effects of various concentrations of the peptide on O 2 production were inhibited by L-thyroxine.
(A)
(B)
(C)
T
9
100
0 0
80+
~
60,
G
A
C
.o ~
"0
40,
P
20.
b
0
T -4
-5
Iodothyronines (IogM)
Fig. 2. Dose-dependent inhibition of the FMLP-induced O~ production by iodothyronines. Neutrophils (2.106 eeils/ml) were treated with L-thyroxine (o) or L-triiodothyronine (e) at various concentrations for 2 rain at 37 ° C, and then 1 ~,M FMLP was added. Each point represents the mean of five experiments. The results are presented as percent of the control value (7.02+0.55 nmol/min per 2. l0 n cells).
Inhibition of the FMLP-induced 0~- production by carious iodothyronines and their structural analogues The effects of various iodothyronines and their structural analogues on the FMLP-induced 0 2 production were examined (Table I). O,L-Thyroxine, at a concentration of 10 #M, inhibited 0 2 production completely and the same concentration of O,L-triiodothyronine inhibited the production by 60-70%. The related compounds including L-diiodothyronine, thyronine, L-diiodotyrosine and O,L-tyrosine, however, had no inhibitory effects on the O~- production. There was no difference between the abilities of the o and L forms to inhibit the O~ producticm, but there was a marked difference between triiodc~thyronine and diiodothyronine, suggesting that the inhibitory potencies depend mainly on the number of iodines. Reversibility of the inhibitory effect When neutrophils were incubated with thyroxine and then washed with the Hepes-buffered saline to
0,02 A
i
A
~ f . - - ' ' "
"6 4. U
;:
1 pM FMLP
::
2 rain
t
20 ng/ml CSa
I
10 ng/rnl PMA
Fig. 1. Effect of L-thyroxine on the O~- production by human neutrophils stimulated with FMLP, C5a or PMA. Neutrophils, as indicated cell counts, were preincubated with 1 mM CaCI: for 10 rain at 37 ° C. The cells were treated with or without L-thyroxine for 2 rain at 37 ° C and then stimulated. (A) 1/zM FMLP was added to the cell suspension (106/ml) (curve l), 1 /~M FMLP was added 1 min prior to 5/xM L-thyroxine (arrow T) (curve 2), 5/~M L-thyroxine was added 2 rain prior to 1 /zM FMLP (curve 3). (B) Either 20 ng/ml C5a alone (. . . . . . ) or 5 p M L-thyroxine 2 rain prior to 20 ng/ml CSa ( ) was added to the cell suspension (4.106/ml). (C) Either 10 ng/ml PMA alone (. . . . . . ) or 5/zM L-thyroxine 2 rain prior to 10 n g / m l PMA ( ) was added to the cell suspension (106/ml).
=% 0 'ID "
0 •8
-7
-6
-5
FMLP (logM)
Fig. 3. Effect of L.thyroxine on O~ production by human neutrophils exposed to different concentrations of FMLP. Neutrophils (2.10 +' cells/ml) were treated with the indicated concentrations of L-thyroxine: none (O), 1 /~M (o), 2.5 p.M (t:3) and 5 p M (Ill), and 2 rain later different doses of FMLP were added. Each point represents the mean of duplicate determinations.
226 TABLE I
o lOO-
Inhibition of FMLP-induced 0 2 production by iodothyronines and their structural analogues Neutrophils (2-106 cells/ml) were incubated with iodothyronines or their analogues at a concentration of 10 /~M for 2 rain at 37°C followed by the addition of 1 tzM FMLP. Each value represents the mean+S.D, of four experiments. The results are presented as a percent of the control value (5.43+_0.43 nmol/min per 2.106 cells). lnhibitors
% of control
L-Thyroxine D-Thyroxine t.-Triiodothyronine D-Triiodothyronine t.-Diiodothyronine Thyronine L-Diiodotyrosine t.-Tyrosine D.Tyrosine
0.6+ 0.4 0.7+ 0.3 36.9+ 5.4 32.5 + 7.5 106.5 :l: 4.5 93,1 + 6.2 100.6 + 6.2 96.3 + 6.7 93.8 ± 10.4
remove L-thyroxine, inhibition of 0~" production revealed restoration (Fig. 4). By washing three times, the rate of the O~ production recovered to approximately 80% of the control.
[ 3H]FMLP binding studies To determine the site at which L-thyroxine and L-triiodothyronine interrupt the signal transduction induced by FMLP, we examined the effects of the iodinated compounds on the specific binding of [3H]FMLP to the neutrophils. As shown in Fig. 5, the compounds ir/,i;ited the binding of [aH]FMLP to the cells. [aH]FMLP binding was reduced by 50% at about 10 -6 M for L-thyroxine and at about 10 -5 M for L-triiodothyronine. L-Thyroxine reduced [3H]FMLP binding with a similar dose response at 37 °C as that at 4 °C (results not shown). A representative result in the Scatchard plots [22] showed the affinity of the receptor
+ ~
6o-
.c: ,,Q
40 -
.~_
o. ,,.I
z,o-
.~
o
.,+
.;
.~
,
+5
.4
Iodothyronlnes (IogM) Fig. 5. Effects of various concentrations of iodothyronines on the binding of ['~H]FMLP. Neutrophils (10~ cells/200/zl) were preincubated with various concentrations of L-thyroxine (O) or L-triiodothyronine (e) for 2 rain at 4°C followed by the addition of [3H]FMLP (40 riM). The cells were incubated for 60 rain at 4°C, and then separated from unbound [3H]FMLP, Specific [aH]FMLP binding in the presence of various concentrations of the iodothyronines was expressed as a percent of the binding observed in the absence of the iodothyronines. Each point represents the mean of triplicate determinations.
to the ligand changed by L-thyroxine (Fig. 6). Binding affinities and the numbers of surface FMLP receptors per cell in the control and L-thyroxine-pretreated cells were 36.2 nM and 69.3 nM, and 22700 and 22200, respectively. Thus, the inhibition of [aH]FMLP binding to neutrophils by L-thyroxine seems mainly to be due to a decrease in the affinity of the receptor to the ligand.
Molecular sieve chromatography The possibility remains that L-thyroxine directly interacts with the peptide leading to a decrease in the formation of the ligand-receptor complex. This possibility was ruled out by the analyses using gel filtration
s
lOO(
~ 0
o
.
~
~.
i 0
,
0
Number o f wash
Fig. 4, Inhibition by L-thyroxine of FMLP-induced O~ production was reversed by washing, Neutrophiis (2. ]06 cells/m]) were incubated with (e) or without (o) 10 #M L-thyroxine for 2 rain at 37 ° C. The cells were washed once, twice or three times with ice-cold Hepes-buffered saline to remove L-thyroxine, and then the O~ production by FMLP (1 p,M) was determined. Each point represents the mean of three experiments. The results are presented as a percent of the control value (5.70+0.25 nmol/min per 2.106 cells).
.
,
1000
2000
Bound (CPM)
Fig. 6. Scatchard analysis of the specific binding of [3H]FMLP to human neutrophils. Neutrophils (106 cells/200 p,I) were pteincubated with (e) or without (o) L-thyroxine (1 p,M) for 2 rain at 4°C followed by the addition of various concentrations of [3H]FMLP. The cells were incubated for 60 min at 4 ° C and then separated from unbound [3H]FMLP. Each point represents the mean of triplicate determinations. The data are representative of three separate experiments.
227 chromatography (results not shown). When [3H]FMLP alone was applied to a Bio-Gel P-2 column, the radioactivity eluted at the No. 11 fraction as a single peak. When L-thyroxine alone was applied and monitored at 325 nm, no peak was detected - probably due to the hydrophobic interaction of the compound with the gel. If L-thyroxine binds with [3H]FMLP, the elution profile of the radioactivity would be expected to change compared to that of [3H]FMLP alone: the peak of the radioactivity may be retarded because the complex interacts with the gel or may be accelerated because the complex possesses a larger molecular weight than [ 3H]FMLP alone. The same count of radioactivity eluted as a single peak at the No. 11 fraction when the mixture of labelled FMLP and L-thyroxine was applied to the column, indicating that FMLP does not form a complex with L-thyroxine. This is supported by the finding that non-specific binding of [3H]FMLP to neutrophils was not affected by L-thyroxine. Discussion
In the present work we demonstrated that thyroxine and triiodothyronine inhibited the respiratory burst of human neutrophils stimulated with FMLP, but not with C5a or PMA. L-Thyroxine also prevented the FMLP-induced Increase in intracellular free Ca 2+ concentrations using the fluorescence probe fura-2 [23] (results not shown). Additionally 10 /~M L-thyroxine reduced the lysozyme release in response to 1 p.M FMLP by 85.1 _+3.6% (mean + S.D.; n -- 6). FMLP-induced O~- production ceased within 1 min after the addition of L-thyroxine (Fig. 1A), and the observation is consistent with the findings [24] that continuous new interactions between FMLP and receptors are required for maintaining the NADPH-O z generating oxidase in the activated state. C5a, like FMLP, has been shown to elicit various biological responses, such as ehemotaxis and 0 2 production, by acting through specific cellular receptors on the plasma membrane of neutrophils [2527]. Of two similar receptor-mediated responses, only that induced by FMLP was prevented by L-thyroxine. The order of the inhibitory potency of the iodothyronines was thyroxine > triiodothyronine, which is different from that of the binding activity to the nuclear thyroid hormone receptor (triiodothyronine > thyroxine), and there was no difference between the L- and the/)-forms. It is unlikely that protein synthesis mediated through nuclear receptors is involved in the inhibitory effects, because the inhibitions occurred within I min after the addition of FMLP and were restored by washing. A similar order of potency of iodothyronines has been reported in the regulation of certain enz'yme~. Hagiwara et al. [28] observed that O,L-thyroxine and O,L-triiodothyronine inhibit myosin light chain kinase of chicken gizzard with IC5o values of about 8/zM and
about 20/~M, respectively, with a lack of stereospecificity. It has also been demonstrated that L-thyroxine is more active than L-triiodothyronine in the stimulation of human erythrocyte Ca2+-ATPase [15] and the inhibition of rat cerebrocortical type II iodothyronine 5'-deiodinase [29]. These and our findings suggest that thyroxine acts as a more potcnt modulator of some cellular functions compared with triiodothyronine and that their stereospecifieities are not necessarily required. The evidences obtained show that the receptor for the chemotactic peptide is modulated by iodothyronines. Possible mechanisms by which the compounds could alter receptor affinity are as follows. The first possibility is that the iodinated compounds may bind the FMLP receptor near the binding site. The second is that they modulate the ligand-receptor complex by binding to membrane components other than the receptor. The third is that they may bind with the specific thyroid hormone receptor on the plasma membrane and alter the FMLP receptor function. The fourth is that they may uncouple the receptor by some direct or indirect effect from the transducing proteins such as GTP-binding proteins. Inhibition of FMLP binding to neutrophils by corticosteroids [30,31], drugs such as phenylbutazone [32,33], indomethacin [34], ibuprofen [35], sulfasalazine [20] and dielofenac [36], and ammonia [37] has, so far, been reported, although the precise mechanisms of their inhibitory effects have not been clarified. The FMLP receptor has been partially purified, characterized and shown to be an integral membrane glycoprotein [38-42]. Recently, the FMLP receptor has been cloned and sequenced [43] and its expression in Xenopus oocytes has been documented [44,45]. The molecular nature of the FMLP receptor, however, has as yet been poorly characterized. Becker et al. [46,47] have postulated that this receptor has three hydrophobic regions which interact with the methionyl, the leucyi and the aromatic ring side chain of FMLP. In our study, several structural analogues including diiodothyronine, thyronine, diiodotyrosine and tyrosine had no significant inhibitory effect in contrast to the potency of thyroxine and triiodothyronine. Thus, the number of iodines is critical for the inhibitory effects and the attachment of more than three iodines to thyronine is required for an inhibitory property. Because iodine is bulky and lipophilic, the additional attachment of iodine may alter the structural and hydrophobic character of the compounds, thereby leading to interaction with the FMLP receptor or another component responsible for the receptor affinity. The inhibitory iodothyronines are assumed to be of less physiological significance because the thyroxine concentration required for inhibition (10 -7 to 10 -5 M) was higher than that of total thyroxine (10 -s to 10 -6 M) or that of free thyroxine (10 - ~ to 10 -1° M) in the
228 serum. However, the fact that the number of iodines of the iodothyronine is related to the inhibitory property might be useful for studying the characteristics of FMLP receptors and the regulation of 0 2 production induced by FMLP. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science and Culture, of Japan. References 1 Beckcr, E,L,, Sigman, M. and Oliver, J.M, (1979) Am. J. Pathol. 95, 81-98. 2 Showell, H J., Freer, gJ., Zigmond, S,H., Sehiffmann, E., Aswanikumar, S,, Corcoran, B. and Beeker, E.L. (1976) J. Exp. Med. 143, 1154-1169, 3 Williams, L.T., Snyderman, R., Pike, M.C, and Lefkowitz, RJ. (1977) Prec. Natl. Aead. Sei. USA 74, 1204-1208. 4 Nakagawara, M., Takeshige, K., Sumimoto, H., Yoshitake, J. and Minakami, S, (1984) Biochim. Biophys. Aeta 805, 97-103. 5 Krause, K,H., Schlegel, W., Wollheim, C.B., Andersson, T., Waldvogel, F.A. and Lew, P.D. (1985) J. Clin. Invest. 76, 13481354. 6 Snyderman, R., Smith, C.D. and Verghese, M.W. (1986) J. Leuk. Biol. 40, 785-800. 7 Fujita, !., Irita, K, Takeshige, K. and Minakami, S. (1984) Biochem. Biophys. Res. Commun. 120, 318-324. 8 Fujita, !., Takeshige, K. and Minakami, S. (1986) Biochem. Pharmacol. 35, 4555-4562. 9 Billah, M.M., Eckel, S., Mullmann, T.J., Egan, R.W. and Siegel, M.I. (1989) J. Biol. Chem. 264, 17069-17077. 10 Koenderman, L., Tool, A., Roos, D. and Verhoeven, A.J. (1989) FEllS Lett. 243, 399-403. 11 Segal, J. and lngbar, S,H. (1982)J. Clin. Invest. 70, 919-926. 12 Angel, R.C., Botta, J.A. and Farias, R.N. (1989) J. Biol. Chem. 264, 19143-19146. 13 Sterling, K., Campbell, G.A. and Brenner, M.A. (1984) Acta Endocrinol. 105, 391-397. 14 Mamiya, S., Hagiwara, M., lnoue, S. and Hidaka, H. (1989) J. Biol. Chem. 264, 8575-8579, 15 Davis, P J. and Bias, S,D. (1981) Biochem. Biophys, Res. Cornmen, 99, 1073-1080. 16 Umei, T,, Takeshige, K. and Minakami, S. (1987) Bioehem. J. 243, 467-472. 17 Sumimoto, H., Takeshige, K. and Minakami, S. (V'~4) Biochim. Biophys. Acta 803, 271-277. 18 Yuo, A., Kitagawa, S., Ohsaka, A., Ohta, M., Miyazono, K., Okabe, T., Urabe, A., Saito, M. and Takaku, F. (1989) Blood 74: 2144-2149.
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