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J Biol Chem, Vol. 273, Issue 50, 33166-33173, December 11, 1998


Intracellular Proteolytic Cleavage of 9-cis-Retinoic Acid Receptor alpha  by Cathepsin L-type Protease Is a Potential Mechanism for Modulating Thyroid Hormone Action*

Takashi Nagaya, Yoshiharu Murata, Shunsuke Yamaguchi, Yoshio Nomura, Sachiko Ohmori, Miyuki Fujieda, Nobuhiko KatunumaDagger , Paul M. Yen§, William W. Chin§, and Hisao Seo

From the Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Dagger  Research Institute of Health, Tokushima Bunri University, Tokushima, Tokushima 770-8514, Japan, and the § Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha  (RXRalpha ) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha . The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha .

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Thyroid hormones are essential for normal growth and development as well as for the regulation of a variety of metabolic pathways (1). These effects are mediated by thyroid hormone receptors (TRs),1 which activate or repress the transcription of specific target genes in a ligand-dependent manner (2). TRs belong to members of the family of nuclear receptors which also include several steroid, retinoic acid, and vitamin D3 receptors. TRs are encoded by two genes, TRalpha and TRbeta , as well as alternative splicing products of each (3). For transcriptional regulation by thyroid hormones, TRs bind to a specific DNA sequence (thyroid-hormone responsive element, TRE) of target genes by forming a monomer, a homodimer, and a heterodimer with TR auxiliary proteins (TRAPs) (4). TREs are composed of the arrangement of two common hexameric DNA half-site sequences (AGGTCA). The orientation of two half-sites is head-to-head (palindrome, Pal) or in an opposite orientation as an inverted palindrome (Lap), with the half-sites arranged head-to-tail as a direct repeat with 4 spacing (DR4). The heterodimer formation (TR/TRAP) on these TREs is suggested to be more stable and important for transcriptional regulation (5). Several studies (6-8) identified 9-cis-retinoic acid receptors (RXRs), which are also members of the nuclear receptor superfamily, as TRAPs in most tissues. Also demonstrated was that preferential expression of RXR isoforms in certain cell lines or tissues results in the apparent heterogeneity of TRAP on EMSA (9).

One of the major target organs for thyroid hormones is the liver, in which a number of genes in the metabolic pathways such as spot 14 (10), malic enzyme (11), and type I 5'-deiodinase (12) are regulated by T3 at the transcriptional level. These actions are mediated through TRbeta which is a major form expressed in the liver (13). We have used primary hepatocyte cultures as a model to study thyroid hormone action in the liver, and we demonstrated that response of hepatocytes to T3 was markedly influenced by the culture conditions (14). When hepatocytes were cultured as spherical aggregates (spheroids) on a positively charged polystyrene dish, T3 increased the expression of 5'- type I 5'-deiodinase gene. However, the T3 effect was markedly attenuated in hepatocytes cultured as conventional monolayer on a collagen-coated dish.

The aim of the present study was to elucidate the mechanisms underlying the altered responsiveness to thyroid hormone in the two culture conditions. It was demonstrated that RXRalpha was the major TRAP in hepatocytes cultured either as monolayers or as spheroids. The presence of a protease(s) in the cytosol, but not in the nucleus, cleaving RXRalpha at its amino terminus, was demonstrated by incubating full-length 35S-labeled RXRalpha with each fraction. With the use of specific protease inhibitors, this enzyme was identified as cathepsin L-type protease. This enzyme activity was much higher in hepatocytes cultured as monolayers than those cultured as spheroids. Introduction of E64d, a membrane-permeable cysteine protease inhibitor, in the monolayer of the hepatocytes resulted in the inhibition of RXRalpha -cleaving enzyme and the increase in the nuclear RXRalpha protein level. Furthermore, E64d augmented T3-dependent induction of spot 14 mRNA. We suggest that the altered cathepsin L-type protease activity in hepatocytes cultured in two different conditions could alter the turnover of RXRalpha in the nucleus and might, at least in part, modulate T3 responsiveness in vivo.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Primary Cultures of Rat Hepatocytes-- The isolation of primary cultured hepatocytes was performed as described previously (14). In brief, hepatocytes were isolated from livers of male Wistar rats by collagenase perfusion and plated onto dishes with two different kinds of surfaces, collagen-coated dishes (Iwaki Glass) for monolayer cultures and polystyrene dishes with positively charged surface (Primaria, Falcon and Becton Dickinson Labware) for spheroid cultures. These primary hepatocytes were grown in serum-free defined medium (14) with penicillin and 100 units/ml streptomycin.

Preparation of Nuclear and Cytoplasmic Extracts-- Three days after the plating, the aggregates of hepatocytes called "spheroids" were formed on Primaria dishes, whereas hepatocytes were grown as a monolayer on collagen-coated dishes. At this time, the cells were harvested and subjected to preparation of nuclear and cytoplasmic extract. Two methods were employed to prepare the nuclear extracts. One method described by Schreiber et al. (15) is applicable for small amounts of cells with rapid isolation of nuclear and cytoplasmic extracts. However, the nuclear extracts prepared by this method contain a minor contamination from cytoplasmic fraction. The other method employs the procedure described by Shapiro et al. (16) with minor modification which results in a highly purified nuclear extract. However, this method requires large amounts of cells and cannot be used to extract the cytoplasmic fraction simultaneously. We will refer to the nuclear extract prepared by Schreiber method as "crude nuclear extract" and the one by a modified Shapiro method as "purified nuclear extract."

The minor modification of Shapiro method is as follows. The nuclear pellet after the centrifugation through a sucrose gradient was washed twice with phosphate-buffered saline, resuspended in the nuclear extraction buffer (20 mM Hepes, pH 7.9, 400 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and kept on ice for 10 min. After centrifugation, the supernatant was collected.

The concentration of proteins was determined by Bio-Rad protein assay kit (Bio-Rad). Nuclear pellet after nuclear protein extraction was used to determine the cell DNA contents by the method of Burton (17) using herring sperm DNA as a standard.

Measurement of T3 Binding Capacity-- T3 binding assay was carried out using isolated nuclei according to the method of Ichikawa and DeGroot (18).

Electrophoretic Mobility Shift Assay (EMSA)-- The DNA binding activities of nuclear extracts from hepatocyte cultures were studied by using radiolabeled synthetic thyroid hormone-responsive elements (TREs), in the absence or presence of in vitro translated TRbeta synthesized by the TNT-coupled reticulocyte lysate system (Promega, Madison, WI). The method for EMSA was described previously (19). In vitro synthesized TRbeta (20) and RXRalpha (21) were also used for EMSA. The sequences of TRE oligonucleotides, Pal, Lap, DR4, and malic enzyme, were previously described (22).

Supershift analysis was performed using two anti-RXRalpha antibodies and one anti-TRbeta antibody. The epitopes recognized by one of the anti-RXR antibodies (antibody N) was residues 92-102 (23) and by the other (antibody C) was residues 230-235 (24). Anti-TRbeta antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Analysis of Proteolytic Cleavage of RXRalpha -- In vitro translated RXRalpha was preincubated with the cytoplasmic or nuclear extract derived from either the monolayer or spheroid cultured hepatocytes for 30 min at 37 °C and then used for EMSA with in vitro translated TRbeta . Alternatively, 35S-labeled in vitro translated RXRalpha was incubated with cytoplasmic extract and analyzed on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

To characterize the protease, a series of inhibitors were added before the incubation of RXRalpha with the cytoplasmic extract. The final concentrations of protease inhibitors were as follows: antipain (50 µg/ml), aprotinin (60 ng/ml), bestatin (40 µg/ml), chymostatin (100 µg/ml), E64 (0.5 µg/ml), EDTA (0.2 mg/ml), leupeptin (0.5 µg/ml), Pefabloc® SC (0.1 mg/ml), pepstatin (0.7 µg/ml), phosphoramidon (4 µg/ml) (protease inhibitor set, Boehringer Mannheim), calpastatin (4 units/ml) (Takara,) and ovocystatin (100 µg/ml) (Takara). CA-074 (10-5 M), CLIK-088 (10-4 M), CLIK-112 (10-5 M), and CLIK-121 (10-5 M) (25-27) were also used.

Immunoprecipitation of in Vitro Translated RXRalpha after Proteolytic Cleavage-- 35S-Labeled in vitro translated RXRalpha (5 µl) was incubated with the cytoplasmic extract (10 µg) from monolayer cultures for 1 h at 37 °C in 40 µl of the buffer C (10 mM Hepes, pH 7.9, 10 mM KCl, 2.5 mM MgCl2). Two anti-RXRalpha antibodies (N and C) were used for the immunoprecipitation. The protocol for the immunoprecipitation, SDS-PAGE, and autoradiography was described previously (28).

Assay of Cathepsin L Activity-- Cathepsin L activity in cytoplasmic extracts from monolayer or spheroid cultured hepatocytes was assessed by the Inubushi method (29) using a substrate for both cathepsin B and cathepsin L (Z-Phe-Arg-4-methylcoumaryl-7-amide (MCA), Peptide Institute, Osaka, Japan) (30) in the presence of specific cathepsin B inhibitor, CA-074 (25, 26). Ten micromoles of the substrate was incubated with the cytoplasmic extract from monolayers or spheroids in the cathepsin assay buffer (100 mM sodium acetate, pH 5.5, 8 mM L-cysteine, 1 mM EDTA) in the presence of 10-4 M of CA-074 at 37 °C for 6 min. The reaction was stopped by 100 mM monochloroacetic acid (pH 4.3). The fluorescence of the solution at 370 nm excitation and 460 nm emission was determined according to that from 7-amino-4-methylcoumarin. The cathepsin L activity was expressed as 7-amino-4-methylcoumarin-liberating rate from Z-Phe-Arg-MCA.

Western Blot Analysis-- The nuclear extract from hepatocytes was prepared by the Schreiber method in the presence of leupeptin (1 µg/ml) to protect RXRalpha from the cleavage. Forty micrograms of the nuclear extract was electrophoresed through 8% SDS-PAGE and analyzed using RXRalpha antibody (D-20, Santa Cruz Biotechnology) by the method previously described (31). The intensity of bands was determined using the densitometric analysis program (NIH image version 1.44).

Effect of E64d on the Responsiveness of Monolayer Cultured Hepatocytes to T3-- Monolayer cultured hepatocytes were incubated with the membrane-permeable cysteine protease inhibitor, E-64d (Taisho Pharmaceutical), beginning 1 day after the plating. On the 3rd day of the plating, 100 nM T3 was added, and the cells were harvested after 24 h. Total RNA was extracted by the acid guanidine phenol/chloroform method (32). Ten micrograms of total RNA were separated by 0.8% agarose electrophoresis, blotted on a nitrocellulose membrane (GeneScreen Plus, NEN Life Science Products), and hybridized with a labeled probe for spot 14 (f5-4, a gift from Dr. C. N. Mariash). Autoradiography was performed by exposing the hybridized membrane to Kodak X-AR (Eastman Kodak Co.). Radioactivity of the specific band was determined using Fujix Bioimage Analyzer (BAS 2000, Fuji Photo Film). The T3-dependent activation of spot 14 mRNA was expressed as fold induction (mean ± S.D. of triplicate results). The data were analyzed by one-way analysis of variance, and the significance between groups was determined by Bonferroni's test.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

T3 Binding Capacity in the Hepatocyte Cultured as Monolayers and Spheroids-- To assess whether the altered responsiveness to T3 in the hepatocytes cultured as monolayers and spheroids is due to alteration of TR amount, T3 binding capacity was determined in the nuclei prepared from the hepatocytes cultured as monolayers and spheroids. The T3-binding capacity was 92.5 fmol/100 µg of DNA in the hepatocytes cultured as monolayer and 99.6 fmol/100 µg of DNA in those cultured as spheroids. The determination was repeated twice, giving the similar results. It is thus demonstrated that the amount of TRs is not changed by the culture conditions.

Difference of TRAPs in the Crude Nuclear Extract from Hepatocytes Cultured in Two Different Conditions-- Since TR heterodimerize with TRAPs to exert T3 action, alteration of TRAP could be responsible for the altered responsiveness to T3. Thus, TRAPs in primary hepatocytes were analyzed by EMSA using the crude nuclear extract prepared by the Schreiber method (15). On DR4-TRE (Fig. 1A), in vitro translated TRbeta bound to DR4 as a homodimer (D) and a heterodimer with RXRalpha (HD). The nuclear extract alone from monolayer (M-N) or spheroid cultures (S-N) did not result in any band shifts, suggesting that the nuclear extract may not have enough TRs to exhibit protein-DNA complexes. To analyze endogenous TRAPs, each nuclear extract was incubated with in vitro translated TRbeta . The nuclear extract from monolayer cultures incubated with TRbeta mainly formed a faster migrating complex (F). In contrast, the nuclear extract from spheroid cultures demonstrated two protein-DNA complexes in the presence of TRbeta . A faster complex (F) migrated closely at TRbeta homodimer and a slower one (S) was at the position of TRbeta -RXRalpha heterodimer.


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  		Fig. 1.   Different TRAPs in the crude nuclear extracts prepared from hepatocytes cultured under two different conditions. A, TRAPs in hepatocytes were analyzed by EMSA using DR4-TRE as a probe. In vitro translated TRbeta formed a homodimer (D). The addition of in vitro translated RXRalpha to TRs results in the formation of a heterodimer (HD). The crude nuclear extract from monolayer cultures (M-N) demonstrated a faster migrating complex (F) with in vitro translated TRbeta , whereas the one from spheroid cultures (S-N) exhibited two binding complexes (F and S). B, the protein-DNA complexes by the hepatocyte nuclear extracts were studied in the presence of T3. Although the homodimer of TRbeta was dissociated by T3, two binding complexes from hepatocyte nuclear extract were not changed, suggesting that both complexes are heterodimers consisting of TRbeta and endogenous TRAPs. C, EMSA was performed using different TREs (Pal, Lap, and malic enzyme (ME)) as probes. The protein-DNA complexes migrated in a pattern similar to that using DR4 as a probe. The faster migrating band (F) was a major complex in the nuclear extracts from monolayer cultures, but two complexes (F and S) were observed in these from spheroid cultures. R, in vitro translated RXRalpha ; M, crude nuclear extract from monolayer; S, crude nuclear extract from spheroids; C, control lysate; beta , in vitro translated TRbeta , *, nonspecific binding.

To clarify whether the faster migrating complex includes TRbeta homodimer, EMSA was performed in the presence of T3. As previously reported (33), the addition of T3 eliminated homodimer binding but did not affect the RXRalpha -TRbeta heterodimer formation (Fig. 1B). In the presence of T3, the binding of both faster and slower migrating complexes did not disappear, suggesting that both complexes are heterodimers of TRbeta and TRAPs in hepatocytes. On different TREs such as palindromic (Pal), inverted palindromic (Lap), and malic enzyme-TRE (Fig. 1C), a similar pattern of protein-DNA complexes was demonstrated. Specifically, only the faster band (F) was observed in the crude nuclear extract from monolayer cultures and two retarded bands (F and S) in that from spheroid cultures. These results suggest that the nuclear extracts from monolayer or spheroid cultures contain different species of TRAPs.

To characterize the TRAPs in hepatocytes cultured in the two conditions, antibodies to RXRalpha were added to the EMSA reactions, because a major TRAP in liver was previously reported as RXRalpha (34). The epitopes of two RXRalpha antibodies are schematically depicted in Fig. 2A. When the antibody C, recognizing the hinge region (amino acids 220-235) of RXRalpha (24), was added to the EMSA reaction, the intensity of the faster (F) band in TRbeta  + M-N and TRbeta  + S-N mixture decreased, and a supershifted band was formed (Fig. 2B, arrowheads). The slower band (S) in TRbeta  + S-N also reacted with this antibody. However, the antibody N raised against the amino terminus (amino acids 92-109) of RXRalpha (23) did not decrease the binding of the faster migrating band in TRbeta  + M-N with no formation of the supershifted band. Similarly, the faster band in TRbeta  + S-N was not recognized by this antibody. However, the intensity of the slower band in TRbeta  + S-N decreased to form the supershifted band. Since the slower complex migrated close to TRbeta -RXRalpha and was recognized by two RXRalpha antibodies, TRAP in the slower band is identified as full-length RXRalpha . On the other hand, the faster migrating complex might represent the RXRalpha with an amino-terminal truncation, since only the antibody to hinge region (Ab C) supershifted this band. Based on the epitopes of two RXRalpha antibodies and the requirement of RXR DNA binding domain for DNA binding, the site of truncation is speculated to be located near the junction of the amino-terminal region and the DNA binding domain (Fig. 2C). Supporting these results, the artificially constructed amino-terminal deletion of RXRalpha also formed a faster migrating complex with TRbeta as similar as M-N did (data not shown).


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  		Fig. 2.   Characterization of different TRAPs in hepatocytes. A, two RXRalpha antibodies were used to characterize TRAPs in hepatocytes. Antibody N recognizes the amino-terminal region (amino acids (a.a.) 92-109) of RXRalpha , and antibody C reacts with the hinge region (amino acids 220-235). B, EMSA was performed using two RXRalpha antibodies (Ab) to identify TRAPs in the protein-DNA complexes. The antibody C decreased binding of the faster band (F) in TRbeta  + M-N and TRbeta  + S-N, and also the slower migrating complex (S) in TRbeta  + S-N to form a supershifted band (arrowhead). The antibody N did not decrease the faster band in TRbeta  + M-N and TRbeta  + S-N but reacted with the slower band in TRbeta  + S-N. The upper panel is a part of autoradiogram exposed longer to show the supershifted bands. The lower panel is the autoradiogram of the same gel with shorter exposure. RXRalpha , in vitro translated RXRalpha ; TRbeta , in vitro translated TRbeta ; M-N, monolayer nuclear extract; S-N, spheroid nuclear extract. C, the result presented in B indicates that the slower migrating complex contains full-length RXRalpha and the faster one contains RXRalpha with an amino-terminal truncation.

TRAPs in the Purified Nuclear Extract-- When using the highly purified nuclear extract prepared by the modified Shapiro method (16), the pattern of protein-DNA complexes on EMSA was quite different from that produced by the crude nuclear extract. As shown in Fig. 3, the faster migrating complex was absent, and only the slower migrating complexes were observed in the nuclear extract from monolayer cultured hepatocytes (TRbeta  + M-N). Accordingly, the difference of protein-DNA complexes in the nuclear extracts from the hepatocytes cultured under these conditions was eliminated. These results suggest that the truncated RXRalpha does not exist in the nucleus and that the truncation observed in the crude nuclear extracts may be due to proteolytic cleavage during the preparation of nuclear extract by enzyme(s) present in the cytoplasmic fraction.


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  		Fig. 3.   Absence of faster migrating complex in the purified nuclear extract. When the purified nuclear extract was prepared by the modified Shapiro method, the faster migrating complex including truncated RXRalpha was almost eliminated. Only the slower band was observed in the nuclear extract from both hepatocytes cultured as monolayers and spheroids. RXRalpha , in vitro translated RXRalpha ; TRbeta , in vitro translated TRbeta ; M-N, monolayer nuclear extract; S-N, spheroid nuclear extract.

Cleavage of RXRalpha by Cytoplasmic Protease(s)-- To study further the cleavage of RXRalpha by cytoplasmic protease(s), the nuclear and cytoplasmic extracts were prepared separately using the method of Schreiber et al. (15), without any protease inhibitors. On Pal-TRE (Fig. 4A), the nuclear extract from monolayer cultures (M-N) mainly formed a faster complex with TRbeta (TRbeta  + M-N), whereas the one from spheroid demonstrated two retarded bands (TRbeta  + S-N). The mixture of in vitro translated TRbeta , RXRalpha , and monolayer cytoplasmic extract (TRbeta  + RXRalpha  + M-C) formed a similar TRbeta -RXRalpha heterodimer. However, when in vitro translated RXRalpha was preincubated with the cytoplasmic extract from monolayer cultures at 37 °C for 30 min and then EMSA was performed with TRbeta , the faster migrating complex was demonstrated (Fig. 4A, 7th lane, Inc (RXRalpha  + M-C) + TRbeta ). This faster band was not observed by the preincubation of TRbeta with the cytoplasmic extract (Inc (TRbeta  + M-C) + RXRalpha ). These results suggest that the proteolytic cleavage of RXRalpha , but not TRbeta , generates the faster migrating complex in EMSA. After preincubation of in vitro translated RXRalpha with the same amount (10 µg) of cytoplasmic extract either from monolayer or spheroid cultured hepatocytes, the faster migrating complex was predominantly demonstrated with monolayer but not with spheroid extracts (Fig. 4B). It was thus suggested that RXRalpha cleaving activity was higher in the cytoplasmic extract of monolayer cultures, leading to a decrease of slower migrating complex in the nuclear extract from monolayer cultures.


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  		Fig. 4.   Proteolytic cleavage of RXRalpha by cytoplasmic extract. A, when the in vitro translated RXRalpha was preincubated with the cytoplasmic extract of monolayer cultures at 37 °C for 30 min and then EMSA was performed with TRbeta (Inc (RXRalpha  + M-C) + TRbeta ), the faster migrating complex (F) was demonstrated. This faster band was not produced by the preincubation of TRbeta with the cytoplasmic extract from the monolayer hepatocytes before addition of RXRalpha (Inc (TRbeta  + M-C) + RXRalpha ) or the mixture of these three with no preincubation (TRbeta  + RXRalpha  + M-C). B, to estimate the proteolytic activity of the cytoplasmic extract from the hepatocytes cultured in the two conditions, EMSA was performed after the preincubation of in vitro translated RXRalpha with the same amount (10 µg) of cytoplasmic extract from either monolayer or spheroid cultures. The faster migrating complex was observed by an incubation with the cytoplasmic extract from the monolayer (Inc (RXRalpha  + M-C) + TRbeta ), whereas it was not with that from the spheroids (Inc (RXRalpha  + S-C) + TRbeta ). Control, lysate alone; TRbeta , in vitro translated TRbeta ; RXRalpha , in vitro translated RXRalpha ; M-N, monolayer nuclear extract; S-N, spheroid nuclear extract; M-C, monolayer cytoplasmic extract; S-C, spheroid cytoplasmic extract; S, slower migrating complex; F, faster migrating complex.

To analyze the time course of RXRalpha cleavage by the cytoplasmic enzyme, 35S-labeled in vitro translated RXRalpha was incubated with the cytoplasmic extract from monolayer cultures and analyzed on SDS-PAGE (Fig. 5A). Surprisingly, full-length RXRalpha (54 kDa) was cleaved very rapidly, generating a 42-kDa band after a 1-min incubation. The proteolysis of RXRalpha was not observed with preheated (65 °C, 3 min) cytoplasmic extract (preheated CE + 60 min).


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  		Fig. 5.   Proteolysis of RXRalpha studied by SDS-PAGE. A, 35S-labeled in vitro translated RXRalpha was incubated with the cytoplasmic extract from the monolayer hepatocyte for various length of time and analyzed by 12% SDS-PAGE. The proteolysis of RXRalpha was very rapid and generated RXRalpha molecule with a molecular mass of 42 kDa. This cleavage was not observed with the preheated (65 °C, 3 min) cytoplasmic extract. B, the proteolytic activity in nuclear and cytoplasmic extracts was compared. 35S-Labeled in vitro translated RXRalpha was incubated with same amount (10 µg) of either nuclear or cytoplasmic extract from monolayer hepatocytes for 30 min at 37 °C and was analyzed by 12% SDS-PAGE. The RXRalpha cleaving activity was predominant in the cytoplasmic extract (RXRalpha  + CE). Although the crude nuclear extract prepared by Schreiber method has some proteolytic activity (RXRalpha  + crude NE), no activity was observed in the purified nuclear extract prepared by the modified Shapiro method (RXRalpha  + purified NE).

To compare the proteolytic activity in the nuclear and the cytoplasmic extracts, 35S-labeled in vitro translated RXRalpha was incubated with each extract (Fig. 5B). The RXRalpha -cleaving activity was predominantly in the cytoplasmic extract (RXRalpha  + CE). Although the crude nuclear extract prepared by the Shreiber method had minor proteolytic activity (RXRalpha  + crude NE), no RXR cleaving activity was observed in the highly purified nuclear extract by the modified Shapiro method (RXRalpha  + purified NE). It is thus concluded that RXR proteolytic activity exists mainly in the cytoplasm and that the presence of proteolytic activity in the nuclear extract could be due to contamination by the cytoplasmic fraction.

To characterize the product after RXRalpha cleavage, an immunoprecipitation experiment was performed using the two RXRalpha antibodies employed in the supershift analysis (Fig. 6). 35S-Labeled full-length RXRalpha (54 kDa) was immunoprecipitated with either anti-RXRalpha antibody N or C. The 42-kDa RXRalpha cleaved by the cytoplasmic extract was recognized by antibody C but not by antibody N, indicating that this RXRalpha form lacks the amino terminus. This result is consistent with our EMSA results, which suggest the faster migrating complex contains RXRalpha with an amino-terminal truncation (Fig. 2).


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  		Fig. 6.   Immunoprecipitation of cleaved RXRalpha by two RXRalpha antibodies. To characterize the cleaved RXRalpha molecules, 35S-labeled RXRalpha was incubated with the cytoplasmic extract from the monolayer and spheroid hepatocytes and subjected to immunoprecipitation by anti-TRbeta antibody (Ab T) or two anti-RXRalpha antisera (Ab N or C). Undigested full-length RXRalpha (55 kDa) was immunoprecipitated by both anti-RXRalpha , whereas the cleaved RXRalpha (42-kDa band) was only precipitated by the antibody C, indicating that the cleaved form of RXRalpha lacks the amino-terminal region. RXRalpha , in vitro translated RXRalpha ; RXRalpha  + M-C, in vitro translated RXRalpha incubated with the cytoplasmic extract from monolayer hepatocytes; Ab T, TRbeta antibody; N, antibody against RXRalpha amino terminus; C, antibody against hinge region of RXRalpha .

Characterization of the Cytoplasmic Enzyme Cleaving RXRalpha -- To characterize the enzyme responsible for RXRalpha cleavage, a series of protease inhibitors were added to the preincubation step, and then EMSA was performed with in vitro translated TRbeta (Fig. 7A). Without any protease inhibitors, the preincubation of in vitro translated RXRalpha with monolayer cytoplasmic extract demonstrated the faster migrating complex with TRbeta on Pal-TRE in EMSA (4th lane, Inc (RXRalpha  + M-C) + TRbeta ). The generation of the faster band (RXRalpha proteolysis) was inhibited by antipain, chymostatin, E64, and leupeptin, suggesting that the enzyme responsible for RXRalpha proteolysis belongs to the family of cysteine proteases. To identify further the protease species among the cysteine proteases, calpastatin or ovocystatin was added to the incubation mixture of 35S-labeled in vitro translated RXRalpha and the cytoplasmic extract from the monolayer hepatocytes. As shown in Fig. 7B, the presence of ovocystatin, but not calpastatin, inhibited proteolysis, suggesting that the cathepsin family of proteases, but not the calpain family, cleaves RXRalpha . When specific inhibitors for cathepsin B or L (cathepsin B-specific, CA-074 (26), and cathepsin L-type-specific, CLIK-088 and -112 (27)) were added to the reaction, cathepsin L-type-specific, CLIKs (CLIK-088 and -112), but not cathepsin B-specific, CA-074, inhibited the RXRalpha cleaving activity in the cytoplasmic extract (Fig. 7C). These results indicate that cathepsin L-type protease in the cytoplasm of hepatocytes cleaves RXRalpha at its amino terminus.


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  		Fig. 7.   Characterization of protease present in the cytoplasm of the hepatocytes cultured as monolayer. A, a series of protease inhibitors were added before EMSA. The generation of faster migrating band due to the proteolysis of RXRalpha by the cytoplasmic extract from monolayer hepatocytes (Inc (RXRalpha  + M-C) + TRbeta ) was inhibited by antipain, chymostatin, E64, and leupeptin, indicating the RXRalpha -cleaving enzyme belongs to the family of cysteine proteases. TRbeta , in vitro translated TRbeta ; RXRalpha , in vitro translated RXRalpha ; M-N, monolayer nuclear extract; S-N, spheroid nuclear extract; M-C, monolayer cytoplasmic extract; S, slower migrating complex; F, faster migrating complex. B, to further characterize RXRalpha -cleaving protease, 35S-labeled in vitro translated RXR was incubated with the cytoplasmic extract from monolayer hepatocytes in the presence of ovocystatin or calpain and analyzed in SDS-PAGE. Only ovocystatin inhibited the RXRalpha cleavage, suggesting that the enzyme is one of the cathepsin family proteases. C, when cathepsin B (CA-074) or L-type-specific inhibitors (CLIK-088 and -112) were added to the incubation reaction, cathepsin L-type-specific inhibitors (CLIK-088 and CLIK-112), but not cathepsin B-specific CA-074, inhibited proteolytic cleavage of RXRalpha by cytoplasmic extract, indicating that cathepsin L-type protease is the enzyme that cleaves RXRalpha at its amino terminus.

Activity of Cathepsin L-type Protease Is Higher in the Cytoplasmic Extract of Monolayer than That in Spheroids-- Since the RXRalpha -cleaving enzyme in the cytoplasm was identified as cathepsin L-type protease, this enzyme activity was measured in the cytoplasmic extract of monolayer and spheroid cultured hepatocytes. As shown in Fig. 8, the (7-amino-4-methylcoumarin) liberating activity from Z-Phe-Arg-MCA (Z-Phe-Arg-4-methylcoumaryl-7-amide, a substrate for both cathepsin B and L) in the presence of CA-074 was greater in hepatocytes cultured as monolayer than those as spheroid. These results correlate well the higher RXRalpha cleaving activity in the monolayer hepatocytes (Fig. 4B).


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  		Fig. 8.   Cathepsin L activity in monolayer and spheroid-cultured hepatocytes. Ten micromolar of Z-Phe-Arg-MCA was incubated with different amounts of cytoplasmic extract from monolayer or spheroid hepatocytes in the presence of cathepsin B-specific inhibitor, CA-074. The amount of 7-amino-4-methylcoumarin liberated from substrate per min/ml was plotted. The data were expressed as mean ± S.D. from triplicate determinations. The activity in the hepatocytes cultured as monolayer was higher than that in spheroids.

Inhibition of RXRalpha -cleaving Enzyme by E64d Augments T3-dependent Induction of Spot 14 Gene-- To study the physiological significance of RXRalpha -cleaving protease in cytosol, a membrane-permeable cysteine protease inhibitor, E64d, which also inhibits cathepsin L-type protease activity, was added to the hepatocytes cultured as a monolayer. The proteolytic activity in the cells was monitored by the incubation of in vitro translated RXRalpha with the cytoplasmic extract cultured in the absence or presence of E64d. The RXRalpha cleaving activity was decreased with the addition of E64d in a dose-dependent manner (Fig. 9A). Under these conditions, the amount of RXRalpha present in the nuclear extract was studied by Western blot analysis. Compatible with inhibition of the cytoplasmic RXRalpha cleaving activity by E64d, the RXRalpha protein level in the nuclear extract from monolayer cultured hepatocytes increased in a dose-dependent manner (Fig. 9B). The intensity of the RXRalpha protein band increased to 1.14- and to 1.22-fold by incubation with 1.0 and 2.5 µM E64d, respectively. The intensity of RXRalpha band from spheroid cultured hepatocytes was 1.16-fold to that from monolayer cultured hepatocytes. Addition of E64d to monolayer cultured hepatocytes did not affect the content of protein or DNA. However, protein per unit of DNA was more in the spheroids than that in the monolayered hepatocytes (data not shown). The level of RXRalpha protein per unit of DNA was thus calculated. As shown in Fig. 9C, the RXRalpha protein level per unit of DNA from spheroid cultured was more abundant (1.57-fold) than that from monolayers, even in the presence of E64d.


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  		Fig. 9.   Inhibition of cytoplasmic RXRalpha cleaving activity by E64d increased nuclear RXRalpha protein in monolayer hepatocytes. A, primary hepatocytes were cultured as monolayer in the absence or presence of membrane-permeable cysteine protease inhibitor E64d. The inhibition of RXRalpha cleaving activity by E64d was monitored by an incubation of in vitro translated RXRalpha with the cytoplasmic extracts. The activity cleaving in vitro translated RXRalpha was decreased by the addition of E64d in a dose-dependent manner. B, the amount of RXRalpha protein in the nuclear extract from the monolayer cultured hepatocytes with E64d incubation was determined by Western blot analysis. The addition of E64d increased RXRalpha protein level in a dose-dependent manner. C, when the RXRalpha protein level per unit of DNA was calculated, the level of RXRalpha protein was more abundant in the nuclear extract from spheroid cultured than in the one from monolayered, even with E64d treatment. The mean ± S.D. of triplicates is shown.

T3-dependent induction of spot 14 mRNA was investigated by Northern blot analysis (Fig. 10A). The induction of spot 14 mRNA was 1.4-fold by 100 nM T3 in hepatocytes under monolayer cultures, whereas it was 2.8-fold in spheroid cultures (p < 0.01 versus monolayer) (Fig. 10B). Incubation of monolayer cultured hepatocytes with E64d increased T3-dependent induction of spot 14 gene 1.9-fold in the presence of 0.2 µM, 2.0-fold with 1.0 µM (p < 0.05 versus no treatment), and 2.1-fold with 2.5 µM of E64d (p < 0.05).


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  		Fig. 10.   Augmentation of T3-dependent activation of spot 14 gene in monolayer hepatocytes by E64d. A, T3-dependent induction of spot 14 mRNA was studied in monolayer cultured hepatocytes with different concentration of E64d by Northern blot analysis. The presence of E64d augmented T3-dependent induction of spot 14 mRNA. B, the radioactivity of spot 14 bands were determined by using Fuji Bioimage Analyzer (BAS 2000), and the fold induction of spot 14 mRNA by T3 was calculated (mean ± S.D.). The induction was 1.4-fold by 100 nM T3 in hepatocytes under monolayer cultures. It increased to 1.9-fold in the presence of 0.2 µM, 2.0-fold at 1.0 µM, and 2.1-fold at 2.5 µM of E64d. In spheroid cultures, T3-dependent induction of spot 14 was 2.8-fold. *, p < 0.05; **, p < 0.01; versus monolayer without E64d.

Thus, the inhibition of RXRalpha cleaving activity in the cytoplasm leads to an increase of nuclear RXRalpha protein and augmentation of T3-dependent transactivation in vivo.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we demonstrated that the mobility of in vitro translated TRbeta -hepatocyte TRAP complex on EMSA could be altered by proteolytic cleavage of endogenous RXRalpha with a cytoplasmic protease. This proteolytic enzyme is present in the cytoplasm and is characterized as a lysosomal enzyme, cathepsin L-type protease. Additionally, the RXR cleaving activity was stronger in hepatocytes cultured as monolayer than those as spheroid cultures. Of note, the cleaved form of RXRalpha is not present in the nucleus. Inhibition of cathepsin L-type protease activity increased nuclear RXRalpha protein and augmented T3-dependent induction of spot 14 mRNA, suggesting that cathepsin L-type protease might alter turnover of RXRs and modulate hormonal responsiveness. If this hypothesis is true, the responsiveness of receptors such as retinoic acid receptor that form heterodimers with RXRalpha should be decreased in the hepatocytes cultured as monolayer. Indeed, retinoic acid receptor beta  expression in response to retinoic acid was also blunted in the hepatocytes cultured as monolayers.2

Heterogeneity of TRAPs by Proteolysis of RXRs-- In the past, TRAPs which enhance TR binding on TREs have been extensively studied in different cell lines or tissues using cross-linking experiments or EMSA (34-38). These studies have demonstrated that the different sizes of proteins ranging from 42 to 65 kDa might exist as TRAPs (34, 37, 38). Later TRAPs, in part, were identified as RXRs after their cloning (7, 8, 39, 40), and specific antibodies against RXRs classified most TRAPs in cells as RXR isoforms (9, 34). Interestingly, TRAPs in some cell lines formed protein-DNA complexes with mobilities that did not correspond to complexes formed with authentic RXRs (9, 34). The mobility of protein-DNA complexes of TRAPs in liver also varied among the reports, even though TRAPs in liver were identified mainly as RXRalpha by a specific antibody (34). One report (35) described two protein-DNA complexes using the liver nuclear extracts, whereas other reports (9, 34) demonstrated only the faster migrating complex whose mobility was close to that of a TR homodimer. These EMSA patterns resembled those observed in the present study using hepatocytes cultured under two conditions. The present study raises the possibility that during the preparation of nuclear extract, contamination with the cytoplasmic fraction leads to the cleavage of RXRalpha , generating TRAPs with different sizes, especially in the cells with higher protease activity such as monolayer cultured hepatocytes. Therefore, the heterogeneity of RXRs reported in different cells could be explained, in part, by the proteolytic cleavage of RXRs during the preparation process of nuclear extract.

Physiological Significance of RXR Proteolysis by Cathepsin L-type Protease-- This study clearly demonstrates that cleaved RXR is not present in the nucleus, because the faster migrating band due to amino-terminal truncated RXRalpha was not demonstrated with the highly purified nuclear extract (Fig. 3). Furthermore, the RXRalpha cleaving activity was hardly detected in the purified nuclear extract (Fig. 5B). In contrast, Matsushima et al. (41) recently reported the presence of RXRalpha cleaving activity in the nuclei of HuH-7 cells derived from a human hepatocellular carcinoma. The enzyme also cleaved RXRalpha at the junction between A/B and C domain. They suggested that the enzyme could be calpain. Although RXR-cleaving protease in the primary cultured hepatocytes belongs to the family of cysteine protease as revealed in our present study, it is not calpain because calpastatin, a specific inhibitor of calpain, did not inhibit proteolysis of RXRs. The discrepancy could be due to differences in species or the nature of cells, i.e. normal hepatocytes versus transformed cells.

It is important to consider the physiological role of RXR proteolysis by cathepsin L-type protease in vivo. Our results that protease inhibitor E64d augmented T3-dependent expression of spot 14 gene in the hepatocytes cultured as monolayer suggest that cathepsin L-type protease may influence responsiveness to T3 by altering the turnover of RXRalpha . The lysosome is a cellular component containing multiple hydrolytic enzymes to degrade proteins or organelles. Its functions are divided into two aspects, heterophagy and autophagy. Heterophagy is concerned with enclosure and degradation of exogenous substances (proteins or bacteria) through endocytosis or phagocytosis. On the other hand, autophagy serves to scavenge obsolete proteins or organelles in cells for regulating their turnover. This is essential to maintain homeostasis of cells and to reconstruct cellular architecture. Thus, as in the case for several cytosolic proteins or organelles (42), nuclear proteins might be degraded in lysosomes.

Two main proteolytic pathways in the cytosol are ubiquitin-proteasome system and calpain. They cleave cytosolic proteins by regulating their turnover. Furthermore, the ubiquitin-proteasome system is well known to degrade and regulate turnover of several nuclear proteins including cyclins (43), NF-kappa B (44, 45), and c-Jun (46). Calpain also cleaves a number of transcription factors such as p53 (47, 48), Jun/Fos (49, 50), RXR (41), NF-kappa B (51), Pit-1 (49), and Oct-1 (49) in vitro. However, the physiological role of calpain proteolysis was only reported in the case of p53 (47, 48).

The present study provides evidence that a lysosomal enzyme, cathepsin L-type protease, could be yet another pathway to regulate the turnover of nuclear proteins. The presence of c-Fos immunoreactivity in rat liver lysosomes could support the role of the lysosomal proteolytic pathway of the action of nuclear factors (52). What are the implications of the lysosomal degradation pathway of nuclear proteins? One can speculate that this pathway might degrade nuclear proteins that are not efficiently degraded by other proteolytic systems. For example, c-Jun can be degraded by ubiquitin-proteasome system, but v-Jun is resistant to the degradation, resulting in its potent oncogenic activity (46). The increased stability of v-Jun is due to a deleted region (delta domain) that alters ubiquitination of protein. However, even without any signal for cleavage, the relatively stable v-Jun protein should also be degraded. The lysosomal degradation system might be an alternative pathway to maintain the life cycle of nuclear proteins. Further studies are required in the future to investigate the RXR molecule from synthesis to degradation.

Alteration of Proteolytic Activity by Culture Conditions-- Since the activity of cathepsin L-type protease to cleave RXR could be one of the determinants for it turnover, the regulation of the protease activity would play an important role in hormonal signaling. The different proteolytic activities in hepatocytes cultured in two different conditions may provide clues to understanding the physiological regulation by this protease. The hepatocytes in the conventional monolayer cultures are reported to enter the rapid growth phase (G1), whereas the cells forming a spherical aggregate are retained in the G0 phase of cell cycle (53). These differences were also supported by results showing that AP-1 proteins that serve as an oncogenic marker were highly expressed, and the differentiation markers such as C/EBPalpha or -beta were repressed in monolayer cultures (54). Reciprocally, AP-1 proteins were low and C/EBPs were high in the hepatocytes on Englebreth-Holm-Swarm gel (basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor), in which cells formed small aggregates similar to spheroids. Cathepsin L mRNA has been reported to be induced by several growth factors such as platelet-derived growth factor (55), 12-O-tetradecanoylphorbol-13-acetate (56), fibroblast growth factor (57), or cell density (58). Furthermore, the promoter region of rat cathepsin L contains two AP2 sites through which 12-O-tetradecanoylphorbol-13-acetate could increase its transcription (59). The higher cathepsin L activity in the growing cells such as those cultured in monolayer could impair responsiveness to T3 through enhanced degradation of RXRalpha .

Since our results showed that the inhibition of cathepsin L-type protease by E64d did not completely restore the responsiveness to T3 in monolayer hepatocytes up to the level in spheroids, other factors rather than cathepsin L-type protease may be responsible for the preservation of responsiveness to T3 in spheroids. The three-dimensional cyto-architecture under spheroid cultures may also be critical for hepatocytes to maintain differentiated function of liver in vivo.

    ACKNOWLEDGEMENTS

We are grateful to Dr. R. M. Evans for providing the TRbeta and RXRalpha cDNAs and RXRalpha antibody (the hinge region) and Dr. C. N. Mariash for the spot 14 cDNA. Taisho Pharmaceutical Company is acknowledged for the provision of E64d. We thank to Dr. T. Maki for helpful discussions.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Tel.: 81-52-789-3867; Fax: 81-52-789-3887; E-mail: hseo{at}riem.nagoya-u.ac.jp.

The abbreviations used are: TR, thyroid hormone receptors; RXRalpha , 9-cis-retinoic acid receptor alpha ; TRE, thyroid-hormone responsive element; TRAP, TR auxiliary proteins; DR, direct repeat; EMSA, electrophoretic mobility shift assay; MCA, methylcoumaryl-7-amide; PAGE, polyacrylamide gel electrophoresis; Z, benzyloxycarbonyl; Pal, palindrome; Lap, inverted palindrome.

2 M. Menjo, Y. Murata, T. Nagaya, and H. Seo, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Oppenheimer, J. H., Schwartz, H. L., Mariash, C. N., Kinlaw, W. B., Wong, N. C., and Freake, H. C. (1987) Endocr. Rev. 8, 288-308[Abstract/Free Full Text]
  2. Ribeiro, R. C., Apriletti, J. W., West, B. L., Wagner, R. L., Fletterick, R. J., Schaufele, F., and Baxter, J. D. (1995) Ann. N. Y. Acad. Sci. 758, 366-389[Medline] [Order article via Infotrieve]
  3. Lazar, M. A. (1993) Endocr. Rev. 14, 184-193[Abstract/Free Full Text]
  4. Glass, C. K. (1994) Endocr. Rev. 15, 391-407[Abstract/Free Full Text]
  5. Hermann, T., Hoffmann, B., Piedrafita, F. J., Zhang, X. K., and Pfahl, M. (1993) Oncogene 8, 55-65[Medline] [Order article via Infotrieve]
  6. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J. Y., Staub, A., Garnier, J. M., Mader, S., and Chambon, P. (1992) Cell 68, 377-395[CrossRef][Medline] [Order article via Infotrieve]
  7. Zhang, X. K., Hoffmann, B., Tran, P. B., Graupner, G., and Pfahl, M. (1992) Nature 355, 441-446[CrossRef][Medline] [Order article via Infotrieve]
  8. Marks, M. S., Hallenbeck, P. L., Nagata, T., Segars, J. H., Appella, E., Nikodem, V. M., and Ozato, K. (1992) EMBO J. 11, 1419-1435[Medline] [Order article via Infotrieve]
  9. Sugawara, A., Yen, P. M., Darling, D. S., and Chin, W. W. (1993) Endocrinology 133, 965-971[Abstract/Free Full Text]
  10. Zilz, N. D., Murray, M. B., and Towle, H. C. (1990) J. Biol. Chem. 265, 8136-8143[Abstract/Free Full Text]
  11. Petty, K. J., Desvergne, B., Mitsuhashi, T., and Nikodem, V. M. (1990) J. Biol. Chem. 265, 7395-7400[Abstract/Free Full Text]
  12. Toyoda, N., Zavacki, A. M., Maia, A. L., Harney, J. W., and Larsen, P. R. (1995) Mol. Cell. Biol. 15, 5100-5112[Abstract]
  13. Hodin, R. A., Lazar, M. A., and Chin, W. W. (1990) J. Clin. Invest. 85, 101-105
  14. Menjo, M., Murata, Y., Fujii, T., Nimura, T., and Seo, H. (1993) Endocrinology 133, 2984-2990[Abstract/Free Full Text]
  15. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Free Full Text]
  16. Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988) DNA (N. Y.) 7, 47-55[Medline] [Order article via Infotrieve]
  17. Burton, K. (1956) Biochem. J. 62, 248-260[Medline] [Order article via Infotrieve]
  18. Ichikawa, K., and DeGroot, L. J. (1987) Mol. Cell. Endocrinol. 51, 135-143[CrossRef][Medline] [Order article via Infotrieve]
  19. Nagaya, T., and Jameson, J. L. (1993) J. Biol. Chem. 268, 24278-24282[Abstract/Free Full Text]
  20. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., and Evans, R. M. (1986) Nature 324, 641-646[CrossRef][Medline] [Order article via Infotrieve]
  21. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229[CrossRef][Medline] [Order article via Infotrieve]
  22. Nagaya, T., Nomura, Y., Fujieda, M., and Seo, H. (1996) Biochem. Biophys. Res. Commun. 226, 426-430[CrossRef][Medline] [Order article via Infotrieve]
  23. Sugawara, A., Yen, P. M., Qi, Y., Lechan, R. M., and Chin, W. W. (1995) Endocrinology 136, 1766-1774[Abstract]
  24. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. M. (1992) Genes Dev. 6, 329-344[Abstract/Free Full Text]
  25. Towatari, T., Nikawa, T., Murata, M., Yokoo, C., Hanada, K., and Katunuma, N. (1991) FEBS Lett. 280, 311-315[CrossRef][Medline] [Order article via Infotrieve]
  26. Katunuma, N., and Kominami, E. (1995) in Biothiols: (Paker, L., ed), pp. 382-397, Academic Press, San Diego
  27. Katunuma, N. (1997) J. Bone Miner. Metab. 15, 1-8
  28. Murata, Y., Sueda, K., Seo, H., and Matsui, N. (1989) Endocrinology 125, 1424-1429[Abstract/Free Full Text]
  29. Inubushi, T., Kakegawa, H., Kishino, Y., and Katunuma, N. (1994) J. Biochem. (Tokyo) 116, 282-284[Abstract/Free Full Text]
  30. Olbricht, C. J., Cannon, J. K., Garg, L. C., and Tisher, C. C. (1986) Am. J. Physiol. 250, 1055-1062
  31. Kikumori, T., Kambe, F., Nagaya, T., Imai, T., Funahashi, H., and Seo, H. (1998) Endocrinology 139, 1715-1722[Abstract/Free Full Text]
  32. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[Medline] [Order article via Infotrieve]
  33. Yen, P. M., Darling, D. S., Carter, R. L., Forgione, M., Umeda, P. K., and Chin, W. W. (1992) J. Biol. Chem. 267, 3565-3568[Abstract/Free Full Text]
  34. Lazar, M. A., Berrodin, T. J., and Harding, H. P. (1991) Mol. Cell. Biol. 11, 5005-5015[Abstract/Free Full Text]
  35. Murray, M. B., and Towle, H. C. (1989) Mol. Endocrinol. 3, 1434-1442[Abstract/Free Full Text]
  36. Lazar, M. A., and Berrodin, T. J. (1990) Mol. Endocrinol. 4, 1627-1635[Abstract/Free Full Text]
  37. O'Donnell, A. L., and Koenig, R. J. (1990) Mol. Endocrinol. 4, 715-720[Abstract/Free Full Text]
  38. Glass, C. K., Devary, O. V., and Rosenfeld, M. G. (1990) Cell 63, 729-738[CrossRef][Medline] [Order article via Infotrieve]
  39. Yu, V. C., Delsert, C., Andersen, B., Holloway, J. M., Devary, O. V., Naar, A. M., Kim, S. Y., Boutin, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 67, 1251-1266[CrossRef][Medline] [Order article via Infotrieve]
  40. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J., and Evans, R. M. (1992) Nature 355, 446-449[CrossRef][Medline] [Order article via Infotrieve]
  41. Matsushima-Nishiwaki, R., Shidoji, Y., Nishiwaki, S., Moriwaki, H., and Muto, Y. (1996) Biochem. Biophys. Res. Commun. 225, 946-951[CrossRef][Medline] [Order article via Infotrieve]
  42. Lee, H.-K., and Marzella, L. (1994) Int. Rev. Exp. Pathol. 35, 39-147[Medline] [Order article via Infotrieve]
  43. King, R. W., Deshaies, R. J., Peters, J. M., and Kirschner, M. W. (1996) Science 274, 1652-1659[Abstract/Free Full Text]
  44. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785[CrossRef][Medline] [Order article via Infotrieve]
  45. Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van, A. D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735[Free Full Text]
  46. Treier, M., Staszewski, L. M., and Bohmann, D. (1994) Cell 78, 787-798[CrossRef][Medline] [Order article via Infotrieve]
  47. Kubbutat, M. H., and Vousden, K. H. (1997) Mol. Cell. Biol. 17, 460-468[Abstract]
  48. Pariat, M., Carillo, S., Molinari, M., Salvat, C., Debussche, L., Bracco, L., Milner, J., and Piechaczyk, M. (1997) Mol. Cell. Biol. 17, 2806-2815[Abstract]
  49. Watt, F., and Molloy, P. L. (1993) Nucleic Acids Res. 21, 5092-5100[Abstract/Free Full Text]
  50. Carillo, S., Pariat, M., Steff, A.-M., Roux, P., Etienne-Julan, M., Lorca, T., and Piechaczyk, M. (1994) Oncogene 9, 1679-1689[Medline] [Order article via Infotrieve]
  51. Liu, Z. Q., Kunimatsu, M., Yang, J. P., Ozaki, Y., Sasaki, M., and Okamoto, T. (1996) FEBS Lett. 385, 109-113[CrossRef][Medline] [Order article via Infotrieve]
  52. Aniento, F., Papavassiliou, A. G., Knecht, E., and Roche, E. (1996) FEBS Lett. 390, 47-52[CrossRef][Medline] [Order article via Infotrieve]
  53. Yuasa, C., Tomita, Y., Shono, M., Ishimura, K., and Ichihara, A. (1993) J. Cell. Physiol. 156, 522-530[CrossRef][Medline] [Order article via Infotrieve]
  54. Rana, B., Mischoulon, D., Xie, Y., Bucher, N. L., and Farmer, S. R. (1994) Mol. Cell. Biol. 14, 5858-5869[Abstract/Free Full Text]
  55. Okamura, N., Tamba, M., Uchiyama, Y., Sugita, Y., Dacheux, F., Syntin, P., and Dacheux, J. L. (1995) Biochim. Biophys. Acta 1245, 221-226[Medline] [Order article via Infotrieve]
  56. Atkins, K. B., and Troen, B. R. (1995) Cell Growth Differ. 6, 713-718[Abstract]
  57. Nilsen, H. M., Jang, Y. J., Delgado, M., Shim, J. K., Bruns, K., Chiang, C. P., Fang, Y., Parfett, C. L., Denhardt, D. T., and Hamilton, R. T. (1991) Mol. Cell. Endocrinol. 77, 115-122[CrossRef][Medline] [Order article via Infotrieve]
  58. Karzai, A. W., and Wright, W. W. (1992) Biol. Reprod. 47, 823-831[Abstract]
  59. Ishidoh, K., Kominami, E., Suzuki, K., and Katunuma, N. (1989) FEBS Lett. 259, 71-74[CrossRef][Medline] [Order article via Infotrieve]

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