Analysis of Differentiation-Induced Expression Mechanisms of Thyrotropin Receptor Gene in Adipocytes

Hiroki Shimura, Asako Miyazaki, Kazutaka Haraguchi, Toyoshi Endo and Toshimasa Onaya

The Third Department of Internal Medicine Yamanashi Medical University Yamanashi 409–3898, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Autoantibodies to the TSH receptor (TSHR), which are present in patients with Graves’ disease, increase thyroidal cAMP levels and cause hyperfunctioning of the thyroid gland (1). In addition, Graves’ disease patients with a high titer of thyroid-stimulating antibodies often exhibit exophthalmos and pretibial dermopathy (2). Although TSH has been thought to act solely in the control of thyroid function, extrathyroidal TSH binding has also been documented (3, 4, 5, 6, 7, 8, 9, 10, 11), suggesting that expression of TSHR in these cells may account for the extrathyroidal manifestations of Graves’ disease.

Although several extrathyroidal cells, such as lymphocytes and fibroblasts, have been shown to express TSHR mRNA detectable only by RT-PCR (12, 13, 14, 15), functional TSHR has been detected in adipocytes (3, 16, 17, 18) and may be involved in the regulation of adipose function. We have recently shown that rat adipose tissue (19), as well as cultured rat preadipocytes (20), express amounts of TSHR mRNA similar to that detected in the thyroid gland. The full-length TSHR cDNA, which we isolated from rat fat cells (19), differed by only one amino acid from the rat thyroid TSHR cDNA. Moreover, when transfected into Chinese hamster ovary (CHO) cells, rat adipocyte TSHR cDNA was functionally indistinguishable from thyroid TSHR cDNA (19). These observations suggest that the presence of functional TSHR in adipocytes is related to the extrathyroidal manifestations of Graves’ disease.

A suitable model for the study of adipocytes is 3T3-L1 cells, a fibroblast-like cell line derived from the Swiss mouse embryo, which, upon reaching confluence, can be induced to differentiate into adipocytes by the hormonal pulse with insulin, isobutylmethylxanthine, and dexamethasone (21, 22). In addition, we recently showed that this differentiation was accompanied by the expression of TSHR mRNA (23). This cultured cell system is therefore useful for studying the regulation of TSHR gene expression in adipocytes.

Our recent report (24) showed that, in 3T3-L1 adipocytes, TSH induces cAMP-mediated down-regulation of TSHR gene transcription. The mechanism of TSHR mRNA regulation in 3T3-L1 cells, however, was different from that in thyroid cells. For example, in the former, TSH-induced down-regulation of TSHR mRNA is evident within 1 h and peaks within 4 h; the transient increase in TSHR mRNA detected in the rat thyroid cell line, FRTL-5, is not observed in 3T3-L1 adipocytes. While the down-regulation of TSHR gene expression in FRTL-5 cells was found to be cycloheximide sensitive, that in 3T3-L1 cells is cycloheximide resistant. Furthermore, while insulin or serum was found to be required for TSH-induced regulation of TSHR mRNA in FRTL-5 cells, neither was required for down-regulation in 3T3-L1 cells.

The 5'-flanking region of the rat TSHR gene has recently been cloned (25), and a minimal promoter region, -220 to -39 bp, exhibiting thyroid-specific expression and TSH/cAMP regulation, has been identified (26, 27, 28, 29, 30). The region between -220 and -177 bp was shown to act as a thyroid-specific enhancer (28, 29, 30). Within this region, a regulatory element from -189 to -175 bp was observed to bind thyroid transcription factor-1 (TTF-1) (28, 31, 32), and single-strand DNA-binding proteins (SSBPs) were found to interact with an element contiguous with the 5'-end of the TTF-1 element (30, 33). TTF-1 and the SSBPs have been observed to function jointly in the regulation of TSHR gene expression in thyroid cells (28, 30, 32, 33). The region between -220 and -192 bp was found to act as an insulin-response element, the activity of which appears to be thyroid specific (29). A sequence between -139 and -132 bp, TGAGGTCA, which is homologous to a consensus cAMP-response element (CRE), TGACGTCA, was found to function as a constitutive enhancer and to be responsible for efficient expression of this gene (26, 27, 28).

Our previous findings, that adipose cells do not express TTF-1 and that hormonal regulation of TSHR gene expression in adipose cells is different from that in thyroid cells (24), led us to hypothesize that the TSHR gene expression in adipocytes may be regulated by a different set of transcription factors from that in thyroid cells, or may be directed by an alternative promoter. We have therefore dissected the 5'-flanking region of the rat TSHR gene The present study shows that the TSHR mRNAs in rat adipocytes are transcribed from almost identical transcriptional start sites to those used in thyroid cells. Using 3T3-L1 adipocytes, we identify an element responsible for the TSHR gene expression in adipocytes. This element includes the cAMP response element (CRE)-like site and Ets-binding motifs and differs from the thyroid-specific enhancer element containing TTF-1-binding sequence. We further demonstrate that repressing factors whose binding to DNA are diminished in differentiated 3T3-L1 adipocytes interact within the fat-specific element.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Determination of Transcriptional Initiation Sites in Rat Adipose Tissue
We performed primer extension to identify the transcriptional initiation sites in the 5'-flanking region of the TSHR gene in rat adipose tissue. The extension products observed with rat adipose tissue RNA were identical to those detected in RNA from FRTL-5 thyroid cells (Fig. 1AGo). The major sites of transcriptional initiation mapped at -83, -72, -69, and -68 bp (arrows in Fig. 1Go, A and B), which almost concurred with those in FRTL-5 cells identified in a previous report (25).



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Figure 1. Primer Extension and Anchored PCR Analyses of the Transcriptional Initiation Sites of the Rat TSHR Gene in Rat Fat Tissue

A, The transcriptional initiation sites were determined by primer extension analysis. As described in Materials and Methods, the end-labeled primer was hybridized to 50 µg total RNAs from rat epididymal fat tissue, FRTL-5, rat liver, and yeast tRNA as control. The primer was extended by reverse transcriptase, and the products were analyzed on PAGE. A sequencing ladder, using the same primer, was run in parallel. Major transcriptional initiation sites are indicated by arrows (in A and B). B, Dots indicate the 5'-end of the TSHR cDNA clones obtained by the anchored PCR. Nucleotide numbering is relative to the translational initiation site, which is designated 1. In addition, elements important for the TSHR gene expression in thyroid cells are indicated. Thus, the insulin-response element (29 ) and TTF-1-binding sequence (28 ) determined in FRTL-5 thyroid cells are indicated by single or double underlines, respectively. The dashed line indicates the SSBP-1-binding site (30 33 ). Horizontal arrows indicate the tandem repeat sequences in the repressive element that appear to be active ubiquitously (27 43 ). The CRE-like sequence is boxed.

 
When we also assayed transcriptional initiation sites by anchored PCR, we detected the 5'-end of clones at -110 (one clone), -106 (one clone), -91 (one clone), -86 (one clone), -83 (two clones), -81 (two clones), -79 (one clone), -72 (one clone), -68 (two clones), -64 (one clone), -62 (one clone), -45 (one clone), and -34 bp (one clone) (Fig. 1BGo). None of these clones contained alternative splicing in the 5'-untranslated region. Data from anchored PCR and primer extension experiments indicate that TSHR mRNAs in adipose tissue are transcribed from multiple sites almost identical to those in thyroid cells.

Identification of Regulatory Elements in the TSHR Promoter Required for Its Expression in Adipose Cells
Using electroporation, we transfected chimeric constructs of 5'- deletion mutants of the TSHR promoter (Fig. 2AGo), ligated to the chloramphenicol acetyltransferase (CAT) reporter gene, into 3T3-L1 cells before and after the induction of differentiation. In differentiated 3T3-L1 cells (adipocytes), the full-length plasmid, pTRCAT5'-1707 (5.3 ± 1.4%), expressed significantly higher CAT activity than the shortest construct, pTRCAT5'-90 (0.8 ± 0.2%). In undifferentiated 3T3-L1 cells (preadipocytes), however, there was no significant difference in CAT activity between pTRCAT5'-1707 and 5'-90. The differentiation of 3T3-L1 cells into adipocytes resulted in a 4.3-fold increase of CAT activity expressed by pTRCAT5'-1707.



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Figure 2. CAT Activities of Chimeric Constructs Containing 5'-Deletion of the 5'-Flanking Region of the TSHR in 3T3-L1 Cells before and after Differentiation

Regulatory elements of the TSHR promoter determined in FRTL-5 thyroid cells are diagrammatically presented in panel A. 3T3-L1 cells before (preadipocytes, open bars) or after (adipocytes, solid bars) were cotransfected with the noted plasmids and pCH110 by electroporation. Conversion rates were normalized to ß-galactosidase activities and are presented relative to the activity expressed by the pSV2CAT positive control. A statistically significant (P < 0.05) increase induced by differentiation is noted by an asterisk (*). The significant (P < 0.05) effect of 5'-deletion from pTRCAT5'-1707 on the CAT activity in the undifferentiated or differentiated cells is denoted by an open circle ({circ}) or a solid circle (•), respectively.

 
When we compared the activity of 5'-deletion mutants that lacked the sequences from -1707 to -1191 (5'-1190), -908 (5'-907), -639 (5'-638), and -420 (5'-419) bp in 3T3-L1 adipocytes, we observed no significant changes in CAT activity (Fig. 2BGo). The deletion mutant, pTRCAT5'-190 (9.8 ± 0.3%), however, exhibited significantly (P < 0.05) higher CAT activity than either pTRCAT5'-1707 (5.3 ± 1.4%) or 5'-220 (6 .5 ± 1.1%) (Fig. 2BGo). While deletion of the sequences from -190 to -178 bp, containing the TTF-1- and SSBP-1-binding sites determined in thyroid cells, and from -146 to -91 bp, containing the CRE-like element between -139 and -132 bp (Fig. 2AGo), significantly (P < 0.01) decreased CAT activity, deletion of the sequence from -177 to -147 bp, in which the TSHR suppressor element binding protein-1 (TSEP-1) interacted (Fig. 2AGo), increased CAT activity 22-fold (Fig. 2BGo). These data suggest that the regions of the TSHR gene between -190 to -178 bp and between -146 to -91 bp constitute positive regulatory elements, whereas sequences between -220 to -191 bp and between -177 to -147 bp constitute suppressor elements.

In 3T3-L1 preadipocytes, only the plasmid pTRCAT5'-146 exhibited significantly higher CAT activity than did pTRCAT5'-1707 (Fig. 2BGo). The CAT activity of pTRCAT5'-146, however, was 7.5-fold higher in 3T3-L1 adipocytes than in preadipocytes while no significant difference in adipocyte and preadipocyte activity was observed with 5'-90 construct (Fig. 2BGo), i.e. relative CAT activities were 0.8 ± 0.2% vs. 0.5 ± 0.1%, respectively, suggesting that the region between -146 and -91 bp is a major positive regulatory element in 3T3-L1 adipocytes.

To identify nuclear factor-interacting sequences in the major positive regulatory element, we performed deoxyribonuclease I (DNase I) protection analysis with nuclear extracts from 3T3-L1 preadipocytes and adipocytes, as well as with extracts of FRTL-5 thyroid cells (Fig. 3Go). In all three nuclear extracts, the region from nucleotides -146 to -127 bp, centered on the CRE-like element, was protected. In addition, nuclear extracts of 3T3-L1 adipocytes and FRTL-5 cells demonstrated protection of the region between -112 to -106 bp on the coding strand, which is located downstream of the CRE.



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Figure 3. DNase I Protection Analysis of the Positive Regulatory Element in the TSHR Gene

Genomic fragment spanning -220 to -50 bp was labeled at 3'-end of the coding strand with [{alpha}-32P]dATP and Klenow fragment. Lane 1 is A+G ladder determined by the Maxam and Gilbert sequence reaction (57 ); lane 2 is control digestion pattern in the absence of added nuclear extract. Other lanes contain the probe preincubated with 30 µg of nuclear extracts from 3T3-L1 cells before (preadipocytes) or after (adipocytes) differentiation, or from FRTL-5 cells. The open bar diagrammatically denotes the region where all three extracts protected; the solid bar indicates the sequence protected by the extracts from differentiated 3T3-L1 and FRTL-5 cells.

 
Activation of the TSHR Promoter by the CRE-Like Site and the Element Downstream of the CRE
To analyze the role of the protected regions in TSHR promoter activity, we introduced mutations into the pTRCAT5'-146 construct (Fig. 4Go). In differentiated 3T3-L1 cells, while mutation of the CRE-like element, TGAGGTCA, to that of the CRE consensus sequence, TGACGTCA (pTRCAT5'-146CRE), did not alter promoter activity, mutation to a nonpalindromic sequence, CGAGGACA, disrupting the dyad symmetry (pTRCAT5'-146NP), significantly decreased promoter activity. In addition, deletion of the CRE-like site, pTRCAT5'-131, led to loss of promoter activity. Mutation of the downstream sequence (pTRCAT5'-146DSM1) also showed reduced promoter activity, almost identical to that of pTRCAT5'-146NP. The downstream sequence contains the complementary sequence of the consensus DNA motif for the Ets family, GGAA (Figs. 4AGo and 5AGo). In addition, this sequence, TTCC, also exists between -100 and -97 bp (Fig. 5AGo).



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Figure 4. Effect of Mutations in the Region Protected by the Nuclear Extract from Differentiated 3T3-L1 Cells on the CAT Activity

The mutated plasmid constructs are noted in panel A. The open and solid bars denote the region protected by the extract from differentiated 3T3-L1 cells (see Fig. 3Go). The CRE-like sequence and the Ets-binding motif are boxed. The differentiated cells were cotransfected with the TSHR CAT mutants and pCH110, conversion rates were normalized to ß-galactosidase activities, and CAT activity was presented relative to pSV2CAT positive control. The significance (P < 0.05) of the effect of the mutation or deletion of pTRCAT5'-146 on the CAT activity is denoted by an asterisk.

 


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Figure 5. CAT Activity in the Differentiated 3T3-L1 Cells Transfected with Chimeric Plasmids Containing One, Two, or Three Copies of the CRE-Like Sequence (CRE, open bars), the Downstream Sequence (DS, hatched bars) or the CRE Plus the Downstream Sequence (CRE+DS, solid bars) Ligated to the 3'- End of an SV40 Promoter-driven CAT Gene

Panel A depicts the sequences of the coding strands of the ligated oligonucleotides. The CRE-like element and putative Ets-binding sites are indicated by double or single underlines, respectively. In panel B, the arrows depict the number of copies and direction of each construct; an right arrow denotes 5' to 3'. As in Figs. 2Go and 4Go, the differentiated 3T3-L1 cells were cotransfected with plasmid pCH110, and conversion rates were normalized to ß-galactosidase activities. The CAT activities are presented relative to that of the pCAT-promoter control with no CRE-like or Ets sites. An asterisk (*) or a solid circle (•) denotes a statistically significant increase relative to pCAT-promoter-CRE or -DS construct, respectively.

 
To confirm the synergism between the CRE-like site and the downstream sequence, we tandemly ligated oligonucleotides containing the CRE-like site alone (CRE), two putative Ets-binding sequences (DS), or the CRE site plus two Ets sites (CRE+DS) to the downstream of SV40 promoter-driven CAT gene (Fig. 5AGo). CAT activity was measured after transient transfection into differentiated 3T3-L1 cells (Fig. 5BGo). We found that the SV40 promoter was not activated when up to three copies of either oligonucleotide containing the CRE-like site or the Ets sites were inserted. In contrast, the presence of both the CRE and the downstream sequence significantly enhanced the SV40 promoter activity.

Interactions of Nuclear Proteins with the Protected Elements
Gel mobility shift analyses were performed to characterize the protein-DNA interactions involving the positive regulatory elements of the TSHR gene in 3T3-L1 cells. When a double-stranded oligonucleotide encompassing the sequence from -146 to -114 (CRE) was used as a probe (Fig. 6AGo), we observed multiple protein-DNA complexes in nuclear extracts from 3T3-L1 preadipocytes, similar to those detected in FRTL-5 extracts. In contrast, nuclear extracts from 3T3-L1 adipocytes formed a specific protein/DNA complex not observed in the other cell (Fig. 6Go, A and B, solid arrow) and exhibited less of the slowly migrating complexes (Fig. 6AGo, dashed arrows). These complexes, however, were not formed with the oligonucleotide probe containing the nonpalindromic mutation (CRENP). Competition analysis (Fig. 6BGo) showed that the formation of adipocyte-specific protein-DNA complex was self-competed by incubating with unlabeled oligonucleotide, as well as by an unlabeled, double-stranded oligonucleotide containing the CRE consensus sequence but with a surrounding sequence different from that of the TSHR promoter (somatostatin CRE, 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3', Promega, Madison, WI). Formation of this complex, however, was not competed by incubation with an unlabeled oligonucleotide spanning the region from -127 to -93 bp (DS1) and lacking the CRE-like sequence or with unlabeled CRENP. These results indicated that the differentiation of 3T3-L1 cells into adipocytes induced a new protein interacting with the CRE-like octamer sequence. The slowly migrating complexes (Fig. 6CGo, dashed arrows), formation of which were decreased after the differentiation, were shown to be formed with activating transcription factor-2 (ATF-2) since these complexes were abolished and supershifted (Fig. 6CGo, arrowhead) by antiserum to ATF-2. Inhibition of complex formation by antiserum to CRE-binding protein (CREB) was evident only in protein-DNA complexes formed with nuclear extracts from FRTL-5 cells (Fig. 6CGo). The adipocyte-specific protein-DNA complex, however, was not altered by antiserum to CREB or ATF-2 (Fig. 6CGo).



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Figure 6. Ability of the CRE-Like Element to Form Protein-DNA Complexes with Nuclear Extract with 3T3-L1 Cells before and after Differentiation

A, Radiolabeled, double-stranded oligonucleotide spanning -146 to -114 bp with (CRENP) or without (CRE) the nonpalindromic mutation (see Fig. 4Go) was incubated with nuclear extracts from 3T3-L1 cells before (PA) and after (A) differentiation, as well as from FRTL-5 cells (F) maintained in medium without TSH for 7 days. Dashed arrows depict the protein-DNA complexes diminished by the induction of differentiation of 3T3-L1 cells. A solid arrow denotes the protein-DNA complexes induced by differentiation. B, The double-stranded oligonucleotide CRE was radiolabeled and incubated with nuclear extracts from differentiated 3T3-L1 cells (A) and unlabeled oligonucleotides, CRE (self competition, a 250-fold excess over probe in molar amount), CRENP (250-fold), DS1 (-127 to -93 bp, 250-fold), or S-CRE (50-fold), which contains the CRE and its surrounding sequence of the somatostatin gene. C, The radiolabeled oligonucleotide CRE was incubated with nuclear extracts from 3T3-L1 cells before (PA) and after (A) differentiation or FRTL-5 cells (F) in the presence of an antiserum against CREB or ATF-2 or a control normal rabbit IgG (NRIgG). An arrowhead denotes a supershifted complex formed by antiserum to ATF-2. Protein-DNA complexes inhibited by antiserum to CREB are indicated with bars.

 
When we performed gel mobility shift analysis using an oligonucleotide probe spanning the region from -118 to -80 bp, including the protected element containing the putative Ets-binding site and the additional Ets site, we found that nuclear extracts from 3T3-L1 preadipocytes formed a faint protein/DNA complex (Fig. 7AGo, complex B). Formation of this complex was increased (Fig. 7AGo, lane 2), and an additional complex (Fig. 7AGo, complex A) became detectable by the differentiation of 3T3-L1 cells to adipocytes. Nuclear extracts from FRTL-5 and Buffalo rat liver cells (BRL 3A) also formed greater amounts of complexes than did the preadipocyte extracts. These protein-DNA complexes were shown to be specific, since they could be self-competed by unlabeled oligonucleotide, but not by an unlabeled oligonucleotide spanning the region from -146 to -114 bp (Fig. 7BGo). Nuclear extracts from FRTL-5 cells also formed an additional complex (complex C) exhibiting the highest mobility. This complex, however, appeared to be nonspecific since it was not self-competed (data not shown).



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Figure 7. Ability of the Putative Ets Site to Form Protein-DNA Complexes with Nuclear Extract with 3T3-L1, FRTL-5, and BRL Cells

A, Radiolabeled, double-stranded oligonucleotide spanning the region from -118 to -80 bp (DS2) with or without mutations was incubated with nuclear extracts from 3T3-L1 cells before (PA) and after (A) differentiation, as well as from FRTL-5 (F), or BRL (B) cells. Mutants are denoted in the lower panel. Putative Ets-binding sites are boxed, and the black bar indicates the sequence protected by nuclear extracts from differentiated 3T3-L1 cells and FRTL-5 cells. B, The double-stranded oligonucleotide, DS2, was radiolabeled and incubated with nuclear extracts from differentiated 3T3-L1 cells and a 250-fold excess of unlabeled DS2 or CRE as a competitor. C, Increasing amounts of unlabeled, double-stranded oligonucleotide were incubated with nuclear extracts from 3T3-L1 cells after differentiation and the radiolabeled oligonucleotide probe DS2. The data were subjected to quantitative densitometry and plotted as percent of B complex in the absence of competitor. The amount of each unlabeled oligonucleotide is presented in fold excess over probe.

 
To identify the site in the promoter region involved in the formation of differentiation-induced protein-DNA complexes, we introduced mutations into the protected sequence (DS2DSM1) (e.g. pTRCAT5'-146DSM1 in Fig. 4AGo). We observed decreased formation of these protein/DNA complexes (Fig. 7AGo, lanes 5- 8), a result consistent with the decreased CAT activity of pTRCAT5'-146DSM1 in 3T3-L1 adipocytes. Since this mutant, DS2DSM1, retained detectable binding activity, we introduced additional mutations into another putative Ets-binding element (DS2DSM2). Disruption of both Ets sites resulted in complete loss of the binding activity (Fig. 7AGo, lanes 9–12). In competition analysis (Fig. 7CGo), significant inhibition of the B complex formation was already evident at a 32-fold excess of unlabeled homologous oligonucleotide (DS2), but not by both mutants at the same amount. Higher amounts of oligonucleotide (~500-fold excess over probe) were required for the 50% inhibition of the complex formation by unlabeled DS2DSM2 in comparison with DS2DSM1 (120-fold). These results suggested that the putative Ets-binding sites in the downstream sequence of the CRE-like site interacted with the differentiation-induced activating factors. However, nuclear extracts from BRL 3A liver cells, expressing no TSHR gene, formed protein-DNA complexes similar to those by extracts from 3T3-L1 adipocytes (Fig. 7AGo). We therefore pursued an interaction of another nuclear factor within the positive regulatory element.

Interaction of Differentiation-Reduced Nuclear Proteins with TSHR Promoter
To analyze the region between the CRE-like site and the putative Ets sites, we performed gel mobility shift analyses using the oligonucleotide, DS1, spanning the region from -127 to -93 bp, as a probe. While nuclear extracts from 3T3-L1 preadipocytes formed multiple protein/DNA complexes, two of these complexes were diminished in nuclear extracts from 3T3-L1 adipocytes (Fig. 8AGo). The proteins forming these complexes appeared to be ubiquitously expressed, since they were also detected in nuclear extracts from FRTL-5 and BRL 3A cells (Fig. 8AGo). These complexes were also found to be specifically formed with double-stranded DNA since formation of these complexes was self-competed, but not by double-stranded unlabeled oligonucleotide containing somatostatin CRE (S-CRE, Fig. 8BGo). In addition, neither unlabeled coding nor noncoding strand of DS1 oligonucleotide competed the formation of these complexes (Fig. 8BGo). All extracts also formed two minor complexes (Fig. 8Go, B and C complexes). While the B complex was nonspecific, since it was not reduced by competition with a 250-fold excess of unlabeled oligonucleotide (Fig. 8BGo), formation of the C complex was competed out not only by unlabeled double-stranded oligonucleotide, but also by its coding or noncoding strand.



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Figure 8. Ability of the Sequence between -127 to -93 bp to Form Protein-DNA Complexes with Nuclear Extract with 3T3-L1, FRTL-5, and BRL Cells

A, Radiolabeled, double-stranded oligonucleotide between -127 to -93 bp (DS2) was incubated with nuclear extracts from 3T3-L1 cells before (PA) and after (A) differentiation, as well as from FRTL-5 (F), or BRL (B) cells. B, The double-stranded oligonucleotide, DS1, was radiolabeled and incubated with nuclear extracts from undifferentiated 3T3-L1 cells and unlabeled double- or single-stranded DS1 as a competitor. A double-stranded oligonucleotide containing a consensus CRE site in somatostatin gene (S-CRE) was also used as a nonspecific competitor. The amount of each unlabeled oligonucleotide present in each assay, in fold excess over probe, is noted.

 
To define the binding site of the proteins forming the A complexes, the G residues on both strands of the oligonucleotide in contact with protein were determined by methylation interference (Fig. 9AGo). While methylation of G residues from -117 to -110 bp on the noncoding strand interfered with formation of the major A complexes (Fig. 9CGo), methylation of G residues on the coding strand had no effect (data not shown). This result is supported by DNAase I protection analysis using a probe labeled on the noncoding strand (Fig. 9BGo). Nuclear extracts from 3T3-L1 preadipocytes and FRTL-5 cells incompletely protected the region between -120 and -110 bp, in which extracts from adipocytes showed no protection.



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Figure 9. Identification of the Sequence Interacting with Differentiation-Reduced Nuclear Proteins by Methylation Interference and DNase I Protection Analyses

In panel A, the dimethyl sulfate-modified, double-stranded DS2 oligonucleotide 32P-labeled in the coding strand was incubated with 25 µg nuclear extract from undifferentiated 3T3-L1 cells. Protein-DNA complexes and free DNA probe were separated on a preparative native gel as described in Materials and Methods. Free DNA and the upper complex of the A complexes shown in Fig. 8AGo were eluted from the gel and cleaved at the modified residues. The cleavage products were resolved by electrophoresis through a 12% denaturing gel. Open circles define the bases whose modification reduces the proportion of DNA in the bound fraction; X defines bases whose methylation does not alter the protein binding. In panel B, genomic fragment spanning -220 to -50 bp was labeled at the 3'-end of the noncoding strand. Lane 1 is A+G ladder determined by the Maxam and Gilbert sequence reaction (57 ); lane 2 is the control digestion pattern in the absence of added nuclear extract. Other lanes contain the probe preincubated with 30 µg of nuclear extracts from 3T3-L1 cells before (Preadipocytes) or after (Adipocytes) differentiation, or from FRTL-5 cells. The black bar diagrammatically denotes the region where all three extracts protected; the hatched bar indicates the sequence incompletely protected by the extracts from undifferentiated 3T3-L1 and FRTL-5 cells. In panel C, results of methylation interference and DNase protection analyses are summarized. The CRE-like site and the putative Ets-binding element are boxed.

 
Based on the results of methylation interference analyses, we again introduced mutations into both the TSHR promoter-CAT chimeric construct and the oligonucleotide probe and performed gel mobility shift analysis (Fig. 10AGo). Mutations in the oligonucleotide probe (DS1DSM3) almost abolished the formation of both the major and minor bands of the A complexes (Fig. 10BGo), thus suggesting that the proteins constituting the major and minor A complexes interact with the same sequence. When these constructs were transiently transfected into 3T3-L1 cells, we observed activation of the TSHR promoter, an effect more evident in preadipocytes (5.8-fold) than in adipocytes (1.9-fold) (Fig. 10CGo). This observation is consistent with the difference in the activity of A complex formations between 3T3-L1 preadipocytes and adipocytes (Fig. 8AGo). In sum, these results indicate that the nuclear factors in which binding is decreased after the differentiation of 3T3-L1 cells act as repressive factors on the TSHR promoter.



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Figure 10. Effect of a Mutation in the Binding Site of Differentiation-Reduced Nuclear Proteins on an Ability of the Complex Formation and on the CAT Activity

The sequences in panel A depict the mutation (DS1DSM3) and the wild-type (DS1) sequence. Open circles indicate the bases whose methylation interferes with the protein binding. Putative Ets sites are boxed. In panel B, each oligonucleotide was used as radiolabeled probe and incubated with nuclear extracts from undifferentiated 3T3-L1 cells. In panel C, 3T3-L1 cells before (Preadipocytes) and after (Adipocytes) the induction of differentiation were transfected with the TSHR-CAT plasmids with or without the mutation. The CAT activities are normalized with ß-galactosidase activities expressed by pCH110 cotransfected and are presented relative to that of positive control plasmid, pSV2CAT, or as the ratio of activity of the mutant vs. the wild type.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown previously that the regulation of TSHR gene expression in 3T3-L1 adipocytes is distinct from that in FRTL-5 thyroid cells (24). We were therefore somewhat surprised to find that the gene is initiated at almost identical sites in adipose and thyroid cells. The rat TSHR promoter has been shown to exhibit features of a house-keeping gene, including the absence of a TATA box, a high G+C content, and the presence of multiple transcriptional initiation sites (25). At typical TATA-less promoters, binding of SP1 to a GC-rich element is required for expression (34, 35, 36). In addition, a transcription factor IID (TFIID) complex containing TATA box-binding protein is thought to be essential for transcription even at a promoter lacking a TATA box, and SP1 bound to a GC can recruit a tethering factor and TFIID to lie along the DNA next to the GC box (34, 35). In some TATA-less genes, the SP1-interacting GC box has been found to act as a start site selector at about the same distance, 36–53 bp (36). It has been shown, however, that the minimal promoter region of the TSHR gene is not protected by purified SP1 protein in DNAase I protection analysis (26). Nevertheless, the common element activated in both adipocytes and thyroid cells may be important for the initiation of gene transcription. Since the major start sites are 44–59 bp downstream from the protected sequence centered on the CRE, CREBs may be a candidate for the initiation of transcription of the gene.

In 3T3-L1 cells, deletion analysis of the 5'-flanking region of the TSHR gene indicated that a 56-bp region between -146 and -90 bp was a positive regulatory element whose activity was induced by the induction of differentiation. Within this 56-bp region, we found that nuclear proteins from differentiated 3T3-L1 cells protected two elements. The upstream element between -146 and -127 bp contains a CRE-like element that was necessary for gene expression in thyroid cells (26, 27, 28). The downstream element spanning the region between -112 to -106 bp includes a putative Ets family-binding site. Both of these elements were found to be indispensable for the enhancer activity in adipocytes, suggesting that the differentiation-induced nuclear factors interact with these sequences, thus contributing to the promoter activity in adipocytes. In fact, we found that differentiation of 3T3-L1 cells induced the formation of a complex between the upstream element and an as-yet-unidentified protein. Competition and mutation analyses, however, suggested that it was a member of the CREB family. The downstream element was also observed to interact with nuclear proteins, the binding of which was increased by the induction of differentiation. This downstream element or the CRE-like site, however, cannot by itself activate the TSHR promoter, since either deletions or mutations of each sequence resulted in loss of promoter activity.

The synergism between the CRE site and putative Ets-binding elements in activating the TSHR promoter has been observed in other genes (37, 38, 39, 40). Members of the Ets family often show functional synergism with other transcription factors to achieve efficient activation of target genes through an Ets-binding site and adjacent DNA sequences (37, 38, 39, 40, 41, 42). For example, in the gene encoding mitochondrial uncoupling protein, Ets motifs have been identified as brown adipocyte regulatory elements (39). Synergistic cooperation of the brown adipocyte regulatory elements and the CRE is essential for the expression of the uncoupling protein gene in brown fat cells (39).

Although differentiation-dependent expression of the TSHR gene may be due to synergistic activation by the CRE-like and Ets sites, a third element may also be involved. This possibility was suggested by lines of evidence indicating that the complexes with the downstream sequence, including Ets sites, were formed by nuclear extracts of BRL liver cells, which did not express the TSHR, and that nuclear proteins from BRL cells and the CRE-like site have been shown to form multiple complexes other than the differentiation-induced complex (26, 27), whereas the construct containing both elements, pTRCAT5'-146, had slight activity in BRL cells (26, 27). We have identified nuclear factors with diminished binding to DNA only in differentiated 3T3-L1 cells. These nuclear proteins appear to suppress the promoter activity of the TSHR gene. The high activity of the complex formations in FRTL-5 and BRL cell nuclear extracts may, therefore, account for the weak activity of pTRCAT5'-146 in these cell lines (27, 28). Methylation interference analysis showed that these factors bind to a sequence contiguous to the 5'-end of the Ets-binding element, thus suggesting that binding of these factors may interfere with the binding of Ets family members to the TSHR promoter.

Transient expression analysis of 5'-deletion mutants revealed that the region between -177 and -146 bp acts as a repressive element in both differentiated and undifferentiated 3T3-L1 cells. This sequence contains the binding site of TSHR suppressor element-binding protein-1 (TSEP-1), a member of the Y-box family (27, 43), which binds to the coding strand of this region in a sequence-specific manner and suppresses the TSHR gene expression in thyroid cells (43). The TSEP-1 mRNA is also detected in both 3T3-L1 cells before and after differentiation (data not shown), suggesting that TSEP-1 represses the TSHR promoter activity in 3T3-L1 cells and that this repression may overcome the weak, but significant, enhancing activity of the region between -146 and -90 bp observed in undifferentiated 3T3-L1 cells.

The sequence between -190 and -177 bp was found to exhibit enhancing activity only in differentiated 3T3-L1 cells. In FRTL-5 cells, this region has been shown to interact with both TTF-1 and the SSBP-1 (30, 33). TTF-1 mRNA, however, was not detected in 3T3-L1 cells before or after differentiation (24). The interactions of nuclear proteins, including SSBP-1, to this element in differentiated 3T3-L1 cells are under current investigation.

Accumulating evidence has revealed that the differentiation of preadipocytes into adipocytes is regulated by at least three transcription factors, CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) (44), adipocyte determination- and differentiation-dependent factor 1 (ADD1) (45), and peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2) (46, 47), all of which appear to be important in regulating the expression of adipose-specific genes (45, 48, 49). The enhancer elements in the TSHR promoter, however, contain neither the CCAAT box nor the E-box motif, the binding sites of C/EBP{alpha} and ADD1, respectively. The third transcription factor, PPAR{gamma}2, has been found to form a heterodimeric complex with RXR{alpha} (50), which then binds to a sequence known as DR-1 [direct repeat with one nucleotide spacer (46, 49, 50)]. Interestingly, the CRE-like site in the TSHR promoter contains a half-site of the DR-1 sequence, AGGTCA. Although we were unable to detect binding of the PPAR{gamma}2/RXR{alpha} heterodimer, produced in the reticulocyte lysate system, to an oligonucleotide probe containing the CRE-like site (data not shown), these adipogenic transcription factors may indirectly regulate the expression of the TSHR gene in adipocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Primer Extension
The 5'-end of the rat TSHR gene transcript was determined as previously described (25), using a 24-bp oligonucleotide primer complementary to 12–35 bp of the rat TSHR cDNA (5'-AGCAGAGTGAGCTGGAGCAGGGAC-3') (25) end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase. The primer (3 x 105 cpm) was hybridized with 50 µg total RNA or yeast tRNA at 30 C overnight and extended with SuperScript II reverse transcriptase (GIBCO BRL, Rockville, MD). Resulting products were analyzed on an 8% polyacrylamide-7M urea gel in parallel with a sequencing reaction generated with the extension primer.

Anchored PCR
Anchored PCR (5' rapid amplification of cDNA ends) was used to clone the 5'-end of the TSHR cDNA. Total RNA (1 µg) from rat epididymal fat tissue was used to synthesize the first-strand cDNA using SuperScript II reverse transcriptase (GIBCO BRL) and 2.5 pmol of primer (5'-AAATTGTAGAAAGAATGTGGC-3'; complementary to 279–299 bp of the TSHR cDNA) (51). After first-strand cDNA synthesis, the original mRNA template was destroyed with ribonuclease H (RNase H), and the first-strand product was purified. An anchor sequence was then added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase (GIBCO BRL) and dCTP. Tailed cDNA was amplified by PCR using a nested primer containing dUMP residues for uracil DNA glycosylase cloning (52) [5'-CAUCAUCAUCAUTCGATGAGCTTCAGAGT-CTGG-3'; complementary to 162–182 bp of the rat TSHR cDNA (51)], and a deoxyinosine-containing anchor primer (5'-CUACUACUACUAGGCCACGCGTCGACTACGGGIIGG-GIIGGGIIG-3'). The amplified products were cloned into pAMP1 plasmid (GIBCO BRL) by the uracil DNA glycolase cloning method (52), and clones containing the amplified DNA were sequenced.

Cell Culture
3T3-L1 cells (CCL 92.1; ATCC, Rockville, MD) were grown in high-glucose DMEM and supplemented with 10% calf serum. Confluent 3T3-L1 preadipocytes were induced to differentiate into adipocytes as previously described (23, 24). Briefly, 1 day after confluence, cells were treated with media containing 10% FBS, 10 µg/ml insulin, 0.2 µg/ml dexamethasone, and 0.5 mM isobutylmethylxanthine. After 3 days, this medium was replaced by media supplemented with 10 µg/ml insulin and 10% FBS, and 2 days later, the media were replaced with media containing 10% FBS. Cells were used for studies 9 or 10 days after induction of differentiation.

FRTL-5 rat thyroid cells (CRL 8305 from ATCC) were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum and a mixture of six hormones containing bovine TSH (10 mU/ml), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml) (53). Buffalo rat liver cells (BRL 3A; ATCC No. CRL 1442) were grown in Ham’s F-12 supplemented with 5% FBS.

Plasmid Construction
Chimeric constructs containing 5'-flanking region of the rat TSHR gene and chloramphenicol acetyltransferase (CAT) gene, pTRCAT5'-1707, 5'-1190, 5'-907, 5'-638, 5'-419, 5'-220, 5'-190, 5'-177, 5'-146, 5'-90, and a plasmid for positive control, pSV2CAT, were kindly provided by Dr. Leonard D. Kohn (Metabolic Diseases Branch, NIDDK, NIH, Bethesda, MD 20892). Mutations of promoter sequences were generated by PCR using forward primers that had the mutated sequence and the ON-8L primer described previously as a reverse primer (25). Amplified fragments were cloned into plasmid p8CAT and sequenced to ensure nucleotide fidelity.

To generate pCAT-promoter-DS or -CRE+DS plasmids, both sense and antisense strand oligonucleotides containing the sequence from -118 to -80 or from -146 to -80 bp, respectively, as well as the XbaI recognition site on their 5'-end were synthesized, annealed, and inserted into the XbaI site of the pCAT-promoter plasmid (Promega). The inserts were sequenced to confirm copy number and direction. pCAT-promoter-CRE plasmids were kindly provided by Dr. Leonard D. Kohn.

All plasmid preparations were purified by CsCl gradient centrifugation (54).

Transient Expression Analysis
3T3-L1 cells before (preadipocytes) and after (adipocytes) differentiation were transfected by electroporation (Gene Pulser, Bio-Rad, Richmond, CA). When undifferentiated 3T3-L1 cells were transfected, cells being grown to 80% confluency in medium with 10% calf serum (3T3-L1 preadipocytes) were used. After the initiation of differentiation, 3T3-L1 cells on the 9th or 10th day (3T3-L1 adipocytes) were transfected. Cells were harvested, washed, and suspended at 5 x 106 cells/ml in 0.8 ml PBS. Either 50 µg pTRCAT5'-1707, equivalent molar amounts of the deletion mutants, or 5 µg pSV2CAT (positive control) were added with 5 µg pCH110 (ß-galactosidase expression plasmid; Pharmacia Biotech, Uppsala, Sweden). Cells were pulsed (300 V; capacitance, 960 µfarads), plated on one (adipocytes) or two (preadipocytes) 10-mm culture dishes, and cultured for 72 h. ß-Galactosidase was measured as described (55). After ß-galactosidase assay, cell extracts were heated at 65 C for 10 min for CAT assays (56). CAT activities were normalized to ß-galactosidase activities and then presented as the activities relative to that expressed by pSV2CAT, which remained in quantitatively measurable range of the assay.

Nuclear Extracts
Nuclear extracts were prepared using a procedure previously described (26, 27, 28, 29, 30) with minor modifications. 3T3-L1 extracts were either from cells after being grown to 80% confluency in medium with 10% calf serum (preadipocytes) or from cells on the 9th or 10th day after initiation of differentiation. FRTL-5 cells maintained in 5H medium (-TSH) for 7 days after near-confluency in 6H medium was achieved. BRL cells were grown until near-confluency in Coon’s modified Ham’s F-12 supplemented with 5% FBS. Cells were harvested, washed with PBS, and after centrifugation at 500 x g, suspended in 5 pellet volumes of 0.3 M sucrose and 3% Tween 40 in Buffer A [10 mM HEPES-KOH, pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml leupeptin, and 2 µg/ml pepstatin A]. After freezing, thawing, and gently homogenizing, nuclei were isolated by centrifuging at 25,000 x g on a 1 M sucrose cushion containing the same buffer. Nuclei were lysed in Buffer B [10 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A]; after centrifugation at 100,000 x g for 1 h, the supernatant was dialyzed in Buffer C [20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 0.1 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A].

DNase I Protection Analysis
Genomic fragment between -220 and -50 bp was synthesized by PCR and subcloned into the p8CAT vector as described previously (25, 26, 27, 28, 29, 30). The plasmid was cut with either HindIII or BamHI, end labeled with [{alpha}-32P]dATP and Klenow fragment, and recut with either BamHI or HindIII. The probe was purified on an 8% native polyacrylamide gel. Initial incubations were for 15 min at room temperature in 25 mM HEPES-KOH, pH 7.6, containing 5 mM MgCl2, 34 mM KCl, 1 µg poly(dI-dC), and 30 µg nuclear extracts. Incubations continued 20 more min in the presence of the probes (50,000 cpm) and in a reaction volume of 20 µl. DNA probes were digested with 0.5 U DNase I (Promega) for 5 min on ice, and the reaction was terminated with 80 µl stopping solution (20 mM Tris HCl, pH 8.0, 250 mM NaCl, 20 mM EDTA, 0.5% SDS, 10 µg proteinase K, and 4 µg sonicated calf thymus DNA). After incubation at 37 C for 15 min, the digested products were phenol extracted, ethanol precipitated, and separated on a 8% sequencing gel. The Maxam and Gilbert A+G sequencing reaction (57) was used to locate the footprinted regions.

Gel Mobility Shift Analyses
Gel mobility shift analyses were performed as described previously, with minor modifications (24, 26, 27, 28, 29, 30). Synthesized double-stranded oligonucleotides were end labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase and then purified on an 8% native polyacrylamide gel; 2.5 µg nuclear extract were incubated in a 20 µl reaction volume for 20 min at room temperature, with or without unlabeled competitor oligonucleotides as noted in individual experiments, and in the following buffer: 10 mM Tris HCl, pH 7.6, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 12.5% glycerol, 0.1% Triton X-100, and 1 µg poly(dI-dC). Labeled probe, 50,000 cpm (~0.5 ng DNA), was added and incubated an additional 20 min at room temperature. DNA-protein complexes were separated on 5% native polyacrylamide gels. In experiments using antiserum to CREB or ATF-2 (Santa Cruz Biotechnology, Santa Cruz, CA), the antiserum or normal rabbit IgG was added to nuclear extracts in the same buffer subsequent to addition of the labeled probe and then incubated for 40 min at room temperature.

Methylation Interference Assays
Methylation interference assays were performed as previously described (30). Single-stranded oligonucleotide spanning the region from -146 to -80 bp was end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase and then purified on an 8% native polyacrylamide gel. To obtain double-stranded probes that had the coding or noncoding strand labeled, end-labeled single-stranded oligonucleotides were annealed with their cold complementary strand and then purified on an 8% native polyacrylamide gel. The double-stranded probes were modified with dimethyl sulfate as described by Maxam and Gilbert (57) for 20 min on ice. For the preparative mobility shift, 5 x 105 cpm of modified oligonucleotides were incubated with 10 µg poly(dI-dC) and 25 µg nuclear extract. The undried gel was exposed to an imaging plate of a BAS2000 image analyzer (Fuji Film Co., Tokyo, Japan) for 10 min, and the regions corresponding to the protein-DNA complex and unbound probe in the gel were excised, eluted, and then precipitated. Base elimination and strand scission reactions at guanines were performed (57). The samples were then purified by butanol extractions and analyzed on a 12% sequencing gel.

Other Procedures
Protein concentration was measured using a Bio-Rad kit; recrystallized BSA was the standard. All experiments were repeated at least three times with different batches of cells. Where noted, values are the mean ± SE of these experiments; significance (P < 0.05) between experimental values was determined by the Student’s t test.


    FOOTNOTES
 
Address requests for reprints to: Toshimasa Onaya M.D., Ph.D., Professor and Chairman, The Third Department of Internal Medicine, Yamanashi Medical University, 1110 Tamaho, Yamanashi 409-3898, Japan. E-mail: onayat{at}res.yamanashi-med.ac.jp

Received for publication February 11, 1998. Revision received June 15, 1998. Accepted for publication June 16, 1998.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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