From the Department of Biochemistry and Integrative
Medical Biology, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582 and
Minophagen Pharmaceutical Co.,
2-2-3 Komatsubara, Zama, Kanagawa 228-0002, Japan
Received for publication, February 11, 2003
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ABSTRACT |
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A cDNA encoding a
secretory protein hitherto unknown was cloned from mouse adrenocortical
cells by subtractive hybridization between the cells without and with
expressing steroid 11 The adrenal cortex of mammals consists of three major zones that
contain both functionally and morphologically distinct cells; they are
the zona glomerulosa (zG),1
the zona fasciculata (zF), and the zona reticularis (1). The cells in
zG, the outermost zone of the cortex, secrete aldosterone, the
strongest mineralocorticoid; and those in zF, the middle zone, produce
glucocorticoids such as corticosterone in most rodents and cortisol in
other mammals including human. Finally, the cells in the zona
reticularis, the innermost portion of the cortex, also secrete
glucocorticoids in many mammals including rats and mice and produce
adrenal androgens in human and some other mammals. In addition to these
three, a zone composed of 3-4 layers of small round cells has been
recognized between zG and zF in various animals (2-9). By using rats
we showed that the cells of the zone were devoid of the two enzymes
(10) determining the distinct steroidogenic functions of zG and zFR,
i.e. aldosterone synthase (Cyp11b-2) and steroid
11 Development and maintenance of the adrenocortical zones require many
cellular processes including regulation of the steroidogenic gene
expression and regulation of cell renewal and arrangements. Steroidogenic factor 1 (19) (SF-1, also referred to as Ad4BP (20)) is a
transcription factor essential for embryonic development of
steroidogenic organs including adrenal cortex and gonads (21). SF-1
also plays a pivotal role in the earlier steps of the adrenocortical steroidogenesis over the entire adrenal cortex by controlling expression of cholesterol side-chain cleavage enzyme (Cyp11a) and
steroid 21-hydroxylase (22). Based on these features, SF-1 is unlikely
to be a key regulator for the zonal differentiation of the
steroidogenesis. Regarding factors regulating expression of the
steroidogenic genes for the last steps of the syntheses, we previously
suggested that AP-1 transcription factors were necessary for the
spatially restricted expression of Cyp11b-1 in zFR (23, 24). Other
regulatory factors playing a crucial role in the zone-specific
steroidogenesis of zFR have been unknown. Furthermore, no regulatory
factor for the functional differentiation of the rest of the cortex, zG
and zU, has been identified so far. Therefore, molecular mechanisms
underlying the zonal differentiation of the adrenocortical
steroidogenesis remain to be solved.
The goal of this study was to explore unidentified factors that control
the functional differentiation of adrenocortical cells. To this end, we
used the mouse adrenocortical cell lines that we established recently
(25). They are derived from the adrenal glands of transgenic mice (26,
27) carrying a temperature-sensitive large T-antigen gene of simian
virus 40, being at different degrees of differentiation from one
another. In the present study, a subtractive cDNA cloning employing
the adrenocortical cell lines, named AcA101 and AcA201, as well as a
conventional cell line Y-1, was carried out. Among the cell lines,
AcA101 is the most undifferentiated one which expresses neither
Cyp11b-1 nor Cyp11b-2, whereas Y-1 is the most differentiated one that
expresses Cyp11b-1 with a responsiveness to ACTH stimuli (28). A
cDNA cloned from AcA101 cells by a subtractive hybridization
encodes a protein termed adrenocortical zonation factor-1 (AZ-1), the
subject of this paper. AZ-1 is a unique secretory protein with a
preprocathepsin B-related structure carrying epidermal growth hormone
(EGF) motifs. This study demonstrates that expression of AZ-1 in
adrenocortical cells is inversely correlated with expression of
Cyp11b-1 in vitro and in vivo.
Cell Culture--
Mouse adrenocortical AcA101 and AcA201 cells
were cultured at 33 °C, a permissive temperature for the mutant SV40
T-antigen protein, under the conditions described previously (25).
Mouse adrenocortical Y-1 cells (28) were cultured at 37 °C with
Ham's F-10 medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 10% heat-inactivated horse serum
(Invitrogen), 200 units/ml penicillin, and 200 µg/ml streptomycin.
The cells were incubated under a humidified atmosphere containing 5%
CO2.
Subtractive cDNA Cloning--
Total RNA was extracted from
AcA101, AcA201, and Y-1 cells with a modified single-step isolation
method employing Trizol reagent (Invitrogen).
Poly(A)+ RNA was prepared with an
oligo(dT)-cellulose column (Amersham Biosciences) and treated with
RNase-free DNase (Promega, Madison, WI).
Poly(A)+ RNA (2 µg) from AcA101 cells was
converted into cDNA with a NotI-oligo(dT) primer. A
cDNA library of AcA101 cells was prepared using the Northern Blotting--
Total RNA was prepared from adrenal
glands, whole brains, hearts, kidneys, livers, skeletal muscles,
spleens, and testes of C57BL/6 mice using Trizol reagent as described
above. The RNA preparations (10 µg) were subjected to Northern blot
analysis as described previously (24). Before transfer to positively charged nylon membranes (Roche Diagnostics), ribosomal RNAs were visualized by ethidium bromide to verify that the amounts of RNA loaded
were comparable with each other (<15%) and that degradation of
the RNA preparations was undetectable under our experimental conditions. A cDNA fragment (position 1490-1810 of Fig.
1A) was labeled with [ Southern Blotting--
Genomic DNA was prepared from the liver
of CL57BL/6 mice and was digested with EcoRI,
BamHI, and HindIII. The digests (10 µg) were
subjected to electrophoresis through 0.8% agarose and blotted on a
positive charged nylon filter (Roche Diagnostics). A cDNA fragment
of AZ-1 (position 1490-1810) was isolated and labeled with DIG-dUTP
(Roche Diagnostics). Hybridization, stringent washing, and detection
with color development were carried out according to the
manufacturer's instructions (Roche Diagnostics) and as described
previously (29, 30).
In Vitro Transcription-Translation--
To construct a plasmid
for expression of recombinant proteins, oligonucleotide primers for PCR
were prepared. A forward primer including the putative translational
initiation codon was designed to contain an artificial BstXI
site at position 48 of the sequence shown in Fig. 1A:
5'-CGCCAGTGTGCTGGAGGCACCATGTGGGGATGT-3'. A reverse PCR primer
corresponding to the C terminus of the protein was designed to contain
the sequence encoding FLAG epitope followed by an XbaI site:
5'-GCTCTAGACTACTTGTCATCGTCGTCCTTGTAGTCGTGGTGCCCCATGTCCTCC-3'. Another reverse PCR primer 5'-GCTCTAGATCAGTGGTGCCCCATGTCCTCC-3' lacking the FLAG tag sequence was also designed. cDNA fragments (either the presence or absence of the FLAG sequence) that were amplified by PCR were inserted into pRc/CMV (Invitrogen). To avoid incorrect nucleotides incorporated in PCR, the DraIII
fragment (position 69-1280 of Fig. 1A) carrying the
PCR-amplified region was replaced by the authentic fragment prepared
from the original cDNA. The resulting pR/C11.13FD1 and pR/C11.13D1
plasmids were used for templates in in vitro
transcription-translation reaction of T7 TNT-coupled
Reticulocyte Lysate System (Promega) in the presence or absence of
[35S]methionine (1000 Ci/mmol, Amersham Biosciences).
Reaction mixtures were subjected to SDS-PAGE. Detection was carried out
by autoradiography or immunoblotting using anti-FLAG M2 monoclonal
antibody (Sigma) as described below.
Antibody Preparation--
Peptides P1
(NH2-Cys-Ser-Gln-Gly-Arg-Pro-Glu-Gln-Tyr-Arg-Arg-His-Gly-Thr-COOH)
and P2
(NH2-Cys-Gly-Arg-Val-Gly-Met-Glu-Asp-Met-Gly-His-His-COOH), corresponding to amino acid residues 386-398 and 456-466,
respectively, were synthesized and each conjugated at the additional
Cys residue to keyhole limpet hemocyanin (Pierce) through a thioether
bond using maleimide-activated keyhole limpet hemocyanin (Pierce) as described in the instruction manual. The peptide-keyhole limpet hemocyanin conjugates were mixed (1:1 (w/w)) and emulsified with Freund's complete adjuvant and injected into IsaBrown hens 6 times intradermally at 2-week intervals. Blood was collected on the 35th day
or thereafter. Antibodies were purified from antisera (10 ml) with a
peptide-conjugated agarose gel column. The agarose gel was prepared by
coupling a 1:1 (w/w) mixture of the two peptides using a SulfoLink
coupling gel (Pierce) according to the instructions. Based on titration
by an immunoblot analysis using a recombinant AZ-1 protein, the
recovery of the purified antibody after the chromatography was
~70%.
In Situ Hybridization--
The EcoRI-BamHI
fragment (position 1490-1810 of Fig. 1A) of the isolated
cDNA clone was subcloned into pZL1 (Invitrogen). Mouse cDNAs
encoding Cyp11b-1 (position 761-950) or Cyp11b-2 (761-953) (31) were
obtained by PCR with primer pairs as described previously (25) from the
total RNA of Y-1 cells after reverse transcription, and they were
cloned into pGEM4-Z (Promega). DIG-labeled antisense and sense RNA
probes were synthesized with T7 and SP6 RNA polymerases, respectively,
using DIG RNA labeling kit (Roche Diagnostics). Mouse adrenals were
excised after transcardial perfusion with phosphate-buffered saline( Immunohistochemistry--
Paraffin sections of
paraformaldehyde-fixed mouse adrenal glands were treated with
affinity-purified chicken anti-AZ-1 antibody (10 µg/ml) or control
chicken IgY (10 µg/ml, Sigma) overnight at 4 °C. The
concentrations of the antibodies were adjusted after protein
determination with Coomassie Brilliant Blue G-250. The secondary
antibody used was rabbit anti-chicken IgY antibody conjugated with
horseradish peroxidase (1:300; Promega). The peroxidase activity was
visualized with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen
peroxide as described previously (32).
Transfection--
pR/C11.13FD1, which was the same DNA construct
as used for in vitro synthesis of FLAG-tagged AZ-1 (AZ-1F),
or a control vector pRc/CMV was linearized by digestion with
BglII. Y-1 cells (3 × 106) were
transfected with the linearized DNA (10 µg) by calcium phosphate
precipitation method using the Profection kit (Promega). The cells were
selected for resistance to antibiotics G418 (400 µg/ml), and colonies
were isolated and expanded for characterization as described below.
Immunoprecipitation and Immunoblotting--
A mock transfectant
(clone 9) and an AZ-1F vector-transfectant (clone 14; see Fig.
7), which showed the highest level of AZ-1F mRNA, were used for
detection of the AZ-1 polypeptide. Cell extracts from them
(3.6-cm2 well) were prepared with an SDS sample buffer (120 µl) consisting of 62 mM Tris-HCl, pH 6.8, 2% (w/v) SDS,
2% (v/v) 2-mercaptoethanol, and 0.01% bromphenol blue. The extracts
were incubated at 100 °C for 5 min before electrophoresis. For
immunoprecipitation, purified chicken anti-AZ-1 antibody (150 µl) was
bound to anti-chicken IgY-agarose (150 µl of 50% suspension;
Promega) by incubation for 1 h at 4 °C with gentle shaking.
The beads were washed with 1 ml of Tris-buffered saline (TBS) 5 times. The supernatants of the used culture media (1 ml) were prepared
by centrifugation to remove cells and cell debris and incubated with 30 µl of the gel suspension for 18 h at 4 °C with gentle
shaking. The agarose gel beads were washed with TBS containing 0.1%
Tween 20 (TBST) 4 times followed by a wash with TBS. They were
resuspended in an SDS sample buffer and treated at 100 °C for 5 min.
The immunoprecipitates and the cell extracts were subjected to 10%
PAGE in the presence of SDS, and polypeptides were electrophoretically
blotted onto Immobilon-P membranes (Millipore, Bedford, MA) according
to standard procedures. The membranes were treated with anti-FLAG
monoclonal M2 antibody (0.88 µg/ml) overnight at 4 °C. They were
washed with TBST and then incubated with a secondary
antibody solution of rabbit anti-mouse IgG conjugated with horseradish
peroxidase (1:25,000 dilution with TBST) for 3 h at room
temperature. When the affinity-purified chicken anti-AZ-1 antibody
(1:200 dilution) was used for immunoblotting, membranes were treated
with rabbit anti-chicken IgY conjugated with horseradish peroxidase
(1:1000 dilution). Bound secondary antibodies were detected by enhanced
chemiluminescence (Amersham Biosciences).
Post-translational Modification--
An AZ-1F-vector
transfectant (clone 14) was cultured in the presence of 10 µg/ml
tunicamycin (Sigma) or vehicle (0.2% (v/v) methanol in the culture
medium) for 18 h, and cell extracts were prepared as described
above. For treatment with peptide N-glycosidase F, cell
extracts of the transfectant containing 2% SDS were diluted by 10-fold
with 10 mM Tris-HCl, pH 7.5, and incubated with 50 units/ml
of N-glycosidase F (Roche Diagnostics) at 37 °C for
18 h. After the treatment, the SDS sample buffer was added to the reaction mixtures. These samples were analyzed by immunoblotting as
described above.
RT-PCR--
RT-PCR analysis was performed by the methods as
described in the previous paper (25) using primer pairs as follows: (i) AZ-1F: 64f, 5'-ACCATGTGGGGATGTTGGCTGG-3' (position 64-85, see Fig.
1A) and FL1485r, 5'-GTCATCGTCGTCCTTGTAGTCG-3' that
corresponds to the FLAG peptide encoding sequence); (ii) AZ-1: 420f,
5'-GGACAACTGCAATCGATGCACC-3' (420-441); 1003r,
5'-GGCTGTGCATCATACAACGAGG-3' (1003-982); this primer pair was used for
detection of mRNA of both endogenous Az-1 gene and
AZ-1F; (iii) Cyp11b-1 (25); (iv) Cyp11a (25); and (v)
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (25). Amplification
conditions for PCR were 45 s at 94 °C, 45 s at 56 °C,
and 2 min at 72 °C for appropriate cycle numbers followed by 7 min
at 72 °C. Cycle numbers were 20 for AZ-1 (420f and 1003r), Cyp11a,
and GAPDH and 25 for AZ-1F (64f and FL1485r), and Cyp11b-1. Experiments
for comparison of relative amounts of mRNAs among the transfectants
were performed within the exponential phase of the amplification
reactions to obtain the linear response concerning the initial mRNA
amounts. PCR products were analyzed by agarose gel electrophoresis
followed by visualization with ethidium bromide. Intensities of the
visualized products were determined by densitometric analysis and were
normalized with that of GAPDH cDNA.
Cloning and Characterization of AZ-1 cDNA
Among the cDNA clones isolated, one was composed of 1926 nucleotides except for a poly(A) tail as shown in Fig.
1A. The cDNA attracted our
attention because the amino acid sequence of its encoded protein had a
unique structure as described below. The protein was termed as AZ-1
since its expression level was inversely related to the degree of the
functional differentiation of adrenocortical cells as described below.
The cDNA contained an open reading frame encoding a polypeptide
chain consisting of 466 amino acids, which was from a translational
initiation codon at position 67 to a termination codon at position
1465. Several nucleotides preceding the methionine codon were
consistent with the consensus sequence for translational initiation
(33). A typical polyadenylation signal was found at position 1907. The
N-terminal 17-amino acid sequence was predictable as a signal peptide
for the secretory pathway (34). No hydrophobic region large enough to
span a membrane was recognized. Possible N-glycosylation
sites were present at Asn-77 and Asn-160. These features in the
predicted amino acid sequence suggest that AZ-1 is a secretory
protein.
When searched in the GenBankTM/EMBL/DDBJ data base, the
predicted amino acid sequence of the open reading frame had an identity of 89% with human tubulointerstitial nephritis antigen-related protein
(TIN-ag-RP) (35) (Fig. 1B), indicating that the identified cDNA encodes a mouse orthologue of the human protein. As Wex
et al. (35) discussed the features of amino acid sequence of
TIN-ag-RP, the N-terminal one-third of AZ-1 polypeptide was rich in Cys
residues and contained EGF-like repeats. The C-terminal two-thirds of
AZ-1 polypeptide contained procathepsin B-related sequence with a Ser residue at position 228, which replaced a conserved Cys residue in the
active site of cysteine proteinases (Fig. 1A), suggesting that AZ-1 does not have a proteinase activity. These structural features were also found in tubulointerstitial nephritis antigen (TIN-ag) (36-38) in mammals and also in non-mammalian homologues including Caenorhabditis elegans F26E4.3 protein (39).
To verify the size of the polypeptide encoded by the isolated cDNA,
we carried out in vitro synthesis of AZ-1 using its cDNA as a template for a coupled transcription and translation reaction in
the presence of [35S]Met (Fig.
2A, left). As seen, the
reaction with the cDNA insert (lane 2) gave a major
product with an electrophoretic mobility of 46 kDa, which matched with
the predicted molecular mass of the encoded protein. We also
constructed a DNA encoding the AZ-1 polypeptide tagged with a FLAG
peptide at the C terminus. The resulting FLAG-tagged polypeptide,
AZ-1F, showed a band with a mobility of 47 kDa (lane 3). The
increase of 1 kDa in molecular mass was consistent with an addition of
the FLAG peptide of 8 amino acid residues to the native polypeptide.
Additional bands with higher mobilities might be polypeptides where
internal Met residues were used for translational initiation. The
reaction with the control vector gave no signal under the experimental conditions (lane 1).
Transcription-translation reaction products in the absence
of radioactive amino acids were examined by immunoblotting employing an
anti-FLAG antibody (Fig. 2A, right). Because the control
vector gave many bands under the current experimental conditions
(lane 4), the experiment with an AZ-1-encoding template
without FLAG tag resulted in no meaningful signal (lane 5).
When the template carrying the FLAG-encoding sequence (lane
6) was used, the anti-FLAG antibody recognized a 47-kDa band in
addition to the other bands common to lanes 4 and
5. Collectively, the results from in vitro synthesis indicated that the isolated cDNA encoded a 46-kDa polypeptide.
Southern Analysis and Genomic Organization of Az-1
Gene--
Southern blot analysis was performed to ensure the presence
of the Az-1 gene in the mouse genome (Fig. 2B).
When digested with EcoRI (lane 1),
BamHI (lane 2), or HindIII
(lane 3), each of the digests of mouse genomic
DNA gave a single hybridization signal, indicative of a single gene.
Thus, the results demonstrated the presence of a genomic DNA sequence
that corresponded to the isolated cDNA sequence.
To compare the AZ-1 cDNA sequence with mouse genome sequence,
Ensembl Genome Browser (www.ensembl.org/) was used. The cDNA sequence was located on chromosome 4 and was encoded in 12 exons that
spanned 9175 bases from positions 127,681,610 to 127,672,436 of the
chromosome in a reverse direction (Fig.
3, A and B). The genomic sequences were identical to the cDNA sequence. Because the
transcription start site was not determined, the 5'-end of the cDNA
sequence was putatively assigned to the 5'-end of exon 1. The sizes of
the hybridization signals obtained with the Southern blot analysis
(Fig. 2B) agreed well with the restriction sites in the
genomic sequence of the data base.
Distribution of AZ-1 mRNA--
Expression levels of the AZ-1
mRNA in the cells used for the cloning of the AZ-1 cDNA were
examined by Northern blot analysis (Fig.
4A). The greatest intensity of
the mRNA signal (~2 kb) was observed in RNA from AcA101 cells
(lane 1), which were used as the source of the cDNA
library, among the three cell lines. A signal with a lower intensity
was detected in RNA from AcA201 cells (lane 2), whereas no
signal was obtained in RNA from Y-1 cells (lane 3) under the
experimental conditions. Several other cell lines established
simultaneously with AcA101 and AcA201 were also analyzed for AZ-1
mRNA. Levels of the mRNA in such cell lines as AcE60 (25) were
consistent with their phenotypes; cell lines displaying functionally
undifferentiated phenotypes had higher levels of AZ-1 mRNA
expression than those displaying differentiated ones (data not
shown).
RNA preparations from various organs of mouse were then analyzed by
Northern blot analysis using AZ-1 cDNA as the probe (Fig. 4B). The greatest hybridization signal was obtained with RNA
from adrenal glands (lane 5) among the organs examined in
the experiments. Although AZ-1 mRNA was undetectable in the whole
brain (lane 4), it was detectable in other organs as
follows: heart (lane 6), skeletal muscle (lane
7), kidney (lane 8), liver (lane 9), and spleen (lane 10). Despite the ability to execute
steroidogenesis, testis gave no hybridization signal (lane
11).
Subsequently, in situ hybridization analysis was performed
to locate expression of AZ-1 mRNA in the adrenal glands. As shown in Fig. 5A, hybridization
signals of an antisense probe for AZ-1 mRNA were detected in the
outer cortical regions composed of zG and zU. The medulla also
exhibited weak hybridization signals. Nuclei of the zF cells appeared
to give weak nonspecific signals. The faint signals seen in the FR were
nonspecific background comparable with those detected with the sense
probe (Fig. 5B). To compare the location of AZ-1 mRNA
with those of Cyp11b-2 mRNA, in situ hybridization
employing an antisense probe for Cyp11b-2 was performed. Cyp11b-2
mRNA was observed sporadically in zG (Fig. 5C). It
should thus be noted that AZ-1 mRNA signals were detectable
irrespective of the hybridization signals for Cyp11b-2 mRNA in zG.
When an antisense probe for Cyp11b-1 was used, hybridization signals
were detected in zFR (Fig. 5D). As shown, the hybridization
signals for AZ-1 mRNA (Fig. 5A) and those for Cyp11b-1
mRNA (Fig. 5D) did not overlap each other. Therefore,
AZ-1 mRNA signals were present in zG and zU but not in zFR, which
express Cyp11b-1 mRNA.
Fig. 5E illustrates immunohistochemical localization of the
AZ-1 protein in mouse adrenal glands by using an anti-AZ-1 antibody raised against peptide antigens (see "Experimental Procedures"). The immunoreactivities were detected in the outer cortical regions corresponding to zG and zU. The weak signals in zFR seemed to be
nonspecific because the control IgY gave a comparable level of
background (Fig. 5F). The medulla was also immunoreactive. Thus, distribution of the immunoreactivities to anti-AZ-1 antibody and
that of the hybridization signals of the AZ-1 antisense probe were
indistinguishable from each other. Both AZ-1 mRNA and protein were
thus expressed in the outer regions of the adrenal cortex, i.e. the outside of zFR.
Detection of FLAG-tagged AZ-1 Protein with Stably Transfected Y-1
Cells--
We then produced stable transfectants of Y-1 cells with an
expression vector encoding AZ-1F protein. Among the clones obtained, a
clone exhibiting the highest expression level of AZ-1F mRNA was
characterized in detail. When the cell extracts prepared from the clone
were analyzed by immunoblotting, two bands with electrophoretic mobilities of 51 and 54 kDa were detected by the use of an anti-AZ-1 antibody (Fig. 6A) and an
anti-FLAG antibody (Fig. 6B). Cell extracts from a clone
obtained with a control vector gave no significant band in either case.
We also prepared immunoprecipitates from the cultured medium of the
transfected cells by using anti-AZ-1 antibody. As shown in Fig.
6C, immunoblotting of the precipitates using anti-FLAG
antibody showed 51- and 54-kDa polypeptides. A faint 52-kDa band seen
with the medium of the control clone (Fig. 6C, lane
5) was likely to result from a cross-reactivity of the secondary
antibody to the heavy chain of bovine or horse IgG in the culture
medium. These results were consistent with the presence of the signal
peptide sequence at the N terminus of the deduced amino acid sequence
of AZ-1.
The difference in the molecular mass of the AZ-1F protein detected with
transfected cells (54 and 51 kDa) from that of the in vitro
synthesized polypeptide (47 kDa) may be attributable to
post-translational modification. To examine N-glycosylation of the polypeptide produced in the transfected cells, the cells were
incubated with tunicamycin, and the cell extracts were analyzed by
immunoblotting with anti-AZ-1 antibody. As shown in Fig. 6D, a 48-kDa band was detected after the tunicamycin treatment in addition
to the 54- and 51-kDa bands, and N-glycanase treatment of
the cell extracts from the transfected cells also produced a 48-kDa
band (Fig. 6E). These results suggested that AZ-1 protein was post-translationally N-glycosylated in the cells.
Inverse Correlation between AZ-1 and Cyp11b-1 Expression in Clonal
Lines Derived from Y-1 Cells--
The expression levels of AZ-1
mRNA in the cell lines used for the subtractive cloning were
apparently in an inverse correlation to the levels of Cyp11b-1 mRNA
(25). The effects of expression of the AZ-1 protein on the phenotypes
of Y-1 cells then were examined by analyzing mRNA levels of the
steroidogenic genes in the transfectants (Fig.
7). We analyzed 10 clones of mock
transfectants obtained with a control DNA and 11 clones obtained with
an AZ-1F expression vector. Fig. 7A shows that mRNA
encoding AZ-1F was detected in 4 clones (lanes 12,
13, 15, and 18) among the 11 clones isolated after transfection with the AZ-1F expression vector, whereas no AZ-1F
mRNA was detectable among the 10 clones transfected with the
control vector (lanes 1-10). A PCR primer pair that was
able to detect mRNA of both the endogenous Az-1 gene and
AZ-1F was used. Unexpectedly, 7 of 10 control clones were found to
express significant levels of the endogenous Az-1 gene
mRNA (Fig. 7B, left). Among the 11 clones obtained with
transfection of the AZ-1F vector, 6 clones (lanes 12,
13, 15, 18, 20, and
21) expressed mRNA of either the endogenous or exogenous
Az-1 genes, indicating that the endogenous Az-1
gene was expressed in at least two clones (lanes 20 and
21). The reason for the induction of the endogenous Az-1 gene through the selection procedure for the
transfectants was unknown. Fig. 7C shows that Cyp11b-1
mRNA was undetectable only in the clones expressing either AZ-1
from the endogenous gene or AZ-1F. On the other hand, Cyp11a
mRNA was detectable in all of the 21 clones with various levels
(Fig. 7D). The experiments with a GAPDH primer pair
monitored efficiencies of reverse transcription and amplification from
the RNA preparations (Fig. 7E). Fig. 7F depicts
the inverse correlation of the mRNA levels between AZ-1 and
Cyp11b-1 (closed circles); the clones expressing a very low or undetectable level of the endogenous Az-1 gene mRNA
expressed Cyp11b-1 mRNA, whereas those expressing higher levels of
the AZ-1 mRNA did not express Cyp11b-1 mRNA. The same
correlation was observed for the 4 lines expressing AZ-1F (closed
triangles). On the other hand, Cyp11a mRNA was expressed
irrespective of AZ-1 expression (open circles and open
triangles). These results indicated that Cyp11b-1 was expressed in
the clonal lines maintaining little AZ-1 expression and became
undetectable in those expressing AZ-1.
AZ-1 cloned in the present study turned out to be a putative
factor involved in the zonal differentiation of adrenocortical cells,
as judged by the solid inverse correlation between expression of AZ-1
and Cyp11b-1 both in vitro and in vivo. Although
it has not been established if AZ-1 plays a causal role as a direct
signal in repressing the Cyp11b-1 gene, the current results
led us to conclude that expression of AZ-1 is coupled with mechanisms
determining the functional differentiation of the cortex. As compared
with known factors that determine differentiation of adrenocortical cells, AZ-1 showed distinct features. First, AZ-1 is an extracellular protein with unique EGF domains and a catalytically inactive proteinase domain related to cathepsin B. Second, different from SF-1 that is
essential for development of the tissue and occurs throughout the
adrenocortical zones (19, 20), AZ-1 is expressed in a spatially
restricted manner and is implicated in the adrenocortical zonation.
Third, in contrast to AP-1 which we have previously identified as an activator for the Cyp11b-1 gene expression
(23, 24), AZ-1 is apparently involved in mechanisms for repressing the
gene. Topographic distribution of AP-1 in vivo also agrees well with the function of selectively activating the
Cyp11b-1 gene expression (23, 24). Collectively with our
previous studies, the present results suggest that AP-1 and AZ-1 are
expressed in a mutually exclusive manner that topographically
corresponds to the actual functions of the cells to execute the
zone-specific steroidogenesis.
Recent studies from other investigators have suggested the presence of
factors occurring in a zone-specific manner that could simultaneously
regulate the zonal differences in the adrenocortical steroidogenesis.
Preadipocyte factor 1 (40) (also called zona glomerulosa-specific
protein) was reported as such a candidate that is expressed in cells of
zG of rats (41). Based on an inhibitory function of preadipocyte factor
1/zona glomerulosa-specific protein in the differentiation of
preadipocytes (40) together with its expression patterns through the
development of the adrenal cortex, this factor is presumed to have a
role in the functional differentiation of the adrenocortical zones
(42). Inner zone antigen is another candidate occurring in zFR in rat
adrenal cortex that was identified to be a putative membrane
progesterone receptor (43). Because anti-inner zone antigen antibody
inhibited activities of Cyp11b-1 in addition to steroid 21-hydroxylase
in vitro (44), the factor seemed to be implicated in the
steroidogenesis of zFR. For identification of these factors,
functionally differentiated cells in vivo such as cells of
zG and zFR of rats were utilized as the resource materials. Although
such methodologies used in these previous studies turned out to be
useful in obtaining the specific molecular markers for the
adrenocortical zones (41, 45), their functions in the zone-specific
steroidogenesis and/or in the development of the zones still remain to
be elucidated. In order to overcome difficulties to search for
factor(s) involved in the functional differentiation, we herein used
the recently established adrenocortical cell line AcA101 at an
undifferentiated state as the alternative resource material. In
this context, the present study employing a different strategy has
revealed that the novel factor whose expression was accompanied by the
lack of expression of Cyp11b-1 was responsible for the zone-specific
steroidogenesis. Thus, regulation of the Az-1 gene is
interconnected with determining the zonal structures of the adrenal cortex.
At the same time, the current results raise further questions as to the
function of AZ-1 in the zonal differentiation of adrenocortical cells.
Because expression of the endogenous Az-1 gene was induced through the selection processes for stably transfected cells, direct
evidence that cells expressing AZ-1 are devoid of Cyp11b-1 expression
was obtained. Although AZ-1 was shown to be secreted in the culture
medium by using the transfected cells, it is unknown at present whether
the secreted form of AZ-1 could repress expression of the
Cyp11b-1 gene by a signaling through the cell surface. Another important issue for the adrenocortical zonation is
identification of molecules that determine expression of Cyp11b-2 in
cells of zG. Based on the present findings that Cyp11b-2 is expressed
in the AZ-1-positive cells in vivo and that the Cyp11b-1 is
expressed in AZ-1-negative cells in vivo, we speculate that
AZ-1 is a prerequisite for cells of zG to express Cyp11b-2 without
expression of Cyp11b-1. Identification of such factors occurring in
cells of zG that determine Cyp11b-2 expression deserves further studies
provided that a combination of AZ-1 with them could actually determine
the functional differentiation of cells in zG.
The primary structure of AZ-1 protein suggests that it carries the
potential to play a role in formation of the cell arrangements in the
adrenal cortex. Among the novel protein family to which AZ-1 belongs,
functions of TIN-ag have well been characterized (37, 46). TIN-ag has
been shown to be essential for development of glomeruli in embryonic
kidney (37), suggesting that TIN-ag is involved in the
epithelial-mesenchymal interactions. Furthermore, despite the
replacement of the cysteine residue essential for the proteolytic
activity of cathepsin B (47-49), it was shown that purified TIN-ag
binds to laminin and type IV collagen and inhibits polymerization of
laminin (46). Because AZ-1 shares essentially the same structural
features as TIN-ag, AZ-1 could interact with extracellular matrix
proteins. These molecules are generally involved in arrangements of
cells in multicellular systems including tumor invasion. It may not be
unreasonable to postulate that AZ-1 functions to regulate the
arrangement of adrenocortical cells through such cell-to-cell and/or
cell-to-matrix interactions.
Previous studies using chimeric (50) and transgenic (51) animals have
suggested that adrenocortical cells arranging centripetally are clonal
cells; a progenitor cell differentiates into two functionally distinct
cell species, cells of zG and those of zFR. Thus, two patterns of the
cell arrangement are likely to be present in the adrenal cortex: the
centripetal arrangements of clonal cells and the concentric
arrangements of functionally differentiated cells. By analogy with the
structure of TIN-ag, it can be hypothesized that AZ-1 plays a role in
development and maintenance of the cell arrangements. Further
characterization of such an effect of AZ-1 on the cell arrangements
through the interaction with matrix proteins and its functional link
to steroidogenic gene regulation is now underway in this laboratory.
-hydroxylase (Cyp11b-1), a marker for the
functional differentiation of cells in the zonae fasciculata
reticularis (zFR). The deduced protein consisting of 466 amino acids
contained a secretory signal, epidermal growth factor-like repeats, and
a proteolytically inactive cathepsin B-related sequence. The amino acid
sequence was 89% identical with that of human tubulointerstitial
nephritis antigen-related protein. Among the mouse organs examined,
adrenal glands prominently expressed its mRNA. The mRNA and its
encoded protein were detected in the outer adrenocortical zones that do
not express Cyp11b-1, i.e. the zona glomerulosa and the
undifferentiated cell zone, while being undetectable in zFR that
express Cyp11b-1. The new protein was designated as adrenocortical
zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1
expression were established from Y-1 adrenocortical cells that
originally express Cyp11b-1 but little AZ-1. Analyses of the clonal
lines revealed that Cyp11b-1 is detected in the clonal lines
maintaining little AZ-1 expression and becomes undetectable in those
expressing AZ-1. On the other hand, irrespective of the AZ-1
expression, all clones expressed cholesterol side-chain cleavage
enzyme, which occurs throughout the cortical zones. These results
demonstrated that adrenocortical cells expressing AZ-1 do not express
Cyp11b-1, whereas those with little AZ-1 express this zFR marker
in vitro and in vivo, implying a putative role
of AZ-1 in determining the zonal differentiation of adrenocortical cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (Cyp11b-1) responsible for the synthesis of
corticosterone/cortisol, respectively (11). The zone thus has been
named the undifferentiated cell zone (zU) (12) after the functionally
undifferentiated nature of its component cells (13-18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZipLox
system (Invitrogen) according to the instructions from the
manufacturer. Subtractive probes were prepared with the chemical cross-linking subtraction method using an RNA subtraction kit (Amersham Biosciences). Single-stranded cDNA (0.4 µg) of AcA101 cells was subtracted with poly(A)+ RNA (10 µg) of AcA201
or Y-1 cells by hybridization and chemical cross-linking reaction. To
reduce signals from SV40 T-antigen mRNA, RNA encoding T-antigen was
synthesized in vitro by using cloned T-antigen genes (25)
and was added to the hybridization reaction. The resulting subtracted
cDNA was labeled with [
-32P]dCTP (3000 Ci/mmol,
Amersham Biosciences) using Sequenase version 2.0 (Amersham
Biosciences). The AcA101 cDNA library (2 × 105
plaque-forming units) was screened with the subtractive probes under
stringent conditions. Positive plaques were isolated through a second
screening process using digoxigenin-labeled cDNA (Roche Diagnostics) probes (not subtracted) from AcA101, AcA201, and Y-1
cells. Plasmids carrying a cDNA insert were recovered from
phage clones utilizing the cre-lox systems. DNA sequencing was performed by the dideoxy termination method using Thermo
SequenaseTM (Amersham Biosciences).
-32P]dCTP (3000 Ci/mmol, Amersham Biosciences) and High Prime (Roche Diagnostics).
Hybridization signals were detected with a Kodak BioMax film with an
intensifying screen.
)
containing 4% paraformaldehyde and were further fixed with the same
fixative overnight at 4 °C. The adrenals were embedded with
paraffin, and 4-µm sections were prepared using
3-aminopropyltriethoxysilane-coated glass slides. After deparaffinization with standard methods, in situ
hybridization was carried out as described previously (17).
Concentrations of the antisense and sense probes (400 ng/ml) in the
hybridization solutions were adjusted based on analysis by the agarose
gel electrophoresis of the synthesized RNA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
For the
subtractive cDNA cloning to identify genes whose transcripts are
expressed in higher levels in undifferentiated adrenocortical cells
than in differentiated cells, we prepared two probes as described under
"Experimental Procedures"; one was obtained by a subtraction
between AcA101 and AcA201 and the other was between AcA101 and Y-1. A
cDNA library of AcA101 cells was prepared to represent the whole
population of mRNA molecules. It was screened by hybridization of
the two subtracted probes onto plaque lifts. We isolated cDNA
clones whose hybridization signal was detected with both subtractive probes.
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Fig. 1.
Nucleotide sequence of AZ-1 cDNA and the
predicted amino acid sequence. A, the amino acid
sequence is shown in single-letter code below the nucleotide
sequence. The putative cleavage site of the signal peptide is indicated
by a triangle. The residues corresponding to those
comprising the active site conserved in cysteine proteinases are
circled. (Ser-228 in AZ-1 replaces the cysteine residue, and
His-399 and Asn-424 are conserved). Two possible
N-glycosylation sites are underlined. A
polyadenylation signal at nucleotide position 1907-1912 is in
italics. B, comparison of the amino acid sequence
of mouse AZ-1 with that of human TIN-ag-RP
(accession number AF236150). Identical amino acids (89%) are indicated
with asterisk.
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Fig. 2.
In vitro translation of AZ-1
polypeptide and Southern blot analysis. A, in
vitro coupled transcription-translation reaction was performed
using a control vector pRc/CMV (lanes 1 and 4),
pR/C11.13D1 (lanes 2 and 5), and pR/C11.13FD1
(lanes 3 and 6). pR/C11.13D1 and pR/C11.13FD1
carry AZ-1 cDNA and FLAG epitope-tagged AZ-1 cDNA,
respectively. Lanes 1-3, translated products (5 µl/lane)
synthesized in the presence of [35S]methionine were
separated on a 10% polyacrylamide gel containing 0.1% SDS and
detected by autoradiography. Lanes 4-6, products (5 µl/lane) synthesized with non-radioactive amino acids were detected
by immunoblotting using anti-FLAG antibody, a secondary antibody
conjugated with horseradish peroxidase, and reagents for enhanced
chemiluminescence. B, mouse genomic DNA was digested with
restriction enzymes (EcoRI (lane 1),
BamHI (lane 2), and HindIII
(lane 3)). The digests (10 µg/lane) were subjected to
electrophoresis through 0.8% agarose gels and blotted onto a
positively charged nylon filter. An AZ-1 cDNA fragment (position
1490-1810) was labeled using DIG-dUTP. Hybridization, stringent
washing, and detection with color development were carried out as
described under "Experimental Procedures."
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Fig. 3.
Genomic organization of the Az-1
gene. A, schematic representation of
the genomic organization of the Az-1 gene. The nucleotide
sequences of the AZ-1 cDNA were located at chromosome 4 of the
mouse genomic DNA sequence data base Ensembl Genome Browser
(www.ensembl.org/) and span the 9175-bp sequence from
position 127,681,610 to 127,672,436 in a reverse orientation.
Closed and open squares represent the
exons and the introns, respectively. Their sizes (bp) are shown
above (exons) and below (introns) the
squares. Thick bar marked with P
represents the hybridization probe used in the Southern blot analysis
of Fig. 2B. Bars marked with E,
B, or H represent hybridization fragments predicted by
restriction sites for EcoRI, BamHI, and
HindIII in the genomic sequence of the data base, being
consistent with the results of the Southern analysis (Fig.
2B). B, DNA sequences of exon-intron junctions in
the Az-1 gene. Upper and lowercase
letters represent the exon and the intron sequences, respectively.
The encoded amino acid sequence is shown below the
nucleotide sequence. The 5'-end of the cDNA is putatively assigned
to the 5'-end of exon 1.
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Fig. 4.
Northern blot analysis of AZ-1 mRNA.
RNA preparations (10 µg/lane) from cell lines (A) and from
mouse tissues (B) were subjected to electrophoresis through
1% agarose gels and transferred onto a positively charged nylon
filter. Before transfer, rRNAs (28S and 18S) were
visualized by ethidium bromide to verify that amounts of RNA loaded
were comparable with each other and that degradation of the RNA
preparations was undetectable under the experimental conditions. An
AZ-1 cDNA fragment (position 1490-1810) was labeled using
[ -32P]dCTP. Hybridization, stringent washing, and
detection by autoradiography were carried out as described under
"Experimental Procedures."
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[in a new window]
Fig. 5.
Localization of mouse AZ-1 in the
adrenal glands. A, in situ hybridization
analysis of mouse adrenal sections using DIG-labeled AZ-1 antisense RNA
probe shows dark purple staining in the outer cortical
regions composed of zG and zU and the medulla. Note that AZ-1 mRNA
signals are detectable in zG irrespective of Cyp11b-2 mRNA
expression and in zU (compare with the hybridization signals in
C and D). B, in situ
hybridization using labeled AZ-1 sense RNA probe gives a weak
background signal comparable with that detectable with the antisense
probe. C, in situ hybridization using the labeled
Cyp11b-2 antisense RNA probe shows hybridization signals sporadically
in zG. D, in situ hybridization using the labeled
Cyp11b-1 antisense RNA probe shows hybridization signals in zFR.
E, immunohistochemistry using the affinity-purified chicken
anti-AZ-1 antibody shows reddish brown staining in
the outer cortical regions composed of zG and zU and the medulla.
Distribution of AZ-1 immunoreactivity is comparable with that of AZ-1
mRNA (compare with the signals in A). F,
immunohistochemistry using a control chicken IgY gives a weak
background signal comparable with that seen with anti-AZ-1 antibody.
Bar, 50 µm.
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Fig. 6.
Detection of AZ-1 protein produced in stable
transfectants and N-glycosylation.
A-C, Y-1 cells stably transfected with a control
vector pRc/CMV (lanes 1, 3, and 5) or
with an expression vector pR/C11.13FD1 encoding FLAG epitope-tagged
AZ-1 (AZ-1F, lanes 2, 4, and
6) were cultured for 24 h at 37 °C. Whole cell
extracts were analyzed by immunoblotting using chicken anti-AZ-1
antibody (lanes 1 and 2) and anti-FLAG antibody
(lanes 3 and 4). Immunoprecipitates (lanes
5 and 6) from the culture media of the transfectants
with chicken anti-AZ-1 antibody were analyzed by immunoblotting using
anti-FLAG antibody as described under "Experimental Procedures."
D, the AZ-1F-expressing cells were cultured in the presence
of vehicle (lane 7) or 10 µg/ml tunicamycin (lane
8) for 18 h. Whole cell extracts were analyzed by
immunoblotting with anti-AZ-1 antibody. E, whole cell
extracts of the AZ-1F-expressing cells were incubated in the absence
(lane 9) or presence (lane 10) of 50 units/ml
N-glycosidase F (N-glycanase) at 37 °C for
18 h. They were analyzed by immunoblotting with anti-FLAG
antibody. ns, nonspecific signals.
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Fig. 7.
Inverse correlation between AZ-1 and Cyp11b-1
expression in clonal lines derived from Y-1 cells. RNAs from 10 stable transfectants obtained with pRc/CMV (control DNA) and
11 obtained with pR/C11.13FD1 (AZ-1F vector DNA) were
analyzed by RT-PCR. cDNAs synthesized with oligo(dT) primer using
total RNA (1 µg) were amplified by PCR using specific primer pairs
for FLAG-tagged AZ-1 (AZ-1F) (A), both the
endogenous AZ-1 and AZ-1F (B), steroid 11 -hydroxylase
(Cyp11b-1) (C), cholesterol side-chain cleavage
enzyme (Cyp11a) (D), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(E) as described under "Experimental Procedures." PCR
products (15 µl) were analyzed through 2% agarose gels and
visualized with ethidium bromide. Numbers with
arrowheads indicate sizes of the products. ns,
nonspecific band. Amplified conditions were within the exponential
phase of the reactions to obtain the linear dose response concerning
the initial mRNA amounts. F, relationships of normalized
mRNA levels between AZ-1 and Cyp11b-1 and between AZ-1 and Cyp11a.
For the 17 clonal lines without expression of AZ-1F, relationships of
mRNA levels of Cyp11b-1 (closed circle) or Cyp11a
(open circle) to those of the endogenous Az-1
gene are shown. For the other four lines (clones 5, 6, 8, and 14;
AZ-1F-expressing lines), the relationships of the mRNA levels of
Cyp11b-1 (closed triangle) or Cyp11a (open
triangle) to those of the endogenous Az-1 gene plus
AZ-1F are shown.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank M. Kondo and K. Kondo for preparation of adrenal sections and Y. Nagatsuka for assistance in affinity purification of the antibodies.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science, by a national grant-in-aid for the Establishment of High-Tech Research Center in a Private University, and by grants from the Mitsubishi Foundation, the Uehara Memorial Foundation, the Ichiro Kanehara Foundation, and Keio University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB050626.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: 81-3-5363-3752; Fax: 81-3-3358-8138; E-mail: mukaik@sc.itc.keio.ac.jp.
¶ Present address: Dept. of Biological Science and Technology, Faculty of Engineering, the University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
** Present address: Dept. of Biochemistry, the University of Texas Health Science Center, San Antonio, TX 78229-3900.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M301477200
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ABBREVIATIONS |
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The abbreviations used are:
zG, the zona
glomerulosa;
AZ-1, adrenocortical zonation factor 1;
AZ-1F, FLAG-tagged
AZ-1;
Cyp11a, cholesterol side-chain cleavage enzyme;
Cyp11b-1, steroid
11-hydroxylase;
Cyp11b-2, aldosterone synthase;
DIG, digoxigenin;
EGF, epidermal growth factor;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
TIN-ag, tubulointerstitial nephritis antigen;
TIN-ag-RP, TIN-ag-related protein;
SF-1, steroidogenic factor 1;
zF, the zona
fasciculata;
zFR, the zonae fasciculata-reticularis;
zU, the
undifferentiated cell zone;
TBS, Tris-buffered saline.
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