Members of the Nuclear Factor 1 Transcription Factor Family Regulate Rat 3{alpha}-Hydroxysteroid/Dihydrodiol Dehydrogenase (3{alpha}-HSD/DD AKR1C9) Gene Expression: A Member of the Aldo-keto Reductase Superfamily

Chien-Fu Hung1 and Trevor M. Penning

Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104-6084


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rat 3{alpha}-hydroxysteroid/dihydrodiol dehydrogenase (3{alpha}-HSD/DD; AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, inactivates nearly all steroid hormones by converting 5{alpha}- and 5ß-dihydrosteroids to their respective 3{alpha},5{alpha}- and 3{alpha},5ß-tetrahydrosteroids and protects against circulating steroid hormone excess. It is highly expressed in rat liver comprising 0.5–1.0% of the soluble protein. Previously, we identified a powerful distal enhancer resident at about -4.0 kb to -2.0 kb in the 5'-flanking region of the 3{alpha}-HSD/DD gene. We now report the functional dissection of this enhancer. Transfection of nested deletions of the 5'-end of the gene promoter linked to chloramphenicol acetyltransferase (CAT) into HepG2 cells located the enhancer activity between (-4673 to -4179 bp). Further internal and 5'-end deletion mutants revealed that a 73-bp fragment (from -4351 to -4279 bp) contained a major enhancer element. This fragment spanned two imperfect direct repeats GTGGAAAAACCCAGGAA and GTGGA-AAAAACCCAGGAA and contained three direct repeats of GGAAAAA. This fragment also contained three potential half-nuclear factor 1 (NF1) sites (TGGA-NNNNNGCCA) and a putative CCA-AT-enhancer binding protein (C/EBP) binding site. The 73-bp fragment enhanced CAT activity from the basal 3{alpha}-HSD/DD gene promoter. Recombinant C/EBP{alpha} and C/EBPß did not bind to this fragment. Electrophoretic mobility shift assays showed that HepG2 and rat liver nuclear extracts bound to this 73-bp fragment. The 73-bp protein complex was competed out by a NF1 oligonucleotide and was supershifted by an NF1 antibody. When the 73-bp fragment was fused to an {alpha}1-globin promoter-CAT construct and cotransfected with CCAAT transcription factor 1 (CTF1)/NF1 into Drosophila Schneider SL2 insect cells (which lack NF1-like proteins) trans-activation of CAT activity was observed. These results indicate that members of the NF1 transcription factor family regulate high constitutive expression of the rat 3{alpha}-HSD/DD gene that is responsible for steroid hormone inactivation. The potential role of NF1 in regulating other AKR genes that have protective roles is discussed.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The aldo-keto reductase (AKR) gene superfamily consists of highly related, soluble, monomeric (37-kDa) NAD(P)H oxidoreductases that convert ketones to alcohols. Their endogenous substrates include steroid hormones and PGs while their exogenous substrates include drugs and carcinogens (1, 2). Genes of this superfamily encode for proteins approximately 320 amino acids in length which adopt an ({alpha}/ß)8-barrel motif and bind NAD(P)(H) in an unusual extended conformation (3, 4). Loop structures at the back of the barrel dictate their ligand specificity. Cluster analysis shows that the first family (AKR1)2 of the superfamily includes aldehyde reductases (AKR1A), aldose reductases (AKR1B), and the hydroxysteroid dehydrogenases (HSDs) that comprise the AKR1C subfamily. The majority of mammalian 3{alpha}- and 20{alpha}-HSDs belong to the AKR1C subfamily. Of these, human type 2 3{alpha}-HSD (AKR1C3) has been implicated in the elimination of 5{alpha}-dihydrotestosterone from the prostate (5) while rat ovarian 20{alpha}-HSD (AKR1C8), which inactivates progesterone, has been implicated in the termination of luteal phase pregnancy (6).

Rat liver 3{alpha}-HSD/DD3 (AKR1C9) is one of the best studied AKRs. It catalyzes the second step in the metabolism of nearly all circulating steroid hormones, e.g. androgens, progestins, and glucocorticoids, by converting their 5{alpha}- and 5ß-reduced dihydrosteroids to their corresponding 3{alpha},5{alpha}- and 3{alpha},5ß-tetrahydro-steroids (7, 8, 9, 10). Interestingly, 5ß-reductase (AKR1D), which precedes 3{alpha}-HSD in steroid hormone metabolism, is also a member of the AKR1C subfamily, and a single-point mutation introduces 5ß-reductase activity into 3{alpha}-HSD (11), suggesting that this pathway of steroid hormone metabolism may have arisen by AKR gene duplication. AKR1C9 is also essential for the biosynthesis of bile acids and their transport from the sinusoidal to the canicular pole of the hepatocyte (12, 13, 14).

HSDs in the AKR1C subfamily also have associated dihydrodiol dehydrogenase (DD) activity, which activates polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols by forming reactive and redox-active o-quinones. These events may contribute to the carcinogenic potential of the parent PAH. For example, 3{alpha}-HSD/DD converts trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (a potent proximate carcinogen) to benzo[a]pyrene-7,8-dione with concomitant production of reactive oxygen species (15, 16). Human homologs exist for AKR1C9 in which the diverse functions of the rat enzyme have been distributed across several gene products. Thus, type 1 3{alpha}-HSD (DD4 and chlordecone reductase; AKR1C4) is a hepatic specific 3{alpha}-HSD, type 2 3{alpha}-HSD (DDX and AKR1C3) is expressed in liver and androgen target tissues, and type 3 3{alpha}-HSD (DD2 which is bile acid-binding protein) shows a similar distribution to the type 2 isoform (17, 18).

AKR1C9 is highly constitutively expressed in rat liver, comprising 0.5–1.0% of the soluble protein but it is also regulated. In female rat liver, a 2-fold increase in mRNA and protein expression is observed, and this female pattern of expression can be established by administering estrogens to male rats (19, 20, 21). It is unknown whether this is a direct effect of estrogens or whether this is due to the regulation of GH secretion by gonadal steroids (20, 21). Additionally, dexamethasone increases steady-state levels of 3{alpha}-HSD/DD mRNA in rat hepatocytes (22). These findings suggest that the AKR1C9 gene may be autoregulated by circulating steroid hormones.

Progress has been made in understanding the genomic structure of AKR genes. The human aldose reductase (AKR1B) gene has been cloned and consists of nine exons and eight introns (23). This genomic organization is conserved in both the rat and human 3{alpha}-HSDs (17, 24, 25). However, less is known about the transcriptional regulation of the AKR genes. Our earlier studies have shown that the AKR1C9 gene promoter contains a basal promoter between (-199 and +55 bp), a weak proximal enhancer between (-498 to -199 bp), a powerful silencer (-755 to -498 bp) that binds Oct, and a strong distal enhancer (-4.0 to -2.0 kb) that controls constitutive expression. The promoter also contains multiple steroid response elements (SREs), which may comprise a steroid response unit (26). We demonstrated that in rat hepatocytes the gene was transcriptionally activated by glucocorticoids via an occupied glucocorticoid receptor that bound to the proximal glucocorticoid response element (27). This suggests that the SREs in the gene promoter are likely to respond to high circulating levels of steroid hormones, and subsequent gene transcription may protect against steroid hormone excess. However, the identity of transcription factors that control the high constitutive expression of AKR genes that serve protective roles, e.g. AKR1C9 and aldose reductase, which is osmoprotective (28, 29, 30, 31), is largely unknown.

The aim of this study was to identify the enhancer(s) and transcription factor(s) responsible for the high constitutive expression of the AKR1C9 gene. We report the location of a powerful distal enhancer -4.5 kb upstream from the transcription start site. We have identified a 73-bp fragment within this region containing a triple repeat that drives reporter gene activity. Gel mobility shift and supershift analysis showed that nuclear factor 1 (NF1) transcription factors bind to this region. When the 73-bp enhancer is fused to an {alpha}1-globin promoter-chloramphenicol acetyltransferase (CAT) construct, it can be trans-activated by cotransfection of NF1 in a null-environment (SL-2 insect cells). These results suggest that NF1 transcriptionally activates AKR1C9 gene expression and hence regulates steroid hormone inactivation in rat liver. NF1 transcription factors have not been previously implicated in AKR gene regulation. The possibility that NF1 may trans-activate other AKR genes that play protective roles is discussed.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence of the Upstream 5'-Flanking Region of the Rat 3{alpha}-HSD/DD (AKR1C9) Gene
We previously reported the sequence of 1593 bp of the 5'-flanking region upstream from transcription start site of the AKR1C9 gene (26). In that study we located a powerful distal enhancer between -4673 to -1906 bp. We now report the gene sequence to -4673 bp from the transcription start site. During the sequence determination, several errors were identified in the region between -1593 to -1533, which are now corrected. The entire sequence from -4673 to -1533 is reported and has been deposited5 (Fig. 1Go).



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Figure 1. Sequence of the Distal Promoter of the Rat 3{alpha}-HSD/DD (AKR1C9) Gene

The sequence starts at the 5'-end; the first underline indicates the position of the 73-bp enhancer including its three NF1 binding sites. The second and third underline indicate the sequence homologous to the promoter sequence of the MCPII gene. The fourth underline indicates the position of the GAGA repeat. Numbering is relative to the sequence of the proximal promoter of the rat 3{alpha}-HSD/DD gene (26 ).

 
Identification of cis-Acting Regulatory Elements in the Distal Promoter of the 3{alpha}-HSD/DD (AKR1C9) Gene
To further localize the enhancer within the upstream region, a p(-4673/+61)CAT construct was deleted stepwise, and the resulting constructs were analyzed for CAT activity in HepG2 cells (Fig. 2Go). Deletion from -4673 to -4197 caused a loss of 64% of CAT activity. Further deletion to -3908 resulted in an another loss of 20% CAT activity. Subsequent deletion resulted in modest decreases of CAT activity that ranged from 3–11% of the activity observed in the parent construct. These results indicated that the strongest enhancer element was located between -4673 to -4197 bp. To test whether this region alone can activate the AKR1C9 gene promoter, this region was PCR amplified and ligated into a phsdCAT construct that contained only the basal promoter (-238 to +61) of the AKR1C9 gene linked to pBLCAT3, to yield the p(-4673/-4183)hsdCAT construct. The CAT activity of this construct was 34% that of p(-4673/+61)CAT (Fig. 3Go, construct b). This enhancer region contained 490 bp of upstream sequence and was designated EN490.



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Figure 2. Analysis of the Distal 3{alpha}-HSD/DD (AKR1C9) Gene Promoter by Transient Expression of 5'-Deletion CAT Constructs into HepG2 Cells

A series of 5'-nested deletions of the AKR1C9 gene promoter linked to the CAT reporter gene were generated. The CAT constructs were transfected into the human hepablastoma cell line, HepG2, and CAT activity was determined by the incorporation of the 1,3-butyryl groups into [14C]chloramphenicol. Relative CAT activity is given as a percentage. Transfection with pBLCAT3 represents a negative control since this construct contains no promoter and no enhancer. Experiments were performed in triplicate.

 


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Figure 3. Analysis of Portions of the Distal 3{alpha}-HSD/DD (AKR1C9) Gene Promoter by Transient Expression of CAT Reporter Gene Constructs Containing Internal Deletions

The top panel is the CAT activity of each construct indicated. Rectangles indicate putative C/EBP binding sites; circles indicate NF1 binding sites. An oval indicates a putative HNF1 binding site. Relative CAT activity is given as a percentage of construct a. Experiments were performed in triplicate.

 
Computer analysis4 showed that there were four imperfect CCAAT enhancer-binding protein (C/EBP) binding sites (-4667/-4660, -4536/-4527, -4523/-4514, and -4288/-4279), one putative hepatocyte nuclear factor 1 (HNF1) binding site (-4265/-4249), and three putative NF1 binding sites (-4351/-4288) contained within this region. The three NF1 binding sites were concentrated in a 64-bp core fragment:

GTGGAAAAACCCAGGAAGCACAGTGGAAAAACA-TGTTTATGCCCTCAGGAAAAAACCCAGGAAC. A comparison of the sequences of three putative NF1 binding sites in the 64-bp fragment with the NF1 consensus sequence is shown in Fig. 4BGo. NF1 is known to bind to a palindromic sequence TGGC/A(N)5GCCA (32, 33), and it also binds to one half of the palindrome with a much lower affinity (34, 35). Based on this sequence information, several deletion constructs were made by PCR to identify minimum sequences required to activate the AKR1C9 gene promoter.



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Figure 4. Oligonucleotides Containing Portions of the Distal Enhancer Used for Generating Reporter Gene Constructs and for Gel Mobility Shift Assays

A, NF1 binding sites, E1, E2, and E3, are indicated by underlines. A putative C/EBP binding site is indicated in bold. Restriction sites (HindIII) introduced are indicated in lowercase. B, A comparison of the sequences of E1, E2, and E3 with the NF1 consensus sequence. The underline indicates sequence identity to the NF1 consensus sequence.

 
Deletion of 189 bp from the 5'-end of the EN490 to remove the three putative distal C/EBP sites (Fig. 3Go, -4484/-4183 construct c) gave a CAT activity that was 67% of that driven by the whole promoter in p(-4673/+61)CAT (construct a). These data suggested that the three putative distal C/EBP binding sites were not involved in the activation of the AKR1C9 gene promoter. They also indicate that the dominant enhancer is located in the 301-bp fragment in the -4484/-4183 construct. Deletion of 62 bp from the 3'-end of the EN490 to remove the putative HNF1 site (Fig. 3Go, -4484/-4245, construct d) did not significantly decrease CAT activity, suggesting that this site was also not functional in the activation of the AKR1C9 gene promoter. Deletion of 274 bp (Fig. 3Go, -4399/-4183, construct e) from the 5'-end of the EN490 did not significantly reduce the CAT activity further (Fig. 3Go, compare -4484/-4245, construct d, with construct e). Removal of 274 bp and 62 bp from the 5'- and 3'-ends of EN490, respectively (Fig. 3Go, -4399/-4245, construct f) gave a CAT activity that was 39% of that driven by the whole promoter in p(-4678/+61)CAT. A construct containing two putative NF1 binding sites (Fig. 3Go, -4478/-4320 construct g) maintained only 17% CAT activity of p(-4673/+61)CAT but increased activity of phsdCAT by 3-fold. A construct containing the third putative NF1 binding site, the proximal C/EBP binding site, and the HNF1 site (Fig. 4Go, -4319/-4183, construct h) only maintained 6% CAT activity of p(-4673/+61)CAT and was completely devoid of enhancer activity. When the CAT activity of a construct containing the three putative NF1 sites and proximal C/EBP binding site (Fig. 4Go, -4399/-4245, construct d or f) is compared with either construct g (-4478/-4320) or construct h (-4319/-4183), it is apparent that these binding sites may function as the AKR1C9 gene enhancer. These binding sites are located within a 73-bp fragment.

To test the function of this 73-bp enhancer, the 73-bp E1234 (three putative NF1 binding sites and the proximal C/EBP binding site), E123 (three putative NF1 binding sites), E234 (two putative NF1 binding sites and the proximal C/EBP binding site), E12 (two putative NF1 binding sites), and E1 (one putative NF1 binding site) (Fig. 4AGo) were ligated into phsdCAT to yield p(E1234)hsdCAT, p(E123)hsdCAT, p(E234)hsdCAT, p(E12)hsdCAT, or p(E1)hsdCAT, respectively. The CAT activity of p(E1234)hsdCAT was 38–40% of the entire promoter p(-4673/+61)CAT (Fig. 5Go) and was identical to that observed in -4399/-4245 construct f (Fig. 3Go). These data indicated that the enhancer element in -4399/-4245 (154 bp) was also located in E1234 (73 bp). The CAT activity of p(E1234)hsdCAT was 13-fold higher than that of phsdCAT. The CAT activity of p(E123)hsdCAT, which lacked the proximal C/EBP binding site, was 9-fold higher than that of phsdCAT. p(E234)hsdCAT, which lacked the first putative NF1 binding site, lost almost all of its enhancer activity and had only 0.65-fold activity of phsdCAT. The CAT activity of p(E12)hsdCAT, which contained the first two putative NF1 binding sites, had 3-fold higher CAT activity than phsdCAT. The CAT activity of p(E1)hsdCAT, which contained only the first putative NF1 binding site, was the same as phsdCAT (Fig. 5Go). These results indicated that E1 was the most crucial element of the enhancer in E1234, but it did not act alone and required the presence of E2 and E3 to activate the AKR1C9 gene promoter. They also indicated that the presence of the putative proximal C/EBP binding site may be important, because of the difference in CAT activity between p(E1234)hsdCAT and p(E123)hsdCAT. The same enhancer elements were also subcloned into thymidine kinase promoter-CAT constructs and shown to increase transcription of the reporter gene from the heterologous promoter. Importantly, E1 was found to be the dominant element required for promoter activity (data not shown).



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Figure 5. Analysis of the Enhancer Activity in the 73-bp Fragment Linked to the Basal 3{alpha}-HSD/DD (AKR1C9) Gene Promoter

The top panel is the CAT activity of each construct indicated. The bottom panel is the sequence of enhancers tested in each construct. Underlined sequences represent the first (E1), second (E2), and third (E3) NF1 binding sites. Bolded sequence is a putative C/EBP binding site (E4). Relative CAT activity is given as a percentage of p(-4673/+61)CAT. Experiments were performed in triplicate.

 
Identification of the trans-Acting Factors That Bind to the 73-bp Enhancer
Electrophoretic mobility shift assays were conducted by using HepG2 nuclear extracts and radiolabeled oligonucleotide probes containing E1234, E123, E234, E12, or E1 to assess DNA-protein interactions at these sites. Similar DNA-protein complexes were formed with each of these oligonucleotide probes (Fig. 6Go). When rat liver nuclear extracts were substituted, these oligonucleotide probes formed DNA-protein complexes that had the same mobility (data not shown). The formation of these complexes was abolished by the presence of a 250-fold molar excess of unlabeled oligonucleotide probe identical in sequence with the labeled probe (Fig. 6Go). In subsequent electrophoretic mobility shift assays, a 10- to 50-fold excess of unlabeled oligonucleotide was sufficient to abolish the complexes, indicating high specificity of the DNA-protein interactions. Since the 73-bp fragment E1234 contains three putative NF1 binding sites and one putative C/EBP binding site, we next tested whether a C/EBP oligonucleotide could inhibit the formation of the E1234-protein complexes. These complexes were not inhibited by a 250-fold molar excess of C/EBP oligonucleotide or NFkB oligonucleotide (used as a negative control), whereas these complexes were completely competed out in a concentration-dependent manner by unlabeled E1234 oligonucleotide (50- to 250-fold excess) (Fig. 7Go). To further confirm that C/EBP transcription factors do not bind to E1234, we demonstrated that in vitro transcribed and translated recombinant C/EBP{alpha} or C/EBPß do not bind to E1234. As a positive control, recombinant C/EBP{alpha} or C/EBPß bound to a C/EBP oligonucleotide, and this binding complex was supershifted by C/EBP{alpha} and C/EBPß antibodies, respectively (Fig. 8Go). By contrast, the DNA-protein complexes formed with E1234 were not supershifted with the C/EBP{alpha} and C/EBPß antibodies (data not shown). Thus, the factors that bind to E1234 are not members of the C/EBP transcription factor family.



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Figure 6. HepG2 Nuclear Extracts Bind to Enhancer Elements in the Rat 3{alpha}-HSD/DD (AKR1C9) Gene Distal Promoter

Sequences of the double-stranded oligonucleotides used in the gel shift analysis are listed in Fig. 4Go. HepG2 nuclear extracts (10 µg) were incubated with [32P]-end-labeled double-stranded oligonucleotide in the absence (-) or presence (+) of a 250-fold molar excess of the unlabeled homologous double-stranded oligonucleotide. The arrows indicate the DNA-protein complexes formed.

 


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Figure 7. C/EBP and NFkB Oligonucleotides Fail to Compete with E1234 for HepG2 Nuclear Extracts

E1234 probe was incubated with HepG2 nuclear extracts to form a complex (lane 1). The complex was competed with a 10-, 50-, and 250-fold molar excess of unlabeled E1234 (lanes 2–4), unlabeled C/EBP (lanes 5–7), or unlabeled NFkB (lanes 8–10) double-stranded oligonucleotides.

 


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Figure 8. E1234 Does Not Bind to Recombinant C/EBP{alpha} or C/EBPß

Recombinant C/EBP{alpha} and C/EBPß were expressed via in vitro transcription and translation using a rabbit reticulocyte lysate system. Lane 1, E1234 ({gamma}-32P-labeled probe); lane 2, E1234 probe and rabbit reticulocyte lysate; lane 3, E1234 probe and recombinant C/EBP{alpha}; lane 4, E1234 probe and recombinant C/EBPß; lane 5, C/EBP oligonucleotide and rabbit reticulocyte lysate; lane 6, C/EBP oligonucleotide and recombinant C/EBP{alpha}; lane 7, C/EBP oligonucleotide and recombinant C/EBPß; lane 8, C/EBP oligonucleotide, recombinant C/EBP{alpha} and C/EBP{alpha} antibody; lane 9, C/EBP oligonucleotide, recombinant C/EBPß and C/EBPß antibody.

 
We next tested whether NF1 transcription factors bind to E1234 by electrophoretic mobility shift analysis. When E1234 was used as a probe, the E1234-protein complex was abolished by a 50-fold excess of NF1 consensus oligonucleotide. When an NF1 consensus oligonucleotide was used as a probe, the NF1-protein complex was inhibited by a 50-fold excess of E1234 (Fig. 9Go). Furthermore, the E1234-protein complex was supershifted by the NF1 antibody (Fig. 10Go); no supershift was observed when preimmune serum was substituted. Importantly, identical results were obtained with nuclear extracts from either HepG2 cells or rat liver. DNase I footprinting showed that HepG2 cell and rat liver nuclear extracts protected the E1 fragment consistent with the important role of this element in driving CAT reporter gene activity (data not shown). Our data indicated that a member of the NF1 transcription factor family binds to the E1234 enhancer and that E1 may be the most relevant site.



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Figure 9. The DNA-protein Complex Formed with the 73-bp Fragment Is Competed by NF1 Oligonucleotides and Vice Versa

The E1234 probe (lanes 1–5) was incubated with HepG2 nuclear extracts to form a complex (lane 1). The complex was competed with a 50- and 250-fold molar excess of unlabeled E1234 (lanes 2–3) and NF1 (lanes 4–5), respectively. NF1 probe (lanes 6–10) was incubated with HepG2 nuclear extracts to form a complex (lane 6). The complex was competed with a 50- and 250-fold molar excess of unlabeled NF1 (lanes 7 and 8) and E1234 (lanes 9 and 10), respectively. The NF1 oligonucleotide had the sequence 5'-TTTTGGATTGAAGCCAATATGATAA-3'.

 


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Figure 10. NF1 Antibody Supershifts the 73-bp Fragment-Protein Complex

The E1234 probe was incubated with HepG2 (lanes 1 and 2) and rat (lanes 3 and 4) nuclear extracts, respectively. NF1 antibody was added to the reactions in lanes 2 and 4 (see Materials and Methods for details).

 
NF1 trans-Activates the 73-bp Enhancer in Drosophila Schneider SL-2 Cells
The NF1 transcription family is often ubiquitously expressed and contains many splice variants that could affect the signal observed in cotransfection paradigms using CAT reporter gene constructs in HepG2 cells. We noted that SL2 insect cells are devoid of NF1 transcription factors, which suggested that these cells could be used in cotransfection experiments (36). As a negative control we cotransfected either pADH (Drosophila Adh promoter) or pADH-CTF1 [in which expression of CTF1 (CCAAT transcription factor)/NF1 is driven by the Adh promoter] with p-55{alpha}CAT (pBLCAT3 containing the {alpha}1-globin promoter and devoid of NF1 sites) into SL2 cells and noted no increase in CAT activity (Fig. 11Go). As a positive control we co-transfected either pADH or pADH-CTF1 with p-87(3XAd){alpha}CAT (which contains four NF1 sites upstream from the {alpha}1-globin promoter) and obtained a 7- to 10- fold increase in CAT activity when pADH-CTF1 and p-87(3XAd){alpha}CAT were cotransfected. In the experimental system in which either pADH or pADH-CTF1 was cotransfected with p(E1234)-55{alpha}CAT (in which the {alpha}1-globin promoter contains E1234 substituted for the four NF1 sites) a 2- to 3-fold increase was obtained when pADH-CTF1 and p(E1234)-55{alpha}CAT were cotransfected. These data suggest that E1234 is trans-activated by NF1 transcription factors.



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Figure 11. CTF1/NF1 Transactivates the 73-bp Enhancer in SL2 Insect Cells

A, Diagrams of reporter gene constructs, p-55{alpha}CAT, p-87(3XAd){alpha}CAT, and p(E1234)-55{alpha}CAT. p-87(3XAd){alpha}CAT contains the {alpha}1-globin promoter (-87 to +36) and contains an upstream CCAAT box (NF1 site) and three adenovirus NF1 binding sites in the pBLCAT3 reporter. p(E1234)-55{alpha}CAT contains the E1234 5' upstream to the {alpha}1-globin promoter (-55 to +36) in pBLCAT3. B, The reporter gene CAT constructs [p-55{alpha}CAT, p-87(3XAd){alpha}CAT, and p(E1234)-55{alpha}CAT] were cotransfected with either the expression vector pADH alone or the expression vector for CTF1/NF1 (pADH-CTF1) into SL2 cells. The cells were subsequently lysed and assayed for CAT activity normalized to ß-galactosidase. Experiments were performed in triplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AKR genes play pivotal roles in steroid hormone metabolism and endocrinology yet their gene structures and details on how they are regulated are only just emerging. Genes of the AKR1C subfamily are involved in the hepatic clearance of steroid hormones and include 5ß-reductase and 3{alpha}-HSD. The rat 3{alpha}-HSD/DD (AKR1C9) gene, which is the focus of this study, contains multiple SREs in its promoter. These are likely to respond to high circulating steroid hormones, and subsequent gene transcription will protect against steroid hormone excess. A human homolog, AKR1C1, shares the dihydrodiol dehydrogenase function of the rat AKR1C9 gene and metabolizes PAH trans-dihydrodiols to yield reactive and redox active o-quinones. The AKR1C1 gene is induced by reactive oxygen species possibly through an antioxidant response element to protect against oxidative stress (37). By contrast, genes of the AKR1B subfamily or aldose reductases catalyze the first step in the polyol pathway and convert glucose to sorbitol (28). In diabetes mellitus, high blood glucose is converted to the hyperosmotic sugar, sorbitol. The resultant increase in water uptake contributes to the complications arising from diabetes, e.g. diabetic retinopathy, neuropathy, and nephropathy (28). The rat, rabbit, and human AKR1B gene promoters have been shown to contain osmotic response elements that respond to hyperosmolarity and induce gene transcription to protect against osmotic stress (28, 29, 31). The transcription factors responsible for this response are unknown. What emerges is that AKR genes serve protective roles; they are often constitutively expressed yet the identity of transcription factors that control their expression is unclear. Our studies show that NF1 contributes to the high constitutive expression of the AKR1C9 gene in rat liver (0.5–1.0% of the soluble hepatic protein).

Sequence analysis of the distal promoter (-4673 to -1533 bp) of the AKR1C9 gene led to the identification of three novel elements. First, the promoter contained GAGA repeats (-2401 to -2271), which are implicated in controlling basal and GH-dependent gene transcription (38, 39). When these GAGA repeats were deleted from the 3{alpha}-HSD/DD gene promoter (from -2685 to -1906), transcription of the reporter gene CAT was decreased 5.8-fold (Fig. 2Go). These results suggest that GAGA repeats may contribute to constitutive expression of the 3{alpha}-HSD/DD gene. This gene also displays sexual dimorphic expression, and GAGA repeats are found in the promoters of other steroid-metabolizing enzymes that show a similar pattern of expression. These genes include cytochrome P450 16{alpha}-hydroxylase CYP2C11 [expressed in males (40)] and cytochrome P450 15ß-hydroxylase CYP2C12 [expressed in females, (41)]. Feminization of rat hepatic steroid hormone metabolism (i.e. elevated 15ß-hydroxylase and 5{alpha}-reductase activities and decreased 16{alpha}-hydroxylase and 5ß-reductase activities) has been attributed to the gonadal control of GH release from the anterior pituitary (41, 42, 43, 44). The 3{alpha}-HSD/DD gene, however, is not regulated solely by GH. Thus, hypophysectomy will reduce 3{alpha}-HSD/DD mRNA, protein, and enzyme activity 8-fold, but normal levels are not restored by the daily administration of GH (21). It is known that GH is required to maintain hepatic estrogen receptor expression, and it is likely that GH primes the liver so that the AKR1C9 gene can be induced by estrogens (45). Although, the existence of GAGA repeats in all these genes is intriguing, it is unlikely to be the element that is responsible for their sexual dimorphic expression because it is resident in genes expressed in both sexes.

Second, the distal promoter contains sequences in the complementary strand from -4134 to -3734 and from -3157 to -2838 that are 94% and 86% identical in sequence to that found between -1578 to -1204 of the MCPII gene, which was identified as a mast cell-specific enhancer element (46). The 3{alpha}-HSD/DD gene promoter also contains perfect mast cell-specific transcription factor binding sites defined as GATA binding sites, at -3933/-3928 and -2965/-2960. This suggests that AKR1C genes may be expressed upon mast cell activation. Activated human mast cells are well known to synthesize 9{alpha},11ß-PGF2{alpha} (47). AKR1C9 can function as a 9-, 11-, and 15-hydroxy-PG dehydrogenase (48). It also shares significant sequence homology with PGF2{alpha} synthase (AKR1C 7) (49, 50). Further, human type 3 3{alpha}-HSD (AKR1C2) can efficiently reduce PGD2 to yield 9{alpha},11ß-PGF2{alpha} (51). It will be of importance to determine whether expression of AKR1C1 genes is correlated to mast cell activation to protect against allergens.

Third, we located a triple repeat within a 73-bp fragment which is the cis-element responsible for high constitutive expression of the AKR1C9 gene. The enhancer element contained three NF1 binding sites and one imperfect C/EBP binding site (Figs. 3Go and 4Go). This enhancer was able to increase the activity of the basal hsd promoter by 13-fold (Fig. 5Go). Electrophoretic mobility shift analysis demonstrated that the 73-bp enhancer bound NF1 transcription factors but not C/EBP transcription factors. In cotransfection experiments in SL2 insect cells, CTF1/NF1 was able to trans-activate the E1234 enhancer.

When considering how NF1 mediates hepatic specific expression of the AKR1C9 gene, it is important to consider that NF1 is encoded by at least four genes (NF1A, NF1B, NF1C, and NF1X) that share high sequence homology with CTF (34, 52, 53, 54, 55). Alternative splicing of the NF1 genes adds further diversity to this family (32, 55). NF1 can bind to DNA as a homo- or heterodimer (34, 56). These factors bind with high affinity to the palindromic sequence TGGC/A(N)5GCCA (32, 33) and with lower affinity to the half-site TGGC/A (34, 35). The NF1 proteins are highly conserved in their N-terminal DNA-binding domain but diverge in their C-terminal transactivation domains (34). Although NF1 is known as a ubiquitous transcription factor, some forms of NF1 are restricted to certain tissues, and recent analysis of NF1 expression in human and murine cell lines showed that there are cell line-specific differences in both the isoforms and the amount of NF1 binding activity observed (57). NF1 sites are frequently found adjacent to or overlap with other transcription factor-binding sites, e.g. AP-1 family members, steroid hormone receptors, and HNF3{alpha} (58, 59, 60). It has been suggested that the interaction of NF1 with other transcription factors may ultimately determine cell- or tissue-specific gene expression. NF1 will form a composite element by binding in close apposition with HNF3{alpha} to modulate liver-specific expression of the serum albumin gene (60, 61); NF1 and HNF1 also regulate the liver-specific expression of the {alpha}-fetoprotein gene (62), whereas estrogens and progestins regulate the amount of NF1 that binds to and trans-activates the vitellogenin gene (63). Thus, liver-specific expression of genes regulated by NF1 appears to be governed by its interaction with other factors. The identity of the NF1 family member that binds to the 73-bp enhancer of the AKR1C9 gene and the identity of cooperative transcription factors that govern the liver-specific expression of this gene remain to be determined.

Identification of NF1 as trans-activators of the AKR1C9 gene promoter suggests a model for the regulation of steroid hormone metabolism in rat liver. High constitutive expression of the AKR1C9 gene leads to the conversion of 5{alpha}- and 5ß-dihydrosteroids to 3{alpha}/5{alpha}- and 3{alpha}/5ß-tetrahydrosteroids and is enhanced by NF1 and repressed by Oct-1 (26). Increases in steroid hormones in the circulation are sensed by steroid receptors that increase AKR1C9 gene transcription in an autoregulatory manner. Although this is the case for glucocorticoids (27), more complex paradigms may exist for regulation by gonadal steroids (Fig. 12Go). Knowing that 5ß-reductase (AKR1D) precedes 3{alpha}-HSD in steroid hormone metabolism, it will be of interest to determine what transcription factors regulate the expression of the AKR1D gene especially since it is the rate-limiting enzyme in this metabolic pathway.



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Figure 12. Regulation of AKR Genes Involved in Hepatic Steroid Hormone Metabolism

 
The AKR1C9 gene is also involved in carcinogen metabolism. NF1 has not been previously implicated in the regulation of genes involved in PAH activation. The CYP1A1 gene, which codes for the predominant cytochrome P450 involved in the activation of PAH-trans-dihydrodiols (proximate carcinogens) to PAH-anti-diol epoxides (ultimate carcinogens), is regulated by the occupied Ah receptor binding to a xenobiotic response element (64, 65, 66). In contrast, NF1 binds to the AKR1C9 gene promoter to enhance activation of PAH-trans-dihydrodiols to PAH-o-quinones with the concomitant production of reactive oxygen species. Since these two pathways of PAH activation are differentially regulated, which pathway predominates may depend upon the availability of these trans-acting factors and their cooperative partners.

NF1 is one of the few transcription factors that have been implicated in the regulation of AKR genes. We have been struck by the sequence similarity between the NF1 sites in the AKR1C9 gene promoter and the osmotic response elements found in the promoters of the rat, rabbit, and human aldose reductase promoters (see Table 1Go). Whether NF1 sites are involved in the constitutive or regulated expression of other protective AKR genes remains to be determined. If NF-1 is involved in mediating stress responses, it is likely to be regulated by stress-activated kinases.


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Table 1. Comparison of E1, E2, and E3 Sequences on the AKR1C9 Gene with Putative Osmotic Response Elements

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The human hepatoma cell line, HepG2 (ATCCHB8065), was maintained in continuous culture in Earle’s MEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, 100 U/ml penicillin, and 10 µg/ml streptomycin (growth medium) at 37 C in a humidified atmosphere with 5% CO2. Cells were passaged every 7 days with a 1:4 dilution. Drosophila Schneider SL-2 cells (a gift from Dr. James B. Jaynes at Thomas Jefferson University) were grown in Schneider’s insect medium supplemented with 10% FBS at 25 C.

Generation of pCAT Constructs
Six CAT reporter gene constructs containing variable lengths of the 5'-flanking region of the AKR1C9 gene were generated by 5' deletion of the pBLCAT3 construct containing 6.6 kb upstream of the rat AKR1C9 gene as described previously (26). Briefly, the pBLCAT3 plasmid containing the -6.6-kb insert was linearized with PstI and SphI and subjected to nested deletion with exonuclease III and S1 nuclease. The deletion mutants were recircularized by the addition of the Klenow fragment of DNA polymerase I and T4 DNA ligase and correspond to -4673 to +61 bp; -4197 to +61 bp; -3908 to +61 bp; -3261 to +61 bp; -2685 to +61 bp; and -1906 to +61 bp where the transcription start site is at +1. The internal deletion and further 5'-deletion constructs were produced by PCR using p(-4673/+61)CAT as the template and a combination of oligonucleotides as primers shown in Table 2Go. The resulting PCR products were cloned into a HindIII site of phsdCAT, which contains the basal AKR1C9 gene promoter (-238 to +61) in pBLCAT3 to yield vectors b–f in Fig. 3Go. Vectors g and h (Fig. 3Go) were created via a slightly different strategy. A 301-bp fragment, -4484/-4183, was PCR amplified using the primers in Table 2Go and blunt-end ligated into the PCR-blunt end vector pCRII (Invitrogen, San Diego, CA). The -4478/-4320 fragment was then released by digestion with NcoI and AfIIII, and the -4319/-4183 fragment was released with AfIIII alone. Each fragment was then blunt-end ligated into the HindIII site of phsdCAT. Another strategy was used to generate p(E1234)hsdCAT, p(E123)hsdCAT, p(E12)hsdCAT, and p(E1)hsdCAT. Because of the lack of available restriction sites in the basal promoter construct, phsdCAT (-238 to +61 bp), the basal promoter, was PCR amplified using primers containing 5' and 3' XbaI amd BamHI linkers, respectively, and a phsd-CAT construct as template. The parental pBLCAT3 vector was linearized at the same restriction sites, and the basal promoter was directionally subcloned into the vector. The vector was then digested at the HindIII and XbaI sites in the multiple cloning site upstream from the hsd promoter, and the enhancer elements in Fig. 4Go. were subcloned into these sites using the compatible HindIII site in the oligonucleotides encoding those elements.


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Table 2. Primers for Generating CAT Constructs

 
Constructs for Cotransfection
The constructs for cotransfection in Drosophila Schneider SL-2 cells—pADH, pADH-CTF1, p-55{alpha}CAT, and p-87(3XAd){alpha}-CAT—previously designated as pPADH, pPADH-CTF1, p{alpha}-CAT-55, and p{alpha}-CAT-88–3XAd (36) were gifts from Dr. Nicolas Mermod (University of Lausanne, Lausanne, Switzerland). pADH is a Drosophila expression vector containing the proximal and distal tandem promoters of the Drosophila alcohol dehydrogenase (Adh) gene. pADH-CTF1 is an expression construct in which CTF1/NF1 expression is driven by the Adh promoter. p-55{alpha}CAT is a pBLCAT3 vector in which CAT activity is driven by -55 to +36 bp of the {alpha}1-globin promoter. p-87(3XAd){alpha}CAT is a pBLCAT3 vector in which CAT activity is driven by -87 to +36 bp of the {alpha}1-globin promoter containing a CCAAT box (CTF/NF1 site) and three adenovirus NF1 sites. To construct p(E1234)-55{alpha}CAT, p-87(3XAd){alpha}CAT was digested by EagI and SalI to remove all four NF1 sites and ligated to a E1234 fragment. The EagI site is located at position -55 of the {alpha}1-globin promoter. The fidelity of the constructs was confirmed by dideoxy sequencing.

Transient Transfection
HepG2 cells were seeded at a concentration of 1.5 x 106 cells per 60-mm tissue culture plate for 24 h. Before transfection (4 h), the medium was removed and replaced with a fresh growth medium. For each pCAT construct, DNA-CaCl2 solutions were prepared by mixing 37 µl 2 M CaCl2, 10 µg of pCAT constructs or CAT control plasmids (pBLCAT3 basic promoter and enhancerless, Promega Corp., Madison, WI), plus 2 µg of pSV-ß-galactosidase containing plasmid (Promega Corp.) in a final volume of 300 µl. DNA was precipitated by mixing with an equal volume of 2 x HEPES-buffered saline [50 mM HEPES (pH 7.1), 280 mM NaCl, and 1.5 mM Na2HPO4)] with constant agitation. The precipitants were incubated at room temperature for 30 min before their addition to the cell culture medium. After 36–48 h, the cells were washed twice with PBS (pH 7.1) and harvested using lysis buffer (Promega Corp.) for CAT and ß-galactosidase enzyme assays. SL2 cells were cotransfected by calcium phosphate coprecipitation as described above. Three micrograms of CTF1/NF1 were cotransfected with 1 µg of reporter construct and 0.5 µg of hsp82LacZ (ß-galatosidase reporter, a gift from Dr. James B. Jaynes).

For each ß-galactosidase enzyme assay, a cell lysate (100 µl) was incubated in 60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, and 50 mM ß-mercaptoethanol, containing 0.67 mg/ml o-nitrophenyl-ß-D-galactopyranoside as a substrate in a total volume of 300 µl at 37 C for 2 h. The reaction was terminated by adding 500 µl of 1 M sodium carbonate, and the absorbance of the o-nitrophenol anion was read at 420 nm. This end point assay was validated by showing that the absorbance was in the linear range with respect to time and lysate protein. For CAT activity, the volumes of the cell lysates were adjusted to contain the same amount of ß-galactosidase activity to normalize for transfection efficiency. Lysates (50–100 µl) were then incubated in a final volume of 125 µl containing 25 mM Tris-HCl (pH 7.5), 40 µM (1.0 µCi) chloramphenicol (chloramphenicol D-threo-[dichloroacetyl[-1, 2-14C]) (57 mCi/mmol, Dupont NEN, Boston, MA), and 50 µM butyryl-CoA. The reactions were initiated by the addition of coenzyme and incubated for 8 h at 37 C. The reactions were terminated by extraction with 500 µl ethyl acetate, the extracts were dried, resuspended in 20 µl ethyl acetate, and applied to TLC plates (Silica Gel IB2; J.T. Baker, Inc., Phillipsburg, NJ), which were developed in CHCl3/MetOH, 97:3 (vol/vol). The results were quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis of the butylated and nonbutylated forms of [14C]chloramphenicol and are reported as relative values in arbitrary units. The results for each construct represent the mean ± SE from three separate experiments. All values were normalized for ß-galactosidase activity cotransfected as pSV-ß-galactosidase.

Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared from HepG2 cells as described previously (26). Radiolabeled oligonucleotides were [{gamma}32P]-labeled at 5'-end using T4 polynucleotide kinase and [{gamma}32P]ATP (30 Ci/mmol, Dupont NEN). Nuclear extracts (10 µg) were incubated with 2 fmol of radiolabeled oligonucleotides (50,000 cpm) for 20 min at room temperature in the presence of 0.5 µg sheared salmon sperm DNA and 5 µg poly(dI·dC) (Pharmacia Biotech, Piscataway, NJ) in a binding reaction of 20 µl containing 40 mM KCl, 25 mM HEPES (pH 7.9), 1 mM EDTA, 0.5 mM DTT, 5 mM, and 5% glycerol. The mixtures were electrophoresed on a 4% polyacrylamide gel in 0.5 x TBE buffer (45 mM Tris-borate and 1 mM EDTA). For the competition studies, a 10- to 250-fold molar excess of unlabeled oligonucleotide were added to the incubation. For the supershift assays, 1 µl of antibodies for NF1 (a polyclonal antiserum that recognizes CTF-2 and a C-terminal peptide of CTF-1 was obtained from Dr. Naoko Tanese at New York University) or two C/EBP isoforms, {alpha} and ß (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to the binding reactions and incubated for another 20 min at room temperature before electrophoresis. Eukaryotic expression constructs for C/EBP{alpha} and C/EBPß (in the pSG5 vector) were gifts from Dr. Mitchell Lazar (Department of Medicine, University of Pennsylvania, Philadelphia, PA). Recombinant proteins were expressed using a rabbit reticulocyte lysate system (Promega Corp.) according to the manufacturer’s protocol. Gel shift analyses were conducted using 1 µl of the translation mixture in place of the nuclear extracts.


    FOOTNOTES
 
Address requests for reprints to: Dr. Trevor M. Penning, Department of Pharmacology, 130c John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084.

This work was supported by Grant CA-55711 awarded by the National Cancer Institute (T.M.P.).

1 Current address: Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205. Back

2 3{alpha}-HSD/DD, 3{alpha}-hydroxysteroid: NAD(P)+ oxidoreductase (EC1.1.1.213). A face-specific/dihydrodiol dehydrogenase, trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC 1.3.1.20) now designated as AKR1C92. Back

3 The nomenclature of the AKR superfamily was recommended by the 8th International Symposium on Enzymology & Molecular Biology of Carbonyl Metabolism held in Deadwood, South Dakota, on June 29-July 3, 1996 (2 ). Back

4 The TESS search engine (www.cbil.upenn.edu) was used to search the Transfac database. Back

5 The sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank and given the accession number AF049661. Back

Received for publication January 11, 1999. Revision received June 10, 1999. Accepted for publication July 12, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Jez JM, Bennett MJ, Schlegel BP, Lewis, M, Penning TM 1997 Comparative anatomy of the aldo-keto reductase superfamily. Biochem J 326:625–636[Medline]
  2. Jez JM, Flynn TG, Penning TM 1997 A new nomenclature for the aldo-keto reductase superfamily. Biochem Pharmacol 54:639–647[CrossRef][Medline]
  3. Wilson DK, Bohren KM, Gabbay KH, Quiocho FA 1992 An unlikely sugar substrate site in the 1.65 129 Å structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257:458–460
  4. Bennett MJ, Schlegel BO, Jez JM, Penning TM, Lewis M 1996 Structure of 3{alpha}-hydroxysteroid/dihydrodiol de-hydrogenase complexed with NADP+. Biochemistry 35:10702–10711[CrossRef][Medline]
  5. Lin H-K, Jez JM, Schlegel BP, Peehl DM, Pachter JA, Penning TM 1997 Expression and characterization of recombinant type 2 3{alpha}-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3{alpha}/17ß-HSD activity and cellular distribution. Mol Endocrinol 11:1971–1984[Abstract/Free Full Text]
  6. Mao J, Duan RW, Zhong L, Gibori G, Azhar S 1997 Expression, purification and characterization of rat luteal 20{alpha}-hydroxysteroid dehydrogenase. Endocrinology 138:182–190[Abstract/Free Full Text]
  7. Tomkins GM 1956 A mammalian 3{alpha}-hydroxysteroid dehydrogenase. J Biol Chem 218:437–447[Free Full Text]
  8. Hoff H-G, Schreifers H 1973 Sexuell differenzierte und sexuell undifferenzierte 3{alpha}- and 3ß-hydroxysteroid-aktivaten und ihre intra zellulore lokalisation in der rattenleber. Hoppe-Seyler’s Z Physiol Chem 354:507–513[Medline]
  9. Penning TM, Smithgall TE, Askonas LJ, Sharp RB 1986 Rat liver 3{alpha}-hydroxysteroid dehydrogenase. Steroids 47:221–247[Medline]
  10. Penning TM, Pawlowski JE, Schlegel BP, Jez JM, Lin H-K, Hoog SS, Bennett MJ, Lewis M 1996 Mammalian 3{alpha}-hydroxysteroid dehydrogenases. Steroids 61:508–523[CrossRef][Medline]
  11. Jez JM, Penning TM 1998 Engineering steroid 5ß-reductase activity into rat liver 3{alpha}-hydroxysteroid dehydrogenase. Biochemistry 37:9695–9703[CrossRef][Medline]
  12. Ikeda M, Hayakawa S, Ezaki M, Ohmori S 1981 An NADP+-dependent 3{alpha}-hydroxysteroid dehydrogenase of rat liver active against C19, C20, C23, C24, C25 and C26 steroids. Hoppe-Seyler’s Z Physiol Chem 362:511–520[Medline]
  13. Stolz A, Takikawa H, Sugiyama Y, Kuhlenkamp J, Kaplowitz N 1987 3{alpha}-hydroxysteroid dehydrogenase activity of the Y’ bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance. J Clin Invest 79:427–434[Medline]
  14. Takikawa H, Ookhtens M, Stolz A, Kaplowitz N 1987 Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by 3{alpha}-hydroxysteroid dehydrogenase. II. Studies in the prograde and retrograde single-pass, perfused rat liver and inhibition by indomethacin. J Clin Invest 80:861–866[Medline]
  15. Smithgall TE, Harvey RG, Penning TM 1988 Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase. J Biol Chem 263:1814–1820[Abstract/Free Full Text]
  16. Penning TM, Ohnishi ST, Ohnishi T, Harvey RG 1996 Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase. Chem Res Toxicol 9:84–92[CrossRef][Medline]
  17. Khanna M, Qin KN, Wang RW, Cheng KC 1995 Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3{alpha}-hydroxysteroid dehydrogenases. J Biol Chem 270:20162–20168[Abstract/Free Full Text]
  18. Hara A, Matsuura K, Tamada Y, Sato K, Miyabe Y, Deyashiki Y, Ishida N 1996 Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells. Biochem J 313:373–376[Medline]
  19. Smithgall TM, Penning TM 1985 Sex-differences in indomethacin-sensitive 3{alpha}-hydroxysteroid dehydrogenase of rat liver cytosol. Cancer Res 45:4946–4949[Abstract]
  20. Penning TM, Isaacson K, Lyttle CR 1992 Hormonal regulation of 3{alpha}-hydroxysteroid dehydrogenase in rat liver cytosol. Biochem Pharmacol 43:1148–1152[CrossRef][Medline]
  21. Hou Y-T, Xia W, Pawlowski JE, Penning TM 1994 Rat dihydrodiol dehydrogenase: complexity of gene structure and tissue-specific and sexually dimorphic gene expression. Cancer Res 54:247–255[Abstract]
  22. Straviz RT, Vlahevic ZR, Pandak M, Stolz A, Hylemon PB 1994 Regulation of rat hepatic 3{alpha}-hydroxysteroid dehydrogenase in vivo and in primary cultures of rat hepatocytes. J Lipid Res 35:239–247[Abstract]
  23. Graham A, Brown L, Hedge PJ, Gammack AJ, Markham AF 1991 Structure of the human aldose reductase gene. J Biol Chem 266:6872–6877[Abstract/Free Full Text]
  24. Lou H, Hammond L, Sharma V, Sparkes RS, Lusis AJ, Stolz A 1994 Genomic organization and chromosomal localization of a novel human hepatic dihydrodiol dehydrogenase with high affinity bile acid binding. J Biol Chem 269:8416–8422[Abstract/Free Full Text]
  25. Lin H-K, Hung C-F, Moore M, Penning TM 1999 Genomic structure of rat 3{alpha}-hydroxysteroid/dihydrodiol dehydrogenase (3{alpha}-HSD/DD AKR1C9). J Steroid Biochem Mol Biol, in press
  26. Lin H-K, Penning TM 1995 Cloning, sequencing and functional analysis of the 5'-flanking region of the rat 3{alpha}-hydroxysteroid/dihydrodiol dehydrogenase gene. Cancer Res 55:4104–4113
  27. Hou Y-T, Lin H-K, Penning TM 1998 Dexamethasone regulation of the rat 3{alpha}-hydroxysteroid/dihydrodiol dehydrogenase gene. Mol Pharmacol 53:459–466[Abstract/Free Full Text]
  28. Ruepp B, Bohren KM, Gabbay KH 1996 Characterization of the osmotic response element of the human aldose reductase gene promoter. Proc Natl Acad Sci USA 93:8624–8629[Abstract/Free Full Text]
  29. Ferraris JD, Williams CK, Jung K-Y, Bedford JJ, Burg MB, Garcia-Perez A 1996 ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress. J Biol Chem 271:18318–18321[Abstract/Free Full Text]
  30. Kultz D, Garcia-Perez A, Ferraris JD, Burg MB 1997 Distinct regulation of osmoprotective genes in yeast and mammals. Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells. J Biol Chem 272:13165–13170[Abstract/Free Full Text]
  31. Iwata T, Minucci S, McGowan M, Carper D 1997 Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter. J Biol Chem 272:32500–32506[Abstract/Free Full Text]
  32. Borgmeyer U, Nowock J, Sippel AE 1984 The TGGCA-binding protein: a eukaroytic nuclear protein recognizing a symmetrical sequence on double stranded linear DNA. Nucleic Acids Res 12:4295–4311[Abstract]
  33. Gronostajski RM 1987 Site-specific DNA binding of nuclear factor I: effect of spacer region. Nucleic Acids Res 15:5545–5559[Abstract]
  34. Gounari F, De Francesco R, Schmitt J, van der Vliet PC, Cortese R, Stunnenberg H 1990 Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA-replication. EMBO J 9:559–566[Abstract]
  35. Paonessa G, Gounari F, Frank R, Cortese R 1988 Purification of a NF1-like DNA-binding protein from rat-liver and cloning of the corresponding cDNA. EMBO J 7:3115–3123[Abstract]
  36. Mermod N, O’Neill EA, Kelly TJ, Tjian R 1989 The proline rich transcriptional activator of CTF/NF1 is distinct from the replication and DNA binding domain. Cell 58:741–753[Medline]
  37. Burczynski ME, Lin HK, Penning TM 1999 Isoform-specific induction of human aldo-keto reductase by polycyclic aromatic hydrocarbons (PAHs), electrophiles and oxidative stress: implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenases. Cancer Res 59:607–614[Abstract/Free Full Text]
  38. Granok H, Leibovitch BA, Shaffer CD, Elgin SCR 1995 Chromatin-GA-GA over GAGA factor. Curr Biol 5:238–241[Medline]
  39. Legraverend C, Simar-Blanchet A-E, Sotiropoulos A, Finidori J, LeCam A, 1996 A novel growth hormone response element unrelated to STAT (signal transducer and activator of transcription)-binding sites is a bifunctional enhancer. Mol Endocrinol 10:1507–1518[Abstract]
  40. Morishima N, Yoshioka H, Higashi Y, Sogawa K, Fujii-kuriyama Y 1987 Gene structure of cytochrome P-450 (M-1) specifically expressed in male rat liver. Biochemistry 26:8279–8285[Medline]
  41. Zaphiropoulos PG, Westin S, Strom A, Mode A, Gustafsson J-A 1990 Structural and regulatory analysis of the cyt P450 gene (CYP2C12) expressed predominantly in female rat liver. DNA Cell Biol 9:49–56[Medline]
  42. Mode A, Gustafsson J-A, Jansson J-O, Eden S, Isaksson O 1982 Association between plasma level of growth hormone and sex differentiation of hepatic steroid metabolism in the rat. Endocrinology 111:1692–1697[Medline]
  43. Zaphiropoulos PG, Mode A, Norstedt G, Gustafsson J-A 1989 Regulation of sexual differentiation in drug and steroid metabolism. Trends Pharmacol Sci 10:149–53[CrossRef][Medline]
  44. Westin S, Storm A, Gustafsson J-A, Zaphiropoulos PG 1990 Growth-hormone regulation of the cytochrome P450IIC subfamily in the rat-inductive, repressive and transcriptional effects on P-450F (IIC7) and P-450PB1 (IIC6) gene expression. Mol Pharmacol 38:192–197[Abstract]
  45. Norstedt G, Wrange O, Gustafsson J-A 1981 Multihormonal regulation of the estrogen receptor in rat liver. Endocrinology 122:325–355[Abstract]
  46. Sarid J, Benfey PN, Leder P 1989 The mast cell-specific expression of a protease gene, RMCP-II is regulated by an enhancer element that binds specifically to mast-cell trans-acting factors. J Biol Chem 264:1022–1026[Abstract/Free Full Text]
  47. Liston TE, Roberts II LJ 1985 Transformation of prostaglandin D2 to 9{alpha},11ß-(15S)-trihydroxyprosta-(5Z, 13E)-dien-1-oic acid (9{alpha}, 11ß-prostaglandin F2). A unique biologically active prostaglandin produced enzymatically in vivo in humans. Proc Natl Acad Sci USA 82:6030–6034[Abstract]
  48. Penning TM, Sharp RB 1987 Prostaglandin dehydrogenase activity of purified rat liver 3{alpha}-hydroxysteroid dehydrogenase. Biochem Biophys Res Commun 148:646–653[Medline]
  49. Pawlowski JE, Huizinga M, Penning TM 1991 Cloning and sequencing of the cDNA for rat liver 3{alpha}-hydroxysteroid/dihydrodiol dehydrogenase. J Biol Chem 266:8820–8825[Abstract/Free Full Text]
  50. Watanabe K, Fujii Y, Nakayama K, Ohkubo H, Kuramitsu S, Kagamiyama H, Nakanishi S, Hayaishi O 1988 Structural similarity of bovine lung prostaglandin F synthase to lens {epsilon}-crystallin of the European common frog. Proc Natl Acad Sci USA 85:11–15[Abstract]
  51. Ohara H, Nakayama T, Deyashiki Y, Hara A, Miyabe Y, Tsukada F 1994 Reduction of prostaglandin D2 to 9{alpha},11ß-prostaglandin F2 by a human liver 3{alpha}-hydroxy-steroid dihydrodiol dehydrogenase isozyme. Biochim Biophys Acta 1215:59–65[Medline]
  52. Santoro C, Mermod N, Andrews PC, Tjian R 1988 A family of human CCAAT-box-binding proteins active in transcription and DNA-replication-cloning and expression of multiple cDNAs. Nature 334:218–224[CrossRef][Medline]
  53. Meisterernst M, Rogge L, Foeckler R, Karaghiosoff M, Winnacker E-L 1989 Structural and functional-organization of a porcine gene coding for nuclear factor-1. Biochemistry 28:8191–8200[Medline]
  54. Rupp R, Kruse U, Multhaup G, Gobel U, Beyreuther K, Sippel AE 1990 Chicken NF1/TGGCA proteins are encoded by at least three independent genes: NF1A, NF1B and NF1C with homologues in the human genome. Nucleic Acids Res 18:2607–2616[Abstract]
  55. Kruse U, Sippel AE 1994 The genes for transcription factor nuclear factor-1 give rise to corresponding splice variants between vertebrate species. J Mol Biol 238:860–865[CrossRef][Medline]
  56. Kruse U, Sippel AE 1994 Transcriptional factor nuclear factor-I proteins form stable homodimers and heterodimers. FEBS Lett 348:46–50[CrossRef][Medline]
  57. Goyal N, Knox J, Gronostajski RM 1990 Analysis of multiple forms of nuclear factor-I in human and murine cell-lines. Mol Cell Biol 10:1041–1048[Medline]
  58. Amemiya K, Traub R, Durham L, Major EO 1992 Adjacent nuclear factor-I and activator protein-binding sites in the enhancer of the neurotropic JC virus-a common characteristic of many brain specific genes. J Biol Chem 267:14204–14211[Abstract/Free Full Text]
  59. Cordingley MG, Riegel AT, Hager GL 1987 Steroid dependent interaction of transcription factors within the inducible promoter of the mouse mammary tumor virus in vivo. Cell 48:261–270[Medline]
  60. Jackson DA, Rowader KE, Stevens K, Jiang C, Milos P, Zaret KS 1993 Modulation of liver specific transcription by interactions between hepatocyte nuclear factor-III and nuclear factor-I binding DNA in close apposition. Mol Cell Biol 13:2401–2410[Abstract]
  61. Cereghini S, Raymondjean M, Carranca AG, Herbomel P, Yaniv M 1987 Factors involved in the control of tissue-specific expression of the albumin gene. Cell 50:627–638[Medline]
  62. BoisJoyeux B, Danan J-L 1994 Members of the CAAT/enhancer-binding protein, hepatocyte nuclear factor-1 and nuclear factor-1 families can differentially modulate the activation of the {alpha}-fetoprotein promoter and enhancer. Biochem J 301:49–55[Medline]
  63. Cardinaux J-R, Chapel S, Wahli W 1994 Complex organization of CTF/NF-1, C/EBP, and HNF3 binding sites within the promoter of the liver specific vitellogenin gene. J Biol Chem 269:32947–32956[Abstract/Free Full Text]
  64. Denison MS, Fisher JM, Whitlock Jr JP 1988 The DNA recognition site for the Dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. J Biol Chem 263:17221–17224[Abstract/Free Full Text]
  65. Denison MS, Fisher JM, Whitlock Jr JP 1988 Protein-DNA interactions at recognition sites for the Dioxin-Ah receptor complex. J Biol Chem 264:16478–16482[Abstract/Free Full Text]
  66. Neuhold LA, Shirayoshi Y, Ozato K, Jones JE, Nebert DW 1989 Regulation of mouse CYP1A1 gene expression by dioxin-requirement of 2-cis acting elements during induction. Mol Cell Biol 9:2378–2386[Medline]