(Received for publication, August 2, 1995; and in revised form, December 18, 1995)
From the
The rat aromatic L-amino acid decarboxylase (AADC) gene
contains alternative promoters which direct expression of neuronal and
nonneuronal mRNAs that differ only in their 5`-untranslated regions
(UTRs). We have analyzed the expression of the nonneuronal promoter of
the rat AADC gene in the kidney epithelial cell line LLC-PK and in cells which do not express the nonneuronal form of AADC by
transient transfection. These studies revealed that the first 1.1
kilobases of the nonneuronal promoter, including the
nonneuronal-specific 5`-UTR (Exon 1), contains sufficient information
to direct tissue-specific expression. Serial deletions of this promoter
localized the cis-active element to a region between -52 and
-28 base pairs upstream of the nonneuronal transcription start
site. An A/T-rich sequence, within this region which we have termed
KL-1, was found to bind a kidney and liver-specific factor by DNase
footprint analysis and was capable of directing tissue-specific
expression from a heterologous promoter. Moreover, when the KL-1
sequence was mutated in the context of the entire promoter sequence,
all transcriptional activity was abolished. DNA sequence comparison
revealed that the KL-1 fragment is highly homologous to the binding
site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies
utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the
KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not
express the nonneuronal form of AADC resulted in activation of
transfected AADC nonneuronal promoter constructs. These results
strongly suggest that the transcription factor which regulates the
tissue-specific expression of the nonneuronal form of AADC mRNA is
HNF-1.
Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) ()catalyzes the decarboxylation of L-dopa to
dopamine and 5-hydroxytryptophan to serotonin, as well as the
decarboxylation of the aromatic amino acids tyrosine, tryptophan, and
phenylalanine to the corresponding amines(1, 2) . AADC
is expressed in neuronal cells, where it participates in the synthesis
of neurotransmitters, and in nonneuronal cells, including liver,
kidney, lung, spleen, and endothelial cells, where its function is less
clearly understood(3, 4, 5, 6) .
Rat(7) , human(8) , and as we show in this report, porcine AADC mRNA exist in two different forms. While the coding region is identical in both forms, liver and kidney AADC mRNA contains a short 5`-untranslated region (UTR) which is entirely unrelated to the 5`-UTR found in AADC mRNA expressed in tissues of neuronal origin. We have previously shown that, within the rat AADC gene, dual promoters direct the expression of these tissue-specific forms of AADC mRNA resulting in the alternative use of two untranslated exons(9) . This has also been shown by others for the rat (10) and human (11, 12, 13) AADC gene. In liver and kidney, transcription initiates at the upstream, nonneuronal promoter, which results in an mRNA with exon 1 as its untranslated sequence. In tissues of neuronal origin, such as brain and adrenal medulla, transcription initiates at the downstream, neuronal promoter, incorporating exon 2 as the 5`-UTR.
To investigate the mechanisms which control differential expression from these two promoters, we have initiated analyses of transcriptional regulation. We have previously shown that a region of the neuronal AADC promoter, containing 2.4 kilobases (kb) upstream of the transcription start site and including the untranslated exon 2, was functional in all cell lines tested, including those which do not express AADC endogenously(14) . These studies identified several cis-active elements within the neuronal promoter which controlled the activity of this promoter, but which appeared to be binding sites for ubiquitously expressed transcription factors. The 5`-UTR itself was also found to be required for optimal expression from the neuronal promoter. Since these elements did not appear to contribute to the tissue-specific regulation of the neuronal promoter, additional regulatory mechanisms must restrict this activity to appropriate tissues. To search for additional sources of tissue-specific regulation, we have extended our transcriptional analyses to the nonneuronal promoter of the rat AADC gene.
The
nonneuronal form of the AADC message has been shown to be expressed in
cells of the liver and
kidney(7, 9, 10, 11, 15) .
Although little is known about the function of AADC in the liver, a
large body of evidence has accumulated which suggests that the function
of AADC in the kidney is to produce dopamine from circulating L-dopa(6, 16, 17, 18, 19, 20) .
Dopamine plays an important role in the regulation of renal electrolyte
and water balance(21) . AADC has been localized to the proximal
convoluted and proximal straight tubules in the kidney and appears to
be responsible for the synthesis of the majority of the dopamine
excreted by the
kidney(4, 17, 18, 19, 22) .
Because of the noradrenergic innervation of the kidney, it has been
difficult to demonstrate that dopamine is produced endogenously in the
kidney. However, the isolation of the renal epithelial cell line
LLC-PK, which is devoid of neural input and tyrosine
hydroxylase activity, and does not metabolize dopamine, yet expresses
high levels of AADC, has provided a model system which has been used to
study renal physiology and the regulation of dopamine synthesis and
release(16, 23) .
We have exploited the
LLC-PK cell line, which expresses the nonneuronal form of
AADC mRNA, to analyze transcriptional activation of the nonneuronal
promoter of the AADC gene. In contrast to our findings with the
neuronal promoter, we demonstrate that the first 1.1 kb of the
nonneuronal promoter, including the nonneuronal-specific 5`-UTR, is
capable of directing tissue-specific expression. Transfection
experiments have localized the tissue-specific enhancer element to an
A/T-rich sequence, which we have termed KL-1, located between -49
and -35 bp upstream of the transcription start site, which
appears to be essential for the tissue-specific expression of the AADC
nonneuronal promoter.
Hepatocyte nuclear factor-1 (HNF-1; also known as LFB-1) (24, 25, 26, 27) is a homeodomain transcription factor, which regulates the transcription of genes expressed predominantly in the liver, kidney, stomach, and intestine(28, 29, 30, 31, 32) . Sequence analysis revealed a near perfect homology between the tissue-specific KL-1 element and the HNF-1 consensus binding sequence(25, 27, 30, 31, 33, 34, 35) . In this study, we demonstrate that the KL-1 element is a binding site for HNF-1 and suggest that HNF-1 is the transcription factor primarily responsible for the tissue-specific expression of the nonneuronal promoter of the AADC gene.
All restriction enzymes, media, and cell culture reagents were purchased from Life Technologies, Inc. unless otherwise noted.
The KL-1 mut and U1 mut plasmids were constructed by using PCR primers to create a SalI site or PstI site, respectively, within the KL-1 or U1 region. For KL-1 mut, the Luc 1 primer was used with the KL-1 mut sense primer 5`-AAATGTCGACTTCACCAGAAAACAAGGTTTAAATGC-3`, and the pGL1 primer 5`-GTATCTTATGGTACTGTAACTGAGCTAACATAACCC-3`, located within the polylinker region of basic vector pGL2, was used with the KL-1 mut antisense primer: 5`-GTGAAGTCGACATTTCTGATGGTATAAAGGTCAAGG-3`, using the1.1-kb luciferase construct containing the 5`-UTR as a template (mutated bases are underlined). The KL-1 mut sense PCR product was digested with XbaI and SalI, the KL-1 mut antisense PCR product was digested with SalI and XhoI. The two fragments were ligated into the pGL2 basic vector previously digested with XhoI and XbaI. Similarly, for the U1 mut, the PCR primer U1 mut sense primer, 5`-GACTTCCTCTGCAGGTATACCATCAGAAATTAATG-3` was used with the Luc 1 primer, and the U1 mut antisense primer, 5`-GGTATACCTGCAGAGGAAGTCCAGAGAAGG-3` was used with the pGL 1 primer with the same template. The U1 mut sense PCR product was digested with XbaI and PstI, and the U1 mut antisense PCR product was digested with PstI and XhoI, and both fragments were cloned into the pGL2 basic vector plasmid previously digested with XhoI and XbaI. All plasmid constructs were verified by DNA sequencing as described above.
To construct the transfection
plasmids KL-1/dopamine -hydroxylase (DBH) and KL-1 opposite
orientation/DBH, sense and antisense oligonucleotides sense,
5`-CCAGATCTAAATTAATGTTTAACCAGGGATCCAA-3`, and antisense,
5`-TTGGATCCCTGGTTAAACATTAATTTAGATCTGG-3`, spanning the region from
-49 to -33 bp of the AADC nonneuronal promoter, were
synthesized which included a BglII site on the 5` end and a BamHI site on the 3` end. The sense and antisense
oligonucleotides were annealed and digested with BglII and BamHI and cloned into the BglII site of the -29
DBH construct. The -29 DBH construct contains sequences from
-29 to +10 bp of the rat DBH promoter, which includes the
TATA box and transcription start site (38) inserted into the
pGL2 basic vector as a BglII/HindIII fragment. The
KL-1 mut/DBH plasmids were constructed in the same way using the
oligonucleotides; sense: 5`-CCAGATCTAAATTCCGGTTGAACCAGGGATCCAA-3` and
antisense: 5`-TTGGATCCCTGGTTCAACCGGAATTTAGATCTGG-3` (mutated bases are
underlined). The U1/DBH and U1 mut/DBH plasmids were constructed in the
same way using the oligonucleotides U1 sense:
5`-CCAGATCTGACTTCCTTGACCTTTATACCGGATCCAA-3` and U1 antisense:
5`-TTGGATCCGGTATAAAGGTCAAGGAAGTCAGATCTGG-3`, containing sequences
between -75 and -55 bp, and mutated oligonucleotides U1 mut
sense: 5`-CCAGATCTGACTTCCTCTACATGTATACCGGATCCAA-3` and antisense:
5`-TTGGATCCGGTATACATGTAGAGGAAGTCAGATCTGG3-`. The resultant plasmids
were sequenced as described above to determine orientation of the
inserted fragment.
Plasmids
were linearized by endonuclease digestion with NotI
(-70LucRPA without 5`-UTR) or XhoI (-70LucRPA with
5`-UTR) and transcribed with T7 or T3 polymerase (respectively) in the
presence of [-
P]UTP using the Maxi-script
transcription kit (Ambion). Total RNA was isolated as described above.
RNase protection analysis was performed using the Ambion RPA II kit
using 2
10
cpm probe per reaction.
Figure 1:
LLC-PK cells express the
nonneuronal form of AADC mRNA. Two percent agarose gel showing
fragments resulting from RT-PCR performed on poly(A)
selected RNA isolated from LLC-PK
cells and porcine
adrenal glands. A, 255-bp porcine neuronal-specific AADC cDNA
fragment. B, 264-bp porcine nonneuronal-specific AADC cDNA
fragment. Results are not quantitative. Porcine adrenal and
LLC-PK
RNA is included as a negative
control.
Figure 2:
AADC nonneuronal promoter constructs
direct tissue-specific expression of a luciferase reporter gene. A, map of the 5` end of the AADC gene showing the location of
the first three exons and the 1.1-kb nonneuronal promoter region
analyzed in transfection experiments. B, top shows diagram of
the 1.1-kb nonneuronal promoter constructs which either excluded
(-5`-UTR) or included (+5`-UTR) the
nonneuronal-specific 5`-untranslated region (Exon 1) used for
transfection assays. Graph depicts results of transient transfection
experiments. Five µg of each plasmid construct, co-transfected with
2 µg of the reporter plasmid CMV--gal, were introduced by the
lipofectamine (Life Technologies, Inc.) transfection method into
LLC-PK
, PC12, CA77, and NRK cells. All transfections were
performed in duplicate. Values represent the ratio of luciferase
activity to
-galactosidase activity, expressed as fold induction
over background, which is the activity of the promoterless pGL2 basic
vector (pGL2-Basic). Results shown represent the average of
five independent experiments. Error is expressed as
S.E.
To delineate regions within this promoter which were responsible for
this tissue-specific expression, a series of deletion constructs were
made (Fig. 3A). For each deletion, two constructs were
made which either included or excluded the 5`-UTR. When these
constructs were introduced into LLC-PK cells, as shown in Fig. 3B, a similar pattern of expression was observed
when the 5`-UTR was included (+5`-UTR) or excluded
(-5`-UTR). However, expression levels were 3-10-fold
greater in the presence of the 5`-UTR. While the higher levels of
expression observed with the -510-bp construct suggest the
presence of both positive and negative elements within this 1.1-kb
region, the data show that removal of all sequences upstream of
-70 bp results in levels of expression similar to those observed
with the entire 1.1-kb promoter sequence. Deletion of sequences between
-70 and -28 bp abolished this expression, suggesting that
sequences between -70 and -28 bp contain a positive
cis-active element. Introduction of these deletion constructs into
CA77, PC12, or NRK cells resulted in no appreciable levels of
expression (data not shown).
Figure 3:
Serial
deletion constructs of the AADC nonneuronal promoter reveal a positive
cis-active element. A, diagram of the AADC nonneuronal
promoter constructs used for transfection assays. Serial deletion
plasmids were constructed which either excluded
(-5`-UTR) or included (+5`-UTR) the
nonneuronal-specific 5`-untranslated region (Exon 1). B,
results of transient transfection experiments. Five µg of each
plasmid construct, co-transfected with 2 µg of the reporter plasmid
CMV--gal, were introduced by the lipofectamine (Life Technologies,
Inc.) transfection method into LLC-PK
cells. All
transfections were performed in duplicate. Values represent the ratio
of luciferase activity to
-galactosidase activity, expressed as
fold induction over background, which is the activity of the
promoterless pGL2 basic vector (pGL2-Basic). Results shown
represent the average of five independent experiments. Error is
expressed as S.E. C, RNase protection analysis of total RNA
isolated from LLC-PK
cells transfected with the
-70-bp luciferase construct including (2) or excluding (1) the nonneuronal-specific 5`-UTR. Protected fragments of
101 and 68 nucleotides (nt), repectively, indicate accurate
transcription initiation.
Fig. 3C shows the use
of the correct transcription start site for transfected plasmids. RNase
protection analyses of total RNA isolated from LLC-PK cells
transfected with the -70-bp luciferase constructs with or without
the 5`-UTR, show the appropriate protected fragments of 101 and 68
nucleotides, respectively.
Figure 4:
Kidney/liver-specific binding site within
the AADC nonneuronal promoter. A, a fragment containing 510 bp
upstream and 71 bp downstream of the AADC nonneuronal transcription
start site was end-labeled with [-
P]ATP on
the antisense strand. Footprinting assays were performed with 50 µg
of nuclear extract from PC12, CA77, LLC-PK
, and NRK cells
and rat liver and kidney in the presence of 25 and 50 µg/ml DNase I (left to right). The location of the protein binding
sites and the 5`-UTR are indicated. B, comparison of a portion
of the rat and human AADC nonneuronal promoter sequence showing
homologous regions. Shaded areas indicate footprinted regions
of the rat AADC nonneuronal promoter. The A/T- rich KL-1 element and
the rat TATA box are underlined. The arrow indicates
the rat transcription initiation site; the caret indicates the
human transcription start site. U1, ubiquitous
footprint.
Fig. 4B shows a comparison of sequences around the transcription start sites of the rat and human AADC nonneuronal promoters. The greatest degree of homology is observed within the first 80 bp upstream of the transcription start site of the rat promoter. This region includes both the non-tissue-specific footprint (-75 and -55 bp), which we have termed U1, and the kidney/liver-specific binding site (-49 to -25 bp). Within the kidney/liver-specific binding site is an A/T-rich sequence, AATTAATGTTTAAC, which we have termed KL-1, which is 100% homologous to the human AADC sequence. Interestingly, little if any homology is observed within the 5`-UTR itself.
Figure 5:
The KL-1 element directs tissue-specific
expression. Results of transfection experiments in which the KL-1 and
U1 regions have been mutated. Five µg of each plasmid construct,
co-transfected with 2 µg of the reporter plasmid CMV--gal,
were introduced by the lipofectamine (Life Technologies, Inc.)
transfection method into LLC-PK
and CA77 cells. All
transfections were performed in duplicate. Values represent the ratio
of luciferase activity to
-galactosidase activity, expressed as
fold induction over background, which is the activity of the
promoterless pGL2 basic vector. Results shown represent the average of
five independent experiments. Error is expressed as S.E. A,
transfection analysis of -1.1-kb, -70-bp, and -52-bp
promoter constructs containing the 5`-UTR and mutations of the KL-1 and
U1 elements in the context of the native promoter. KL-1 mut,
-1.1-kb nonneuronal construct with KL-1 mutation, U1
mut, -1.1-kb nonneuronal construct with U1 mutation. Blackened area approximates location of mutated region. B, transfection analysis of plasmids containing the natural
and mutated versions of the KL-1 and U1 elements, in normal and
opposite (opp) orientation, placed upstream of the
heterologous DBH promoter. &cjs2112; and &cjs2113;, natural sequence;
&cjs2089; and &cjs2090;, mutated sequence;
, DBH promoter;
&cjs3613;, orientation.
To further test whether the KL-1 or the U1 sequence could
act as a tissue-specific enhancer element, DNA fragments containing
these elements were placed upstream of the first 29 bp of the DBH
promoter (-29 DBH), in either normal or opposite orientation (Fig. 5B). The -29 DBH construct has previously
been shown to direct no appreciable levels of luciferase expression in
all cells lines tested(38) . Plasmids containing mutated
versions of these fragments were constructed in the same way. These
KL-1/DBH and U1/DBH constructs were then assayed for their ability to
direct tissue-specific expression of the luciferase reporter gene.
Introduction of KL-1/DBH constructs into LLC-PK cells
resulted in luciferase expression levels which were 20-30-fold
greater than -29 DBH expression levels regardless of orientation.
However, mutation of the KL-1 element totally abolished expression from
this reporter gene. On the contrary, the normal U1/DBH constructs
directed barely detectable expression of the luciferase reporter gene
in LLC-PK
cells, regardless of orientation, which was
abolished by the mutation. Introduction of these constructs into CA77
cells resulted in no detectable levels of luciferase expression above
background. These results demonstrate that KL-1 acts as a
tissue-specific enhancer by directing tissue-specific expression from a
heterologous promoter regardless of orientation. Furthermore, since the
U1 fragment has little effect on the activity of a heterologous
promoter and a minimal effect on the activity of its own promoter in
the absence of functional KL-1, these data confirm that KL-1 is solely
responsible for controlling the cell-specific expression of the AADC
nonneuronal promoter.
Figure 6:
HNF-1 binds to the KL-1 element in the
AADC nonneuronal promoter. Mobility shift analyses of the KL-1 and U1
fragments. A, sequences of DNA elements used in mobility shift
assays: rat -fibrinogen promoter HNF-1 binding site
(
-fib)(30) , KL-1 element, mutated KL-1 element (KL-1M), HNF-1 binding consensus sequence, U1 fragment, and
mutated U1 fragment (U1M). Mutated bases are underlined. Results of gel shift studies with B. KL-1
and
-fibrinogen probes. Exposure time, 18 h. C, KL-1 and
-fibrinogen probes with HNF-1
antibody. Exposure time, 18 h. D, U1 probe. Exposure time, 3 days. In C, arrow indicates supershifted complex and
-HNF-1
is
HNF-1
antibody.
Mobility shift studies were also performed to analyze binding
to the U1 region. Fig. 6D shows that the U1 element
binds poorly to a factor which is present in nuclear extracts from
LLC-PK, CA77, and PC12 cells, which is consistent with our
earlier footprinting data. This binding is inhibited in the presence of
100-fold molar excess of the U1 element, but is not competed by equal
amounts of a mutated U1 sequence, demonstrating that this binding is
specific. The weak binding is consistent with the low levels of
activity observed in transfection experiments. The sequence of the
elements used in these experiments are shown in Fig. 6A. The mutated U1 sequence used in these
experiments is the same sequence assayed in earlier transfection
experiments.
Figure 7:
Cotransfection of HNF-1 cDNA into CA77
cells activates AADC nonneuronal promoter constructs. Results of
cotransfection experiments of HNF-1 cDNA with AADC nonneuronal
promoter constructs. Five µg of each plasmid construct,
co-transfected with 2 µg of the reporter plasmid CMV-
-gal,
were introduced by the lipofectamine (Life Technologies, Inc.)
transfection method into CA77 cells. All transfections were performed
in duplicate. Values represent the ratio of luciferase activity to
-galactosidase activity, expressed as fold induction over
background, which is the activity of the promoterless pGL2 basic
vector. Results shown represent the average of three independent
experiments. Error is expressed as S.E. A, analysis of
-1.1-kb, -70-bp, and -52-bp promoter constructs
containing the 5`-UTR and mutations of the KL-1 and U1 elements in the
context of the native promoter. KL-1 mut, -1.1-kb
nonneuronal construct with KL-1 mutation; U1 mut,
-1.1-kb nonneuronal construct with U1 mutation. Blackened
area approximates location of mutated region. B, analysis
of plasmids containing natural and mutated versions of the KL-1
element, upstream of the heterologous DBH promoter. &cjs2112;, natural
sequence; &cjs2089;, mutated sequence;
, DBH promoter; &cjs3613;,
orientation.
Transcription from alternative promoters of the AADC gene, followed by alternative splicing of untranslated exons, leads to the expression of AADC mRNAs with distinct 5`-UTRs in neuronal and nonneuronal cells. While the neuronal promoter directs expression to catecholamine and serotonin producing neurons in the central and peripheral nervous system and the adrenal medulla, the nonneuronal promoter directs high levels of expression in the liver and kidney, as well as lower levels in lung, spleen, and intestine. In this study we demonstrate that the tissue-specific expression of this promoter is directed by the hepatocyte transcription factor, HNF-1.
We have shown that 1.1 kb of the upstream, nonneuronal promoter of the rat AADC gene contains cis-active elements which can direct expression of a reporter gene only in a cell line which expresses the nonneuronal form of AADC mRNA. Transfection experiments and DNase footprint analysis identified an A/T-rich sequence, AATTAATGTTTAAC, which we have termed KL-1, as a binding site for a protein found only in liver and kidney cell nuclear extracts. Mutational analysis further demonstrated that this region is essential for nonneuronal promoter activity, and a DNA fragment containing this element is capable of directing tissue-specific expression of a reporter gene from the heterologous DBH promoter, in an orientation-independent manner.
The A/T-rich KL-1
element contains sequences similar to the TATA box sequence and shares
some similarity with the A/T-rich binding sites of homeodomain
proteins(43, 44, 45) . A comparison of this
region to binding sites for known transcription factors revealed a near
perfect sequence homology to the HNF-1 consensus binding
sequence(25, 27, 30, 31, 33, 34, 35) .
We present several lines of evidence to demonstrate that HNF-1 is the
factor which binds to this sequence and is responsible for the
tissue-specific activation of the nonneuronal AADC promoter. First,
mobility shift experiments demonstrated that a complex of similar size
binds to both the KL-1 sequence and the HNF-1 binding site located
within the -fibrinogen gene, and that both fragments can compete
for binding to a factor which is present only in nuclear extracts of
liver and kidney cells. Second, we show that this complex is recognized
by an antibody to HNF-1
. Finally, we show that cotransfection of
HNF-1
cDNA into CA77 cells, which do not express the nonneuronal
AADC promoter, leads to activation of the nonneuronal promoter, and
that cotransfected HNF-1
can activate transcription from a
construct containing the KL-1 element placed in front of the DBH
promoter.
The transcription factor HNF-1 was first identified as a
regulator of several liver-specific genes, including albumin, -
and
-fibrinogen,
-antitrypsin,
-fetoprotein,
pyruvate kinase, transthyretin, and aldolase B (see Blumenfeld et
al.(46) and references therein). Although originally
thought to be liver-specific, it was later found to be expressed in in
kidney, stomach, intestine, spleen, and
colon(28, 29, 30, 31, 32, 34, 46, 47) .
Thus the expression pattern of HNF-1 is highly consistent with that of
the nonneuronal form of AADC. HNF-1 contains a divergent homeodomain
and sequences homologous to the A box of the POU domain(27) .
Consistent with it being a homeodomain protein, there is also evidence
that HNF-1 plays an important role in the differentiation of the
hepatocyte phenotype(48) , as well as a role early in
embryogenesis(46, 47) .
A comparison of the rat and human nonneuronal promoter sequences encompassing this region reveals a high degree of homology within the region corresponding to -85 to -30 bp of the rat promoter. Moreover, the KL-1 sequence is 100% homologous to the corresponding human AADC sequence (see Fig. 5A). Because the human transcription start site has been proposed to be located 60 bp upsteam from the corresponding rat transcription start site(11) , the human KL-1 element appears to be located within the first exon. It is possible that this cis-active element is located 3` of the transcription start site in the human gene. However, the high percentage of homology exhibited between the human and rat promoters in this region, along with the transfection data presented here, suggests that it is more likely that the human transcription start site is located further downstream than previously reported.
A binding site for an apparently ubiquitously expressed
protein (U1) was also identified in the AADC nonneuronal promoter, in a
second region which shares a high degree of homology with the human
promoter, suggesting that this region may also play an important role
in the regulation of the nonneuronal promoter. Mutation of this region
within the context of the entire 1.1-kb promoter led to a moderate
decrease in activity, while placement of this element in front of a
heterologous promoter resulted in very minimal expression only in
LLC-PK cells. Since this element is capable of binding a
protein present in all cells, and is not necessary for high level
expression in LLC-PK
cells, it does not appear to play a
role in the tissue-specific expression of AADC.
The demonstration of tissue-specific regulation of the nonneuronal AADC promoter is in marked contrast to our previous analyses of the neuronal promoter of the AADC gene(14) . In similar transfection experiments, 2.4 kb of the neuronal AADC promoter was found to direct expression of a reporter gene in both AADC expressing and nonexpressing cells. Taken together, these data suggest that, while tissue-specific expression of the nonneuronal promoter is regulated by binding of HNF-1 to the KL-1 element, the neuronal promoter either requires elements not included in the first 2.4 kb of 5`-flanking sequence, or some other aspect of its context within the AADC gene, to direct appropriate cell-specific expression. These observations are further supported by a recent report by Sumi-Ichinose et al.(49) , in which transgenic studies of the human AADC gene demonstrated a requirement for both promoters to obtain appropriate expression patterns. Specifically, transgenes containing the neuronal promoter alone were expressed in all tissues examined. Conversely, transgenes containing the nonneuronal promoter or both promoters exhibited correct expression patterns, although higher expression levels were observed with transgenes containing both promoters.
There are numerous examples of multiple promoter systems(50) . For the systems whose transcriptional regulation has been studied in great detail, several mechanisms have been identified by which expression from a proximal promoter can be inhibited by a distal one(51, 52, 53) . Although this type of promoter regulation has been demonstrated for promoters separated by short distances, similar mechanisms have been proposed for alternative promoters located as far as 4 kb apart(54) . Our present data cannot rule out the possibility that inhibitors or silencers of neuronal promoter expression lie elsewhere in the gene. However, the demonstration of tissue-specific regulation of the upstream nonneuronal AADC promoter by HNF-1 suggests that transcriptional interference, or a similar mechanism, may control tissue-specific expression from the downstream neuronal AADC promoter.
We have previously shown that inclusion of the neuronal 5`-UTR (Exon 2) in transfection constructs results in increased levels of expression from the neuronal AADC promoter(14) . Here we show a similar effect of the nonneuronal 5`-UTR (Exon 1). Although in the absence of the 5`-UTR expression from the nonneuronal promoter is 5-50-fold over background, the presence of the 5`-UTR increases this expression 3-10-fold. We do not believe that this effect is due to the presence of additional sequences around the transcription start site, since the constructs which did not include the 5`-UTR did contain 25 bp of sequence 3` of the transcription start site. As was the case for the neuronal 5`-UTR, our experiments cannot distinguish between a transcriptional or a postranscriptional function for this untranslated exon. This region could represent a binding site for a cis-active transcription factor, although we did not detect binding to this region in our DNase I footprint assays, nor did we find any homology to the human nonneuronal 5`-UTR. Alternatively, this region could function at the level of the mRNA to stabilize the message or to increase translational efficiency.
The different expression patterns
exhibited by the two AADC promoters raises questions regarding the
function of the alternative promoters of the AADC gene. The presence of
multiple promoters in genes generally allows greater flexibility in the
regulation of expression, providing a mechanism for differential
tissue-specific expression, developmental and hormonal regulation, or
differential regulation of expression levels. Since the two forms of
AADC mRNA differ only in a short 5`-UTR, we can presume that
differential regulation of AADC may occur at the level of the mRNA or
at the level of transcription. While our data does not provide any
evidence for differences in the function of the two 5`-UTRs at the mRNA
level, we have identified major differences in the control of
transcriptional activation of the two promoters. The presence of two
differentially regulated promoters may therefore provide a mechanism
for differential regulation of AADC mRNA in brain versus liver
and kidney where the products of AADC activity may perform different
functions. Although it has long been assumed that AADC was not a
regulated enzyme, recent reports have shown that AADC is modulated at
both the enzyme and mRNA levels. AADC mRNA levels are regulated in
response to various agents including reserpine(55) ,
dexamethasone(56) , dopamine receptor antagonists(57) ,
interleukin 1 and prostaglandin E
(58) , and
AADC (59) and monoamine oxidase B inhibitors(60) .
These experiments have measured AADC mRNA levels in various brain
regions, adrenal medulla, or in PC12 cells. It is not known whether
these agents can cause similar increases in AADC mRNA expression in
liver or kidney.
Until recently, little has been known about the
function of AADC in nonneuronal tissues such as liver and kidney. The
recent recognition of the importance of dopamine as a renal hormone
suggests that AADC may play a role in the regulation of kidney
function. In the kidney, dopamine appears to regulate renal salt and
water balance by modulating both
Na,K
-ATPase activity and the
Na
/H
exchanger via DA
and
DA
dopamine receptors (reviewed in Lee(18) ).
Specifically, it has been shown that AADC activity in the kidney can be
modulated in response to sodium intake(61, 62) . The
association of altered renal dopamine levels with several diseases
including hypertension, diabetes and congestive heart failure (see Lee (18) and references therein) also suggests that modulation of
nonneuronal AADC enzyme or mRNA levels may play a role in maintaining
proper kidney function. In fact, increases in AADC activity in the
kidney have been observed in experimental hypertension(63) .
Further analysis of AADC expression in the kidney is required to
determine whether any of these effects on enzyme activity are due to
alterations in levels of AADC mRNA. The ability to study this
regulation in the renal epithelial cell line LLC-PK
, which
has been shown to be a valuable model for renal cellular physiology,
will contribute to the understanding of renal dopamine production and
function.
Identification of HNF-1 as a transcription factor essential for directing tissue-specific expression of the nonneuronal AADC promoter is an important initial step toward the understanding of the function and regulation of the AADC gene in nonneuronal cells. Because HNF-1 is a major regulator of the expression of liver-specific enzymes, its role in the regulation of the AADC nonneuronal promoter further suggests that AADC also performs important functions in the liver.