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Endocrinology Vol. 140, No. 2 609-617
Copyright © 1999 by The Endocrine Society

ARTICLES

Induction of Rat Aldose Reductase Gene Transcription Is Mediated through the cis-Element, Osmotic Response Element (ORE): Increased Synthesis and/or Activation by Phosphorylation of ORE-Binding Protein Is a Key Step

Kaoru Aida, Masato Tawata, Yukinobu Ikegishi and Toshimasa Onaya

Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We isolated the rat aldose reductase gene and examined the 5'-flanking sequence for the presence of transcription regulatory element responsive to hyperosmolarity. Deletion of aldose reductase gene up to -1047 bp abolished the transcriptional activation in response to osmotic stimuli in transient transfection experiments. A 17-bp sequence [rat osmotic response element (rORE)], which is located in bp -1073/-1057 and contains the TGGAAAATCAC sequence, confers osmotic response on a heterologous promoter. Electrophoretic mobility shift assays using the 17-bp fragment demonstrated that distinct DNA-protein complexes (I and II) were formed predominantly with nuclear extracts from the cells exposed to hyperosmolarity. When the nuclear extracts were preincubated with calf intestinal alkaline phosphatase or protein phosphatase 1, formation of complexes I and II was reduced to the control level. However, incubation with protein tyrosine phosphatase and addition of antiphosphotyrosine antibody had no effect on the complexes. When the nuclear extracts were preincubated with diamide to oxidize the thiols, complexes I and II were not affected. Pretreatment of the cells with cycloheximide abolished the complexes. All of these data indicate that activation by phosphorylation and/or increased synthesis of rORE-binding protein(s) are the key steps in induction of transcription of the rat aldose reductase gene by hyperosmolarity. Furthermore, we showed that glucose was more effective than NaCl in induction of aldose reductase both in transient transfection experiments and by Northern blot analysis. The results suggest the presence of a glucose-specific mechanism of induction in addition to that by NaCl.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH the precise mechanism by which hyperglycemia leads to diabetic microangiopathy is still controversial, the role of the polyol pathway has received much attention (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Sorbitol is formed by the reduction of glucose by an enzyme aldose reductase through this pathway. We have reported that the erythrocyte sorbitol/blood glucose ratios, which reflect aldose reductase activity, are significantly increased in diabetic patients with microangiopathy compared with those in patients without microangiopathy (4).

On the other hand, physiological roles of aldose reductase are not yet completely understood. Renal medullary cells accumulate large amounts of organic osmolytes, including sorbitol, to compensate for the interstitial hypertonicity (11, 12, 13). Hypertonicity elevates the activity and abundance of aldose reductase by increasing transcription of its gene (13, 14, 15, 16, 17). Induction of aldose reductase by hypertonic media was also demonstrated in kidney mesangial cells (18), renal proximal tubule cells (19), Chinese hamster ovary cells (18), retinal pigment epithelial cells (20, 21), aortic smooth muscle cells (22, 23), and arterial endothelial cells (24).

Recently, the sequence of the putative osmotic response element of the canine betain transporter (GBT1) gene, TonE (tonicity-responsive element), was described (25). Then, the 11-bp osmotic response elements (OREs) of the rabbit, human, and mouse aldose reductase genes were reported (26, 27, 28, 29).

In our previous report (22), we showed that aldose reductase messenger RNA (mRNA) levels as well as its activity were induced by glucose in a concentration-dependent manner up to 205.5 mM in a rat aortic smooth muscle cell line, A7r5 cells. Glucose was more effective than NaCl in the induction of aldose reductase mRNA and activity (22).

In this report, we examined the entire 3.5-kb 5'-flanking sequence of rat aldose reductase by deletion mutagenesis and transfection studies for the presence of a transcription regulatory element to hyperosmolarity or ORE. We also analyzed the nuclear proteins that bind to the element.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DMEM was obtained from Life Technologies (Gaithersburg, MD). Pepstatin A, leupeptin, phenylmethylsulfonylfluoride, and dithiothreitol (DTT) were obtained from Wako Pure Chemical (Osaka, Japan). [{alpha}-32P]Deoxy-ATP (~6000 Ci/mmol) was purchased from Amersham (Aylesbury, UK). Promega Corp. (Madison, WI) provided the Dual Luciferase Assay System and luciferase vectors. Calf intestinal alkaline phosphatase (CIP), TaKaRa Taq polymerase, and other restriction enzymes were obtained from Takara (Kyoto, Japan). Protein tyrosine phosphatase (PTPase) and protein phosphatase 1, catalytic subunit (PP1), were obtained from Boehringer Mannheim (Mannheim, Germany).

Genomic cloning
The 1.3-kb rat aldose reductase complementary DNA (cDNA) was obtained by PCR, using a pair of oligonucleotide primers: AR cDNA forward and reverse (Table 1Go). The PCR product was cloned into pCR2.1 (Invitrogen, San Diego, CA) and confirmed that the sequence was identical to that in the previous report (30). A total of 5.2 x 105 clones of rat genomic library in EMBL-3 SP6/T7 (CLONTECH Laboratories, Inc., Palo Alto, CA) were screened with the cDNA, which was labeled with BcaBest labeling kit (Takara). One clone was isolated from the library. The clone contained more than 16 kb of the insert. A 4.5-kb XbaI fragment containing the 3.5 kb 5' of the transcription initiation site was subcloned into pBluescript to give subclone p513–1. The nucleotide sequence of the 3'-part of this clone was identical to the reported 5'-flanking sequence (-1335 to +177) of the rat aldose reductase gene (31).


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  		Table 1. Sequences of primers used in the study

 
Plasmid construction
A fragment spanning from -3.5 kb to +16 bp of the transcription start site was amplified using the subclone p513–1 as a template by PCR with a pair of primers derived from the sequence of subclone p513–1; the distal primer, 513-Mlu, was constructed with a MluI restriction site, and the proximal primer, ARXho6, was constructed with a XhoI site (Table 1Go). The PCR product was digested with MluI and XhoI, isolated from agarose gels, and directionally inserted into the pGL3 basic vector (Promega Corp.), which contains the firefly luciferase sequence downstream from the cloning cassette. The resulting plasmid, pARLuc-3.5k, was digested with exonuclease III using the Takara deletion kit (Takara) following the manufacturer’s instructions, to generate progressively 5'-deleted vectors (pARLuc-2.9k, pARLuc-2.4k, pARLuc-1.9k, pARLuc-1447, pARLuc-1335, pARLuc-1216, pARLuc-1047, pARLuc-946, pARLuc-683, pARLuc-541, and pARLuc-270). Briefly, 20 mg pARLuc-3.5k plasmid DNA were digested with KpnI and MluI, treated with exonuclease III for various periods followed by heat inactivation of the enzyme, then mixed with mung bean nuclease, treated with Klenow fragment, and religated with T4 DNA ligase. The deletion constructs were sequenced to determine the extent of deletions.

To construct aldose reductase gene/reporter plasmids of which the 5'-end of the aldose reductase gene was located between -1216 and -1047 bp (pARLuc-1148, pARLuc-1128, pARLuc-1111, and pARLuc-1073), PCR was used with corresponding primers with a MluI restriction site (-1148Mlu, -1128Mlu, -1111Mlu, and -1073Mlu; Table 1Go) and the primer ARXho6. The PCR products were inserted into the MluI-XhoI site of the pGL3 basic vector.

To investigate the putative rORE identified in this study and a homologous sequence (rpsuedoORE) found 5' to the putative ORE, we made the following oligonucleotides and their complementary oligonucleotides: rORE, 5'-gatcAACTGGAAAATCACCAG-3'; and rpseudoORE, 5'-gatcAAGTGGAAAATATCTGT-3'. The complementary oligonucleotides were annealed and self-ligated to generate concatemers and inserted into a BamHI site of the pGL3 promoter (Promega Corp.), which contains the heterologous simian virus 40 (SV40) promoter upstream of the luciferase reporter gene. The number of oligonucleotides and the orientation and sequences of the constructs were confirmed by sequencing. pSV40Luc(rORE)1, pSV40Luc(rORE)3, pSV40Luc(rORE)4, and pSV40Luc(rpseudoORE)4 contain one, three, and four copies of the rORE sequence and four copies of the rpseudoORE sequence in tandem, respectively.

To subclone the fragment -1220/-1049 into pGL3 promoter vector, the region was amplified by PCR, digested with BamHI, and ligated into a BamHI site of pGL3 promoter vector. The primers used were -1220Bam and -1049Bam (Table 1Go). The sequence of the resulting plasmid (pSV40Luc-1220/-1049) was confirmed.

Cell culture and plasmids transfection
Rat aortic smooth muscle cell line A7r5 cells were maintained in DMEM containing 5.5 mM glucose supplemented with 10% FCS (22). A7r5 cells were seeded into 12-well culture plates and transfected 24 h later using Lipofectamine reagent (Life Technologies) and OptiMEM I (Life Technologies) following the manufacturer’s instructions with 100 ng luciferase construct plasmids and 8 ng pRL-CMV (Promega Corp.), a plasmid containing the Renilla luciferase gene under the control of cytomegalovirus (CMV) immediate early enhancer/promoter, to monitor the efficiency of transfection. Transfected cells were maintained in isotonic medium for 24 h, then switched to hypertonic medium (150 mM glucose or 75 mM NaCl) or maintained in isotonic medium for another 20 h. The osmolarity of those media was 324 ± 13 mosmol/kg H2O (n = 9) for isotonic medium, 474 ± 17 mosmol/kg H2O (n = 8) for glucose-supplemented medium, and 471 ± 12 mosmol/kg H2O (n = 9) for NaCl-supplemented medium, respectively. The transfected cells were harvested and assayed for luciferase activity using Dual Luciferase Assay Systems (Promega Corp.) and a luminometer (Lumat LB9501, Berthold Japan, Tokyo, Japan).

Nuclear extracts preparation
Nuclei were isolated from A7r5 cells cultured for 6 h in the isotonic medium or the hypertonic medium as described above. Nuclear extracts were prepared in the presence of protease inhibitors, pepstatin A (2 mg/ml), leupeptin (2 mg/ml), phenylmethylsulfonylfluoride (0.5 mM), and DTT (0.5 mM), as descried by Shimura et al. (32). Protein concentrations were determined by the Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA). When the cells were treated with cycloheximide (100 µg/ml), it was added 2 h before switching the medium, and nuclear extracts were prepared in the same manner.

Electrophoretic mobility shift assay
The synthetic complementary oligonucleotides (rORE, shown in Fig. 4AGo) were annealed, labeled with [{alpha}-32P]deoxy-ATP by Klenow polymerase, and used as a probe. Nuclear extracts (3 µg) were incubated with the probe (~15,000 cpm) in a total volume of 20 µl binding buffer containing 10 mM Tris-HCl (pH 7.5), 10% glycerol, 50 mM NaCl, 0.5 mM DTT, and 0.05% Nonidet P-40. Binding reactions were carried out in the presence of 1 µg poly(dI-dC)-poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ) and with or without specific competitors, as indicated. In experiments with phosphatases or antiphosphotyrosine monoclonal antibody (PY20, Transduction Laboratories, Inc., Lexington, KY), the reaction mixtures were incubated with phosphatases or antiphosphotyrosine antibody for 20 min at 30 C before addition of the probe. Chemical oxidation of the thiols in the protein was performed with diamide (1 and 3 mM; Sigma Chemical Co., St. Louis, MO), an inorganic catalyst of oxidation of thiols [(SH)2] to generate disulfides (-S-S-) (33). Oxidation was carried out on ice for 5 min in the binding buffer without DTT. Subsequent chemical reduction of the disulfides to thiols was carried out with 3 mM DTT at 25 C for 5 min. Then the probe was added. After a further 20 min at room temperature, the binding mixtures were resolved by electrophoresis on a 4% polyacrylamide gel (acrylamide-bisacrylamide, 30:0.8) in 40 mM Tris-HCl (pH 8.5), 190 mM glycine, and 1 mM EDTA at 150 V at 4 C. The gels were dried and analyzed with a Fuji Photo Film Co., Ltd. Bioimage analyzer BAS 2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan).



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  		Figure 4. Electrophoretic mobility shift assay to identify the nuclear protein-binding site. A, The sequences of oligonucleotides used in the electrophoretic mobility shift assay and the sequences of OREs. The nucleotides in bold type are conserved. The underlined nucleotides diverged from the rORE core. The rat sequences are also compared with the OREs present in aldose reductase genes from rabbit (27 ), human (28 ), and mouse (29 ) and to the TonE described in the canine BGT1 promoter (25 ). B, Electrophoretic mobility shift assay using double stranded rORE as a probe. The probe was incubated with nuclear extracts from untreated cells (C), glucose-treated cells (G), and NaCl-treated cells (Na) in the presence or absence of a 100-fold molar excess of various competitors as indicated. C, The double-stranded rpseudoORE was used as a probe and incubated with nuclear extracts in the presence or absence of a 50-fold molar excess of competitors as indicated.

 
Northern blot analysis
A7r5 cells were grown in 10-cm dishes. Cycloheximide (100 µg/ml) was added 2 h before switching the medium. Northern blot analysis of total RNA was carried out using aldose reductase cDNA as a probe according to the method previously described (34).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a DNA region required for the responses to glucose and NaCl
pARLuc-3.5k reporter plasmid, which contains the DNA fragment from bp -3.5k to bp +16 of the rat aldose reductase gene, responded to glucose and NaCl treatment with 4- and 3-fold increases in luciferase activities in A7r5 cells (Fig. 1Go, A and B). The effect of glucose was significantly greater than that of NaCl (P < 0.001). Other osmolytes (mannose, mannitol, L-glucose, 3-O-methylglucose, galactose, {alpha}-methylglucoside, and LiCl) at the same osmolarity induced luciferase activities similar to those produced by NaCl; the activities again were lower than those induced by glucose (Fig. 1BGo). Figure 1CGo shows the Northern blot analysis. Glucose induced aldose reductase mRNA more than other osmolytes.



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  		Figure 1. 5'-Deletion analysis of the rat aldose reductase gene and effects of various osmolytes on the gene. A, Sequential 5'-deletions of the rat aldose reductase gene were generated by exonuclease III digestion of the pARLuc-3.5k construct. The reporter plasmids were transfected into A7r5 cells and treated with glucose or NaCl for 20 h. Luciferase activities were assayed with the Dual Luciferase Assay System (Promega Corp.), and data are presented as the fold induction of luciferase activity (mean ± SEM) due to glucose or NaCl treatment, calculated by dividing the amount of luciferase activity in glucose- or NaCl-treated cells by the amount of luciferase activity in untreated cells (the number of determinations is shown in parentheses). B, The pARLuc-3.5k construct was transfected into A7r5 cells, then the cells were exposed to medium containing the indicated osmolyte. The osmolarities of the media are the same. The number of determinations is shown in parentheses. C, Northern blot analysis. A7r5 cells were exposed to various osmolytes, and 12 h later, total RNAs (20 µg) were prepared, electrophoresed, transferred to a membrane filter, then hybridized with aldose reductase cDNA. After deprobing, the filter was hybridized with ß-actin. Lane 1, Control: lane 2, glucose; lane 3, NaCl; lane 4, mannitol; lane 5, L-glucose; lane 6, 3-O-methylglucose; lane 7, LiCl.

 
To map the glucose- and NaCl-responsive region, a series of 5'-deletion reporter plasmids was constructed by exonuclease III digestion of the pARLuc-3.5k and tested for activity. When A7r5 cells were transfected with the reporter plasmids containing more than -1216 bp of 5'-flanking sequences, glucose or NaCl treatment of A7r5 cells increased luciferase activities almost comparable to the original pARLuc-3.5k construct (Fig. 1AGo). However, further deletion up to bp -1047 totally abolished the response to both glucose and NaCl.

The sequence of the region between bp -1216 and bp -1047 is shown in Fig. 2AGo. We found four sequences similar to ORE in this region. Therefore, we deleted those ORE-like sequences one by one. A gradual reduction in the induction ratios upon deletion was observed (Fig. 2BGo); however, pARLuc-1073 was still capable of induction of about 2.5-fold. When all four ORE-like sequences were deleted (pARLuc-1047), induction was abolished.



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  		Figure 2. 5'-Deletion analysis of the rat aldose reductase gene. A, A part of the genomic sequence of the rat aldose reductase gene. The boxed sequences are those similar to ORE. B, The luciferase reporter plasmids were constructed by PCR and transfected into A7r5 cells as described in Fig. 1Go. pARLuc-1148 lacks fragment C; pARLuc-1128 lacks fragment C and rpseudoORE; pARLuc-1111 and pARLuc-1073 lack fragment C, rpseudoORE, and fragment D; pARLuc-1047 lacks all of those four sequences. The number of determinations is shown in parentheses.

 
A sequence identical to human and mouse ORE (26, 27, 28) was present between bp -1070 and bp -1060. To demonstrate that this putative ORE sequence (rORE) can act independently of the surrounding sequences in the rat aldose reductase 5'-flanking region, one, three, or four copies of the corresponding sequence from bp -1073 to bp -1057 were placed in tandem in the downstream of the heterol-ogous promoter SV40 linked to the luciferase reporter gene (pGL3-promoter vector) to form pSV40Luc(rORE)1, pSV40Luc(rORE)3, or pSV40Luc(rORE)4, respectively. When pSV40Luc(rORE)4 plasmid was transfected into A7r5 cells and treated with glucose and NaCl, the luciferase activities increased by 3.0- and 2.2-fold, respectively, compared with those after transfection with the parental pGL3-promoter plasmid (Fig. 3Go). The responses of pSV40Luc(rORE)4, pSV40Luc(rORE)3, and pSV40Luc(rORE)1 to the stimuli decreased as the copy number of rORE decreased.



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  		Figure 3. Effects of ORE-like sequences on a heterologous promoter. One, three, or four copies of rORE and four copies of rpseudoORE in tandem and fragment -1220/-1049 were placed in the pGL3-promoter vector to construct pSV40-Luc(rORE)1, pSV40Luc(rORE)3, pSV40-Luc(rORE)4, pSV40Luc(rpseudoORE)4, and pSV40Luc-1220/-1049, respectively. The pGL3 promoter contains the SV40 promoter upstream of the luciferase gene. The constructs were transfected into A7r5 cells and treated with glucose or NaCl. The number of determinations is shown in parentheses.

 
At 75 bp upstream of the rORE sequence, another homologous sequence was found (rpseudoORE, -1148/-1132; Fig. 2BGo). When pSV40Luc(rpseudoORE)4, which contains four copies of the rpseudoORE sequence in tandem, was transfected, glucose and NaCl did not increase the luciferase activity (Fig. 3Go). Deletion of this sequence from the 5'-flanking region also did not affect the luciferase activity (pARLuc-1128, Fig. 2BGo).

When pSV40Luc-1220/-1049, which contains four ORE-like sequences (Fig. 2AGo), was transfected, and the cells were treated with glucose or NaCl, the luciferase activity induction was comparable to that of pSV40Luc(rORE)1 (Fig. 3Go).

Identification of DNA-binding activity in nuclear extract
The probes and competitors used in the electrophoretic mobility shift assay are shown in Fig. 4AGo. Three major complexes (I, II, and III) were formed (Fig. 4BGo). A broad band (complex III) was formed with the nuclear extracts from cells cultured in both isotonic and hypertonic media. Slowly migrating bands (complexes I and II) were formed predominantly with nuclear extracts from hypertonic cells. A 100-fold molar excess of unlabeled (cold) rORE or 11-bp ORE (rORE core) eliminated complexes I and II, but not complex III. The rpseudoORE sequence (-1148/-1132), which was inactive in the luciferase assay (Fig. 3Go), did not compete any of the three bands. As the rpseudoORE sequence differs in only 2 bases from the 11-bp ORE (rORE core), we made mutated, double stranded oligonucleotides to delineate the nucleotide sequence responsible for protein binding (Fig. 4BGo). rORE M1, in which a cytosine at -1062 bp is mutated to an adenine, did compete for complexes I and II. On the other hand, rORE M2, in which an adenine at -1061 bp is mutated to a thymine, did not virtually compete for those complexes. Other nucleotide differences between rORE and rpseudoORE (5', C-G; 3', CAG-TGT) are not involved in DNA-protein interaction (Fig. 4BGo and data not shown).

When rpseudoORE was used as a probe, three bands appeared (a, b, and c, Fig. 4CGo). The upper two bands (a and b) were more intense in nuclear extracts prepared from isotonic and NaCl-treated cells than those from glucose-treated cells. The bands a and b were competed by unlabeled probe in a 50-fold molar excess, but were only slightly competed by rORE in a 50-fold molar excess. Around rORE sequence, there were two other sequences similar to rORE (Fig. 2AGo): fragment C (-1213/-1203) and fragment D (-1125/-1115), which is in the opposite direction. When these sequences were used as probes, no specific band was observed (data not shown).

To investigate whether those proteins bound to the rORE were phosphorylated, we treated the nuclear extracts with phosphatases (Fig. 5Go). Nuclear extracts from glucose- or NaCl-treated A7r5 cells were incubated with CIP (nonspecific phosphatase), PTPase (phosphotyrosine phosphatase), or PP1 (serine/threonine phosphatase) before addition of the labeled probe. CIP and PP1 treatment inhibited the formation of complexes I and II to the unstimulated level, but incubation with PTPase had no effect on the complexes. When we incubated the nuclear extracts with antiphosphotyrosine antibody before addition of the probe, the complex formation was not affected (data not shown).



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  		Figure 5. Effects of phosphatase treatment of the nuclear extracts on DNA binding. Nuclear extracts were incubated with CIP, PTPase, or PP1 for 20 min at 30 C before addition of the labeled rORE, and the incubation proceeded as described in Materials and Methods.

 
As some transcription factors are under redox regulation, we studied the redox state of thiols of the rORE-binding proteins (Fig. 6Go). When 1 mM DTT was added to the nuclear extracts, the intensities of complexes I and II decreased slightly. When the nuclear extracts were preincubated with 1 mM diamide to oxidize the thiols of the nuclear proteins, formation of complexes I and II was not affected, and complex III was totally abolished. Subsequent addition of 3 mM DTT had no effect on complexes I and II and restored complex III.



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  		Figure 6. Effect of diamide on the rORE-binding activity. The nuclear extracts were prepared without DTT. Those extracts were treated with 1 mM diamide or 1 mM DTT. Some of them were subsequently treated with 3 mM DTT. Then, the labeled rORE was added, and the reaction mixtures were proceeded as described in Materials and Methods.

 
We next investigated whether nuclear proteins that bind to rORE are newly synthesized in response to glucose and NaCl or are modified posttranslationally by the stimuli. As shown in Fig. 7AGo, cycloheximide treatment abolished induction of aldose reductase mRNA by glucose and NaCl. When nuclear extracts were prepared from A7r5 cells that were pretreated with cycloheximide and then exposed to glucose or NaCl, the formation of complexes I and II was virtually abolished (Fig. 7BGo).



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  		Figure 7. Effects of cycloheximide on aldose reductase mRNA and rORE-binding proteins. A7r5 cells were treated with cycloheximide (100 µg/ml) for 2 h, then media were switched to isotonic or glucose- or NaCl-containing medium. A, Twelve hours after switching the medium, total RNAs were prepared, and Northern blot analysis was performed using aldose reductase cDNA as a probe. B, Six hours after switching the medium, nuclear extracts were prepared, and the electrophoretic mobility shift assay was performed as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we identified and localized a glucose/NaCl-responsive enhancer element (rORE) in the 5'-flanking region of the rat aldose reductase gene between bp -1073 and -1057 of the transcription initiation site and also characterized the rORE-binding protein.

Deletion of the rORE sequence abolished the promoter activity, and the oligomers of rORE enhanced the heterologous promoter when stimulated by glucose or NaCl. In an electrophoretic mobility shift assay, rORE forms two specific complexes (I and II), which were predominantly observed in the nuclear extracts from glucose- or NaCl-treated cells and were competed by unlabeled rORE and the 11-bp rORE core (TGGAAAATCAC).

The rORE core shares a high degree of homology with other osmolarity-responsive elements; rORE-core is identical to OREs of human and mouse aldose reductase genes (28, 29) and is different from rabbit ORE by one nucleotide (27), a cytosine at the first nucleotide in rabbit and a thymine in rat, human, and mouse OREs (Fig. 4AGo). When a cytosine at bp -1062 is substituted by an adenine (rORE M1) and used as a competitor, formation of complexes I and II is competed, but when an adenine at bp -1061 is substituted by a thymine (rORE M2), complexes I and II are not affected. Therefore, an adenine, but not a thymine, at bp -1061 is required for DNA-protein interaction. Substitution of the first adenine within the 11-bp rabbit ORE by a guanine was shown to delete the osmotic response (27).

As shown in Fig. 2BGo, the magnitude of induction dropped gradually with decreasing fragment size from -1216 to -1073 bp. There are four sequences similar to ORE in this region: fragment C, rpseudoORE, fragment D, and rORE (Fig. 2AGo). Fragment D is in the opposite direction. Recently, Ko et al. (28) suggested that the ORE-like sequences present around human ORE (OreC) cooperatively interact for the enhancer activity. Therefore, we investigated the possibility that other elements located close to rORE may potentiate the response, although rORE is a major cis-element of induction as mentioned above. However, based on transfection analyzes and electrophoretic mobility shift assays (Fig. 4Go, B and C, and data not shown), we think that rpseudoORE and fragments C and D are not involved in the transcriptional activation of the aldose reductase gene. rpseudoORE forms complexes a and b, which are predominant in nuclear extracts from control and NaCl-treated cells (Fig. 4CGo). The meaning of this is unclear at present.

Although osmotic response elements have been identified in aldose reductase gene and betain transporter gene as described above, the protein that binds to the element has not been characterized. We showed here partial characterization of the protein. When nuclear extracts were treated with CIP or PP1, the formation of complexes I and II was inhibited (Fig. 5Go). Incubation with PTPase had no effect on the complexes. Preincubation of the nuclear extracts with antiphosphotyrosine antibody also did not affect the complex formation. Treatment of the nuclear extracts with diamide to oxidize the thiols did not affect the formation of complexes I and II (Fig. 6Go), suggesting that the redox state of thiols is not involved in the interaction of rORE and rORE-binding proteins. Furthermore as shown in Fig. 7Go, pretreatment of the cells with cycloheximide abolished the induction of aldose reductase mRNA and rORE-binding proteins. These results indicate that phosphorylation at serine/threonine residues of rORE-binding proteins is involved in the protein-DNA interaction and that new protein synthesis is required to induce transcription of aldose reductase gene.

In our previous studies (22), we showed that in A7r5 cells, glucose induced aldose reductase mRNA and its activities more efficiently than NaCl. Here we confirmed the results by transient transfection assays. When pARLuc-3.5k was transfected, the luciferase activity induced by glucose was 1.5-fold of that induced by NaCl. Glucose-specific induction became obscure upon shortening of the test fragment in the reporter vectors (Figs. 1AGo and 2BGo) and is not observed in transfection with pARLuc(rORE)4 (Fig. 3Go). Furthermore, as shown in Fig. 1BGo, other osmolytes induced the luciferase activities no more than NaCl, and the results in Northern blot analysis were similar to those in transient transfection assays (Fig. 1CGo). Induction by L-glucose, which is unable to enter the cells, and that by 3-O-methylglucose, which is not phosphorylated and further metabolized, were also less than that by glucose. All of these results suggest that the glucose-specific induction mechanism involves the metabolism of glucose and is not medicated by rORE. Experiments to elucidate its precise mechanism are under way.

Studies in yeast have revealed a two distinct transmembrane osmosensors, SLN1 and SHOP1, that regulate the mitogen-activated protein kinase cascade (PBS2-HOG1 pathway) (35, 36, 37). However, there is little information available regarding how mammalian cells recognize and transduce hyperosmotic stimuli to transcriptional machinery. Hypertonicity stimulates three cascades of MAP kinase homologs: ERKs (38, 39, 40), Jun N-terminal kinase-1/stress-activated protein kinase, and p38 (40, 41, 42, 43, 44, 45). In MDCK cells, however, the induction of the sodium/myo-inositol transporter mRNA by hypertonicity does not involve the ERK pathway (46). Recently, Kültz et al. (45) reported that although p38 and Jun N-terminal kinase-1/stress-activated protein kinase cascades are activated by hyperosmolarity in PAP-HT25 cells, activation of those pathways is not necessary for transcriptional regulation of the aldose reductase gene through the ORE. We also observed that inhibition of ERK does not affect the aldose reductase induction by glucose and NaCl (Aida, K., M. Tawata, and T. Onaya, manuscript in preparation). These results suggest that hyperosmotic stress signals through divergent pathways.

Isolation of the rORE-binding proteins and characterization of their interaction with the rORE should clarify the mechanisms of regulation of aldose reductase gene expression.

Received May 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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