of Nuclear Thyroid Hormone Receptors of Cultured Skin From Patients With Resistance to Thyroid Hormone
Kazuo Ichikawa, I.A. Hughes, Allen L. Horwitz,
and Leslie J. DeGroot
Nuclear thyroid hormone receptors of patients with the syndrome of resistance to thyroid hormone were investigated in cell lines from seven patients in four affected families and compared to results from six normals. Fibroblasts cultured from skin biopsies were used. When binding affinity and capacity for L-triiodothyronine (T,) were examined by incubating whole cells or isolated nuclei, no significant differences were found. The amount of receptor released during the incubation of nuclei (9.3% to 19.0% of total nuclear receptors) was also within the normal range in these patients. When T, binding assays were performed on 0.3 mol/L KCI extracted receptor, a significant decrease in binding capacity (MBC) without a difference in binding affinity (Ka) was observed in four patients and a lower Ka with normal MBC was found in two patients. Recovery of receptors in saline extracts, from patients’ fibroblasts showing a low MBC. was low in comparison to normals. Lability of salt extracted receptors at 38 “C was normal and salt extractability of T, occupied receptors, examined by incubation of [‘26l]-T, labeled nuclei with various concentrations of KCI, was only slightly decreased. This lower salt extractability of receptors was insufficient to account for the low MBC obtained by Scatchard analysis of T, binding to nuclear extracts. Gel filtration and density gradient sedimentation of salt-extracted receptors showed Stokes radius of 34 A, and sedimentation coefficient of 3.4 S in all patients and normals. From these values, molecular weight of 49,000 and total frictional ratio (f /fo) of 1.4 were calculated for nuclear receptors from patients and normals, suggesting a somewhat asymmetrical shape of receptors. Density gradient sedimentation patterns of micrococcal nuclease digested receptors, in which receptors were associated with 6.5,12.5, and 17 S chromatin components, was also indistinguishable from normals. We conclude that in most patients with generalized thyroid hormone resistance, abnormalities of receptor function are evident after salt extraction of fibroblast nuclear receptors. but that the receptors have normal shape and thermolability. These abnormalities were consistent within but heterogeneous between sibships. suggesting heterogeneity of hereditary abnormalities in nuclear receptors in this disorder. B 7987 by Grune & Stratton, Inc.
INCE THE FIRST REPORT of the syndrome of thyroid hormone resistance in 1967,’ more than 100 patients have been detected with this disorder. According to their clinical manifestations, they seem to represent instances of generalized resistance, isolated pituitary resistance, or peripheral tissue resistance to thyroid hormone. In these patients, serum TSH is not suppressed, although thyroid hormone levels are elevated with pituitary or generalized resistance. Patients are clinically and metabolically euthyroid or hypothyroid in peripheral or generalized resistance. Although evidence for hormone unresponsiveness is clear, the cause(s) of these disorders remains poorly defined. Nuclear thyroid hormone receptors are generally believed to mediate biological responses after binding thyroid hormones2 The response of TSH to thyroid hormone, which is impaired in most of these patients, was recently reported to be transcriptionally regulated at the nuclear level.’ From these facts, it is likely that an abnormality in these patients exists at the nuclear level. However, results of studies in nuclear receptors from these patients are not conclusive. In this report, L-triiodothyronine (T,) binding studies were
From the Thyroid Study Unit, Department of Medicine, University of Chicago; and the University of Wales College of Medicine. Cardif, Wales. Supported by United States Public Health Service Grant AM13377 and the David Wiener Research Fund. Address reprint requests to Leslie J. DeCroot, MD, Thyroid Study Unit, Box 138. University of Chicago, 5841 South Maryland Ave. Chicago, IL 60637. o I987 by Grune & Stratton, Inc. 0026-0495/87/3604-OOI7$03.00/0
performed using whole cells, isolated nuclei, and saltextracted receptors from six normal subjects including one normal mother of an affected subject, and seven patients with thyroid hormone resistance from four families. MATERIALS
Subjects The controls were three males (newborn, 8 years old, and 20 years old) and three females (4, 11, and 25 years old). Patient MRG (9%-year-old male) is the first case of generalized resistance to thyroid hormone reported in 1967.’ He was clinically euthyroid and had deaf mutism, stippled epiphyses, goiter, high serum proteinhound iodine, Tr, and T,, normal thyroid-stimulating hormone normal thyroxine binding globulin and normal basal metabolic rate. MNG (16-year-old female) is his younger affected sister.4 In these patients, cultured skin fibroblasts showed an abnormal glycosaminoglycan accumulation response to T,.5 Patients HM (6-year-old female) and KM (&year-old female) are sisters with generalized resistance to thyroid hormone reported in 1982.6 They showed no clinical evidence of hyperthyroidism but had goiter, high serum Tr and T4, normal serum TSH, and a brisk response of TSH to (TRH). Their cultured skin fibroblasts showed an altered low density lipoprotein degradation response to Tr. These results imply possible involvement of fibroblasts in these patients’ illness. Patient RO (6-year-old male) was reported in 1983 to have pituitary resistance to thyroid hormone.’ His younger sister and father were reported to have pituitary and generalized resistance, respectively, to thyroid hormone. His mother, SO (25year-old female), who was normal, was also studied and is included among our controls. VT (6-year-old female) and her father, ET (47-year-old male), are previously unreported cases of generalized resistance to thyroid hormone. They showed elevated total and free T1 and T1 levels in blood in the face of normal TSH levels and brisk response to TRH, and were clinically euthyroid. L-Tr, calf thymus DNA type 1, dithiotreitol (DTT), and microMerabolism, Vol36, No 4 (April),1987: pp 392-399
NUCLEAR T, RECEPTORS
were purchased and
from Sigma serum
(St Louis). from
Island Biochemical Company (Grand Island, NY). [‘*‘I]-T, (2,700 to 3,250 &i/pg) was from Amersham (Arlington Heights, IL). The following buffers were used: SMTD-PMSF buffer [0.25 mol/L sucrose, 1 mmol/L MgCl,, 20 mmol/L Tris-HCl, pH 7.85, 1 mmol/L DTT, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF)]; MTD buffer (I mmol/L M&II, 10 mmol/L Tris-HCI pH 8.0, I mmol/L DTT). Fibroblast cultures were established from skin biopsy samples. Cells were grown in modified Eagle’s medium containing 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 Hg/mL). Experiments were performed between four and I2 passages in cell culture. T, binding characteristics did not change significantly during these passages. Every experiment was done 2 to 3 weeks after replating, when cells were confluent (1.8 to 3.5 x lo6 cells/l00 mm diameter plastic Petri dish). The growth medium was replaced with serumless Ham’s F- 10 medium 48 hours before and changed again 24 hours before the experiments. At the beginning of the experiment, the medium was removed and cells were washed with IO mmol/L phosphate buffered saline pH 7.4 (PBS). Cells were harvested by trypsinization (0.05% trypsin, 0.02% EDTA in PBS for 10 minutes at 37” C), washed once with PBS, and cell number was determined by a hemocytometer. Cells harvested this way were used for whole cell incubation study. For isolation of nuclei, 2 x IO’ cells were suspended in 6 mL of SMTD-PMSF buffer and mechanically homogenized (four strokes) in a Potter-Elvehjem homogenizer (clearance 0.10 to 0.15 mm). Triton X-100 was added to 1% final concentration and further homogenization (two strokes) was performed. The homogenate was then incubated for I5 minutes. After a ten-minute centrifugation at 1,000 x g, the crude nuclear pellet was gently homogenized (two strokes) in SMTD-PMSF buffer containing 0.5% Triton X-100 and centrifuged for ten minutes at 1,000 x g. This pellet. washed once with SMTD-PMSF buffer containing 1 mmol/L CaCI,, was used as purified nuclei. For preparation of nuclear extract, nuclei were incubated in 0.3 mol/L KCI-MTD buffer for 60 minutes with vigorous shaking at 1 S-minute intervals. The residual chromatin was then removed by centrifugation at 135,000 x g for 60 minutes. Nuclear isolation and extraction procedures were carried out at 2O C. This method provided nuclei with a protein/DNA ratio of 2.6 to 3.0 and recovery of 40% to 55% in every cell line. In each preparation, nuclear purity was checked by phase contrast microscopy, which revealed no whole cell and little cytoplasmic contamination in the nuclear preparations. Hormone binding studies were performed using whole cells, isolated nuclei, or nuclear extract. When whole cells were used, cells from 5 to IO plates were harvested by trypsinization, washed with PBS, suspended in Ham’s F-10 medium (0.5 to 2.0 x lO”cells/mL), and 0.5 mL aliquots were incubated for two hours at 37” C with various amounts of [‘*‘I]-T,. Specific nuclear T, binding reached a maximum after 90 minutes and remained unchanged up to 120 minutes. To determine nonspecific binding. duplicate tubes contained in addition 3 x lo-’ mol/L of unlabeled T,. After the incubation cells were immediately cooled in an ice bath, and thereafter all procedures were performed at 0 to 2O C. Cells were collected by centrifugation and the supernatant was saved for free hormone determination. More than 99% of [‘251]-T3 in the supernatant was unbound, as determined by adsorption onto resin. Cells were washed with ice cold PBS, resuspended in 1 mL SMT buffer containing 0.5% Triton X-100 and passed through a 25-gauge needle. The syringe was washed once with 1 mL of the same buffer. Nuclei were collected by centrifugation at 1,000 x g for ten minutes and washed with the same buffer and radioactivity was counted. Cell number did not change before and after incubation with T,. DNA recovery was 5OY0 to 70% and all data were corrected for DNA
at 37O C was optimal
and is the same as that of previous
When isolated nuclei were used, the nuclei of fibroblasts
for whole studies.6*8.9
from ten to 20 culture dishes were suspended in SMTD buffer and 0.5 mL aliquots containing 5 to 25 fig DNA were incubated for two hours at 22” C with various amounts of [‘*‘I]-T3. Specific T, binding reached maximum after 90 minutes and remained unchanged up to 240 minutes, Nonspecific binding was determined by addition of 3 x IO-’ mol/L of unlabeled T, in duplicate tubes. After the incubation, nuclei were immediately cooled in an ice bath and all procedures were thereafter performed at 0 to 2* C. Nuclei were collected by centrifugation and the supernatant was kept in an ice bath for the determination of receptors released during the incubation. Nuclei were washed twice with SMTD buffer containing 0.25% Triton X-100 and radioactivity was counted. For each assay, nuclear extract was prepared from nuclei of fibroblasts recovered from 20 to 40 culture dishes. 40 to 75 fig of extract protein was incubated in 0.5 mL of 0.3 mol/L KCI-MTD buffer for two hours at 22O C with [‘*‘I]-T3 in the absence or presence of 3 x lo-’ mol/L unlabeled T,. Specific T, binding reached a maximum after 90 minutes and was stable throughout 240 minutes. After the incubation, assay tubes were immediately cooled and Dowex I x 8, Cl-, 200 to 400 mesh anion exchange resin (Bio-Rad, Richmond, CA) was added for separation of bound and free hormones as previously described.“. After centrifugation, an aliquot of the supernatant was removed and radioactivity was counted for bound hormone determination. This method was also used for determination of receptors released after the incubation of nuclei with hormone. Incubation temperature of 2Z” C was optimal for TZ binding studies of nuclei and nuclear extract, and binding activity was stable at this temperature for four hours. This is identical to the results obtained with rat liver in a parallel study and in a previous report.“. Scatchard assays were performed on whole cells, isolated nuclei, and nuclear extract using 0.005 to 0.4 x 10e9 mol/L [‘Z51]-T3. The assay was done in duplicate and other duplicate tubes contained in addition 3 x lo-’ mol/L unlabeled T, for the determination of nonspecific binding. Specific binding was calculated by subtracting nonspecific binding from total binding. A straight line best fitted to the data by the criterion of least squares was used for analysis. In all the experiments, the linear correlation coefficient was between 0.87 and 0.99. Experiments were repeated two to five times using different passages of cell cultures. There was no significant correlation between the passage of cell lines and T3 binding parameters (affinity constant [Ka] and binding capacity [MBC]) over the five to 15 passages of cells with which this study was done. Salt extractability of nuclear thyroid hormone receptors was examined as follows: A fibroblast suspension was incubated with 0.5 x 10m9 mol/L [“‘I]-T,. Nuclei were prepared and MTD buffer containing various concentrations of KC1 were added at a nuclear concentration of 1.0 A260 U/mL. After a one-hour incubation in ice, samples were centrifuged and radioactivity in the supernatant was measured and expressed as the percent of total nuclear bound radioactivity. Heat inactivation of receptor was performed on nuclear extracts. Fibroblast nuclear extract was prepared and protein content was adjusted to IO0 to 200 pg/mL. Aliquots of 0.7 mL were incubated at 38” C for zero, ten, 20,30 and 40 minutes. After the heat treatment, samples were immediately cooled and any precipitate was removed by centrifugation. The supernatant was incubated with 0.2 x 10m9 mol/L [‘*‘II-T, in the absence or presence of 3 x lo-’ mol/L unlabeled T, at 22O C for two hours. Specific [‘*‘II-T, binding was determined and t- l/2 was calculated by least square analysis of the semilogarithmic plot. For isokinetic glycerol gradient sedimentation, nuclear extract was incubated with 0.2 x 10m9 mol/L of [IZ51]-T3 and free hormone
ICHIKAWA ET AL
was removed by Dowex resin. 200 PL samples containing 500 rg of ovalbumin as internal standard were applied on 5 mL 8% to 35% wt/voI linear glycerol gradients containing 0.3 moI/L KCl-MTD buffer and centrifuged at O” C at 60,000 rpm for I8 hours with an SW-65 rotor (Beckman Instruments, Palo Alto, CA). The gradient was divided into 33 equal fractions. Radioactivity and absorbance at 280 nm were determined in each fraction. For gel filtration, a Sephadex G-150 column with inner diameter of 1.5 cm and a total volume of 180 mL was used. Nuclear extract (0.5 mL) prelabeled with [‘251]-Tsand containing 2 mg of ovalbumin as internal standard was applied. The column was eluted with 0.3 mol/L KCI, 2 mmoI/L EDTA, 10 mmol/L Tris-HCI, pH 8.0, 10 mmol/L 2-mercaptoethano1 at an elution rate of 6.6 mL/cm*/h. Fractions of 2.4 mL were collected, and radioactivity and absorbance at 280 nm were determined. Digestion of nuclei with micrococcal nuclease and subsequent isokinetic glycerol gradient analysis was performed as follows. After harvesting fibroblasts by trypsinization, cells were incubated with 0.46 x IO-’ mol/L [‘*‘I]-T, in the absence or presence of 3 x lo-’ mol/L unlabeled Ts. Nuclei were isolated, washed twice with the digestion buffer (10 mmol/L KCl, 1 mmol/L CaCI,, 10 mmol/L Tris-HCI, pH 7.4, 1 mmol/L DTT, 0.1 mmol/L PMSF), and suspended in the same buffer. Micrococcal nuclease was added to 750 U/mL and incubated at 0 C. After 120 minutes, EDTA was added to a final concentration of 10 mmol/L to stop the reaction. Undigested chromatin was removed by centrifugation and 0.5 mL of the supernatant was applied on a 3% to 30% wt/wt, linear glycerol gradient and centrifuged at 0’ C at 40,000 rpm for 16 hours in a SW-41 rotor (Beckman Instruments) The gradient was fractionated into 30 equal fractions, and radioactivity and absorbance at 260 nm were determined. Specific [‘*‘I]-T, binding was determined by
subtracting [“‘I]-T, bound in the presence of 3 x IO-’ mol/L unlabeled Tg from total [ “‘I]-T, bound, in each fraction. Special attention was taken in all the experiments to process samples without delay. Cell harvesting and subsequent binding assay, salt extraction, and application to column or gradient centrifugation were done in the same day. Isolated nuclei or nuclear extracts were never stocked for analysis on another day. Protein concentration was determined by the method of Lowry et al,” modified to allow the determination of precise protein concentration with particulate fraction or in the presence of Triton X-1OO.‘2 DNA content was determined by fluorometry.‘3 RESULTS
T3 Binding to Nuclear Thyroid Hormone Receptors in Whole Cells, Isolated Nuclei, and Nuclear Extract Table 1 shows the results of Scatchard analysis of T, binding to nuclear receptors performed by incubating T, with whole cells, isolated nuclei, and nuclear extract, as indicated in Materials and Methods. No difference in binding capacity or affinity was seen in whole cell incubations and nuclear incubation studies. In whole cell studies of patients’ fibroblasts, we found binding capacity of 3,300 to 5,600 sites for TJnucleus (normal, 1,300 to 5,000 sites/ nucleus) and affinity of 1.O to 1.6 x 10” L/mol (normal, 0.8 to 2.0 x 10” L/mol). Binding capacity of 36 to 88 fmol/ 100 fig DNA (normal, 23 to 80 fmol/ 100 pg DNA) and affinity of 1.3 to 3.1 x 10” L/mol (normal, 1.6 to 3.2 x 10” L/mol) were obtained in the nuclear incubation study. The normal
1, Binding Characteristics of Nuclear Thyroid From Patients
With Thyroid Hormone
Hormone Receptors of Cultured Skin Fibroblasts Resistance and Normal Subjects Patients
Whole ceil incubation Ka (x IO” L/mol) MBC (binding sites/cell)
Isolated nuclei Ka 1x IO”
MBC (fmol/ 100 pg DNA)
Percent receptor released
Ka of released receptors
0.8 (0.6, 1.O)
Nuclear extracts Ka (x IO” L/mol)
0.90) MBC (pmol/mg protein)
(0.03, 0.04) Percent recovered
(0.07, 0.04,0.06) 15
0.10 (0.08.0.11) -
Normal values are range of six controls. Each value from one cell line is the mean of three to five separate experiments (whole cell study), or the mean of two to three experiments (nuclear and nuclear extract studies) from different passage of cell culture. Values from each experiment are shown in parentheses for the patients with abnormal results. MBC in the isolated nuclei indicate T3 binding capacity in the nuclear pellet and does not include released receptor. Percent of receptor released was calculated by: MBC of released receptor x 100. MBC in the isolated nuclei + MBC of released receptor When the percent recovered in nuclear extract was determined, Scatchard analysis of Ts binding to isolated nuclei and nuclear extract was performed simultaneously using the same batch of nuclei. The amount of receptor recovered in the nuclear extract was estimated from MBC and expressed as percent of amount of receptor detected in isolated nuclei including released receptor.
NUCLEAR T3 RECEPTORS
Whole Cell Incubation
BOUND kAl) Fig 1. Typical Scatchard plots of T, receptor binding from controls and patients showing abnormal results. Ka, x 10” I/mol; B, binding capacity was expressed as the number of T, binding sites/nucleus in the whole cell incubation study, fmol of T, binding/100 pg DNA in isolated nuclei, and pmol of T, binding/mg protein in nuclear extract. The same abnormalities were obtained for these patients using other passages of cell culture.
values are the range of six studies on normal fibroblasts. During the nuclear incubation, a certain amount of receptor is released into the media, presumably due to endogenous nuclease activity or release of receptors that are loosely associated to chromatin or exist in nucleoplasm. The amount of released receptors was also identical on comparison of normals (12.3 k 5.9% of total nuclear receptors) and patients (12.6 i- 3.8%; mean +- SD of seven patients). Released receptors showed a Ka of 1.2 to 5.3 x 10” L/mol in patients (normal, 0.5 to 4.5 x IO” L/mol). In contrast, when nuclear extracts from patients were used for binding assays, an obvious decrease in binding capacity was observed in MRG, MNG, HM, and KM. Binding capacities of 0.03 to 0.10 pmol/mg protein (normal, 0.11 to 0.32 pmol/mg protein) and affinities of 1.8 to 5.6 x 10” L/mol (normal, 0.8 to 4.5 x 10’OL/mol) were found in these patients. The amounts of receptor detected by binding assay in salt extracts were 27% and 15% of total nuclear receptors in MRG and HM, respectively, whereas two normals showed 45% and 56%
recovery. This lower recovery of receptors in salt extract is in agreement with lower MBC found in the salt extracts. Lower Kas were found in nuclear extracts from two other patients in one family. However, the MBC of nuclear extracts from these patients were within the normal range (ET showed MBC of 0.36 pmol/mg protein, Ka of 0.5 x lOlo L/mol, and VT showed MBC of 0.23 pmol/mg protein, Ka of 0.8 x 10” L/mol). Figure 1 shows typical Scatchard plots of control and patient fibroblasts, nuclei, and nuclear extract studies. We could not show any abnormality in T, binding characteristics using nuclei or nuclear extract of RO’s fibroblast. Although in some preparations the data suggested the presence of two binding sites, other experiments on the same individual showed no evidence for two binding sites. No individual showed consistent evidence for multiple binding sites. In these experiments, nuclear purity was carefully checked in each preparation by protein/DNA ratio and phase contrast microscopy, and no significant difference was seen between individual cells. Salt Extractability of Receptors Because lower binding capacity and lower recovery of receptors were seen in nuclear extract from patients MRG, MNG, KM, and HM, we evaluated the possibility that receptors with these patients associate more tightly to chromatin than normals. Fibroblasts harvested by trypsinization were labeled with [‘*‘II-T3 and salt extractability of [‘251]-T, in isolated nuclei was examined at 0.15, 0.3, and 0.6 mol/L KCI. As shown in Table 2,0 mol/L, 0.15 mol/L, 0.3 mol/L, and 0.6 mol/L KC1 solubilized 15.8 of 2.2%, 51.5 t 6.5%, 84.0 * 3.1%, and 87.8 + 2.9% (mean + SD of four patients) of receptor. Slightly lower extractability was found in MRG, MNG, and KM at 0.3 and 0.15 mol/L KCl, although more receptors were released at 0 mol/L KC1 in patients than normals. Because more than 80% of receptors were released at 0.3 mol/L KC1 in all patients, this slightly lower extractability does not account for the lower MBC observed in nuclear extracts from these patients. Stability of Nuclear Receptors Another possible explanation for lower recovery of receptors in nuclear extract is that receptors are unstable after salt
Table 2. Salt Extractability of Receptors KCI Concentration(mol/Ll 0
Controls (n = 4) (% extractable T, binding)
11.8 zk 1.7%
Salt extractability of nuclear receptor was examined as in the Materials and Methods section. Extractable T, binding activity at each salt concentration was expressed as the percent of total nuclear T, binding activity. Controls are mean + SD of four normal subjects. Each value is the mean of three to five determinations in one experiment. Each determination did not vary by more than 5% of the mean.
ICHIKAWA ET AL
Table 3. t-V* ef7, Binding Activity in Nuclear Receptor at 38°C
t-s at38°C Control
25 k 7
t-I$ of T, binding activity in nuclear receptor at 38°C was examined as in the Materials and Methods section. Control is mean t
SD af +ofowr
normal subjects. Each value is mean of two to three experiments.
extraction. To evaluate this possibility, thermal lability of receptors was tested. After treatment of receptors at 38OC, the remaining binding activities were determined. As shown in Table 3, no evidence suggesting unusual lability of receptors from patients was obtained. In all cases, the velocity of inactivation at any moment of time depends upon the remaining binding activity, suggesting that this is a chemical reaction of the first order. Molecular Size Determination of Salt Extracted and Micrococcal Nuclease Digested Receptors
Molecular size of salt extracted nuclear thyroid hormone receptors from patients with thyroid hormone resistance was
determined by Sephadex G-150 gel filtration and glycerol density gradient sedimentation and compared to that from normals. As is shown in Fig 2, no size difference was observed comparing normals and patients. In every case, Stokes radius of 34 A, and sedimentation coefficient of 3.4 S were obtained. From these values, molecular weight of 49,000, total frictional ratio (f/fo) of 1.4,14 and frictional ratio due to shape (f/fo) shape of 1.3” were calculated in all normals and patients examined. In making these calculations, partial specific volume of 0.735 mL/g was used, because this value was ,determined for nuclear receptors of cultured rat pituitary tumor cells (GH, ce11)16and is close to 0.725 mL/g, which was selected by Martin and Ames” as representative of most proteins for their sucrose density centrifugation studies. Frictional ratio of >l indicates asymmetrical shape of nuclear thyroid hormone receptor, identical to previous reports using rat tissues.‘6.‘8 Because 18 hours and 15 hours were required for gradient centrifugation and gel filtration of receptors, respectively, part of bound T, could dissociate from the receptor and result in an incorrect estimation of receptor peak, even if all procedures were performed at 0“ C. To evaluate this possibility, Dowex resin was used to separate the bound hormone from free in each fraction. No difference was obtained in the pattern of the binding peak. The estimated molecular size of receptors in human fibroblast nuclear extract is in agreement with the molecular weight of rat tissue nuclear receptors examined by x-ray inactivationI and SDS-polyacrylamide gel electropho-
Fig 2. Molecular sire determination of salt-extracted receptors in patients with thyroid hormone resistance. Sephadex G-l 50 gel filtration (left panels) and 8% to 35% continuous glycerol density gradient sedimentaiton (right panels) were performed. I), void volume; I, position of internal standard of ovalbumin (3.5 SJ.
NUCLEAR T, RECEPTORS
Fig 3. Glycerol gradient sedimentation pattern of micrococcal nuclease digested nuclear receptors. Nuclei prelabeled with [‘2sl]-T1 were digested with micrococcal nuclease. 10.7%. 13.0%. 12.3%. and 6.2% of chromatin was rendered perchloric acid-soluble by digestion in the control, MRG, KM, and HM, respectively. 1, position of internal standard of catalase (11.6s).
9 a I
boittom 10 20 I
resis after affinity labeling,*” and considered to represent the size of the receptor protein itself. On the other hand, micrococcal nuclease digestion is believed to produce nuclear receptors associated with other molecules, possibly internucleosomal linker DNA, and nucleosomes.*‘~** To look at the interaction of receptors with chromatin, we did gradient sedimentation analysis of micrococcal nuclease digested receptors from these patients. As shown in Fig 3, we found 6.0 to 6.5, 12.5, and 17 S T3 binding peaks in both normal subjects and patients.
Previous studies dealing with characterization of nuclear thyroid hormone receptors in patients with thyroid hormone resistance report normal numbers of receptors with normal binding affinity to T3 in fibroblasts: normal numbers of receptors with slightly decreased affinity to TX due to decreased nuclear T, uptake in fibroblasts,23 or two binding sites with a normal high T, affinity-low capacity site and an additional lower affinity-higher capacity site in fibroblasts and low affinity to T, in circulating lymphocytes.* All of these studies were done by incubating patients circulating lymphocytes or relatively small numbers of cultured fibroblasts with T,, and examined nuclear binding characteristics using crude nuclear preparations. For further characterization of nuclear receptors in these patients, we have developed an improved isolation method for cultured human fibroblasts nuclei, by modifying a previously reported Triton X-100 method.24 Using this method, we constantly obtain pure
nuclei (protein/DNA ratio of 2.6 to 3.0, and little cytoplasmic contamination seen by phase contrast microscopy) with approximately half recovery of DNA in all normal and patient cells tested. When whole cells or isolated nuclei were incubated with T,, no difference from normal was detected in binding capacity, affinity or amount of receptors released during incubation of isolated nuclei in any patient. We found a higher Ka in nuclear incubation studies than in whole cell incubation studies, both in normals and patients. This difference is possibly due to purer nuclei used in the nuclear incubation study, but suggests that intranuclear-free T, concentration is not higher than extracellular or cytosolicfree T, concentration at equilibrium. Because no significant difference of Ka in whole cell and nuclear incubation study was found between normals and patients, we considered that an abnormality in nuclear or cellular T, uptake at equilibrium was not present in this system. The existence of two binding sites was previously observed in two of the patients studied in this paper (MNG, MRG), but we could not currently find evidence supporting this finding. Neither impurity of nuclear preparations nor an altered range of concentration of [‘*‘I]-T, caused results compatible with two binding sites. Possibly the many improvements in preparation and assay of receptors used in this study account for the difference. Further characterization of nuclear receptor protein was performed using receptors prepared by incubating nuclei with 0.3 mol/L KC1 at pH 8.0. In contrast to the study performed on nuclei or whole cells, Scatchard analysis of T,
ICHIKAWA ET AL
binding to nuclear extracts showed significantly lower binding capacity (expressed as pmol T, bound to 1 mg protein) with normal Ka in patients MRG, MNG, KM, and HM. However, these extracted nuclear receptors in patients with GRTH had normal molecular size as examined by gradient sedimentation (3.4 S) and gel filtration (34 A), and normal lability at 38’ C, suggesting almost identical protein structure of receptors to normal fibroblasts. Salt extractability study showed that more than 80% of T,-nuclear receptor complex was extractable with 0.3 mol/L KC1 in these patients, whereas only 15% to 27% of total nuclear receptors were detected by Scatchard analysis (normal 45% to 56%). There are several possibilities to explain a lower binding capacity in salt-extracted receptors from patients with GRTH, in face of normal salt extractability of salt extracted receptors at 3S” C. There could be (1) a significantly larger loss of patients’ receptors during binding assay of nuclear extracts or during the resin test used for separation of bound and free hormones, (2) unoccupied receptors significantly resistant to extraction in patients, or (3) receptors that are heterogeneous, consisting of unstable and relatively stable types, and patients having a greater abundance of unstable receptors that cannot be detected by heat inactivation because of rapid decomposition before the study. Regarding the first possibility, we recently noticed that Dowex resin, used for separation of bound and free hormone, adsorbs small amounts of receptor-bound hormone. In usual circumstances, this effect is so small that it can be neglected. When a low concentration of receptors or receptor was used after purification, this caused significant underestimation of bound hormone. To prevent this effect, we adjusted starting cell numbers for nuclear extract preparation and protein concentration of nuclear extracts to the same range in all cell lines. Thus, it is unlikely that this difference in MBC in salt-extracted receptors is an erroneous finding caused by different protein content in the nuclear extracts used. We do not have data regarding the second and third possibilities. In two patients from the same sibship, a significantly lower affinity for TJ was found after salt extraction of receptor. Although we did not further study the mechanism of the abnormality in these patients, it is possible that nuclear receptors in these patients are structurally different and
cannot maintain high affinity for T, after dissociation from chromatin. This idea is supported by the normal Ka observed in the released receptor, which is present in a complex with DNA and protein, in contrast to the lower Ka found after salt extraction. It is also possible that the variations in Ka reflect alterations in inapparent, nonspecific TS binding sites that behave as “free T3” in the resin test or on Sephadex chromatography, as discussed by Seelig et al.*’ However, our studies were done at 20° C in contrast to those of Seelig et al at O” C. Further, the uniformity in preparation of extracts and their protein contents would indicate that the observed lower Kas in ET and VT, in comparison to other patients and controls, establishes that their receptors are clearly qualitatively different than controls. Interestingly, altered salt extractability of glucocorticoid nuclear receptors occurs in glucocoid insensitive murine lymphoid cells lines.26 Although we failed to find a large difference in salt extractability of T,-occupied receptors in patients with thyroid hormone resistance, abnormalities in receptor T3 binding were brought out during salt extraction. In a previous report, T, was found to increase the affinity of nuclear receptors for chromatin,27 and chromatinassociated receptor had decreased affinity for T3 in the presence of physiologic concentrations of divalent cations.** Possibly a higher affinity for chromatin of the T,-occupied receptor, compared to the unoccupied receptor, is critical for receptor function. If so, a tight association of unoccupied receptors to chromatin, which is compatible with a low T, binding capacity in salt extracted receptors, could cause dysfunction of the receptor and result in hormone unresponsiveness If a lower affinity for T, in the chromatin-bound receptor than in the free (chromatin unbound) receptor is critical for receptor function, then a very low affinity for T, of the free receptors, which is compatible with lower affinity for T, after salt extraction, may cause dysfunction of receptor, resulting in hormone unresponsiveness. ACKNOWLEDGMENT We thank Dr Samuel Refetoff for providing tibroblasts from patients with thyroid hormone resistance, and Myrna Zimberg for typing the manuscript.
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