Endocrinology Vol. 138, No. 10 4416-4420
Copyright © 1997 by The Endocrine Society
Preputial Sebocyte 5
-Reductase Isoform Specificity1
Dianne Deplewski,
Shutsung Liao and
Robert L. Rosenfield
The University of Chicago, Pritzker School of Medicine, Departments
of Pediatrics (D.D., R.L.R.), Medicine (R.L.R.), and Biochemistry and
Molecular Biology (S.L.), Chicago, Illinois 60637-1470
Address all correspondence and requests for reprints to: Robert L. Rosenfield, M.D., The University of Chicago, Pritzker School of Medicine, Department of Pediatrics and Children’s Research Foundation, 5841 South Maryland Avenue (MC-5053), Chicago, Illinois 60637-1470.
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Abstract
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We determined whether the 5
-reductase (5-R) isozyme specificity
in rat preputial sebocytes is in accordance with that of sebaceous
glands (type 1 isoform) or with that of other genital tract
androgen-dependent organs (type 2). Immunostaining indicated expression
of type 1 5-R in preputial epithelial cells in the gland as well as
in culture. Northern blots and ribonuclease protection assays both
showed the presence of the type 1 isozyme in preputial sebocytes, with
little if any expression of the type 2 messenger RNA. Ribonuclease
protection assay demonstrated that both homogenized preputial gland and
freshly dispersed preputial cells express 2-fold more 5-R type 1
messenger RNA than does skin and 7-fold more than cultured preputial
cells. Since cultured preputial cells do not fully differentiate in
monolayer culture, these findings suggest that 5-R is increasingly
expressed as sebaceous cells differentiate. The predominance of the
type 1 isoform in preputial sebocytes is important because it indicates
programming by a skin- and sebocyte-specific signal in the midst of the
genital tract where the type 2 isoform otherwise predominates.
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Introduction
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THE PREPUTIAL gland is an androgen target
tissue (1, 2, 3) composed primarily of sebocytes, which are specialized
epithelial cells that resemble human sebaceous cells in many ways
(4, 5, 6). As with other sebaceous glands, androgen has been found to
stimulate preputial gland sebocytes to grow and develop (7, 8).
Preputial gland sebocytes express androgen receptor, and this
expression increases with sebocyte maturation (9). The preputial glands
of rats are an attractive model in which to study sebaceous cell
differentiation because they are easy to isolate, and single cell
suspensions can be prepared for primary cell culture (10).
5-Reductase (5-R) is important in androgen action as it converts
testosterone, the major circulating androgen, to the more potent
androgen 5-dihydrotestosterone (DHT) (11, 12, 13, 14). Excessive DHT
formation has been implicated in the pathogenesis of acne vulgaris (15, 16), suggesting that activity of 5-R may play an important role in
sebaceous cell growth and differentiation. Two forms of 5-R exist,
which are differentially expressed in various tissues, likely as a
result of their respective promoters. They have different pH optima and
sensitivity to inhibitors. The two isozymes are approximately 46%
identical in sequence, have similar gene structures, are both
hydrophobic, and share similar substrate preferences (11, 12). The type
1 gene encodes a 255-amino acid protein that is 60% homologous to the
human protein (11, 17). It has a neutral to basic pH optimum and is
more sensitive to finasteride than its human counterpart because of a
specific four-amino acid segment in the amino terminus, which may form
a portion of the substrate-binding domain (18). The type 2 isozyme has
been shown to be 254 amino acids in length and to have an acidic pH
optimum (11).
The type 1 isozyme has been shown in the rat to be the predominant
isozyme expressed in the liver, ovary, and many somatic tissues,
whereas the androgen target tissues of the genital tract, such as the
epididymis and vas deferens, have been found to express mainly the type
2 isozyme (11). Because of these differences in tissue distribution, as
well as substrate affinities, it has been postulated that the type 1
isozyme plays a catabolic role (such as inactivating testosterone in
the peripheral tissues), and the type 2 isozyme plays an anabolic role
in steroid hormone metabolism (11, 19). However, this is clearly an
oversimplification. For example, in humans the type 1 isozyme has been
found to be the major form of 5-R in the sebaceous cells of the
scalp (13, 20, 21, 22). To further understand sebaceous gland development,
we have determined whether 5-R isozyme expression in the sebocytes
of the rat preputial gland is in accordance with that reported for
other androgen-dependent genital tract organs or with other sebaceous
cells.
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Materials and Methods
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Tissues and cells
Tissue samples from male (preputial gland, epididymis, and
prostate), and female (preputial gland, ovary, and liver) young adult
Sprague-Dawley rats were minced in 4 C PBS, and dorsal skin (from males
and females) was snap frozen in liquid nitrogen and powdered on dry
ice. Homogenates were made with a polytron in 4 M
guanidinium thiocyanate, 25 mM Na-Citrate, 0.5% sarcosyl,
and 0.1 M 2-mercaptoethanol (23). Epithelial cell
suspensions were prepared from the preputial glands and shaved dorsal
skin as previously reported (6). Cell culture was carried out as
previously reported on a 3T3-J2 mitomycin-C treated feeder layer in an
epithelial cell culture medium containing 10% FCS (6). Epithelial
cells were harvested after incubation with EDTA and trypsin.
Immunostaining
Rat preputial glands were frozen in liquid nitrogen,
sectioned on a cryostat, and fixed to gelatin-coated slides using a
paraformaldehyde-picric acid fixative. Cells grown on plastic slides or
cell smears were fixed in methanol for 20 min at -20 C. The sections
were probed with control Igs purified from serum of nonimmunized
rabbits and a rabbit antirat polyclonal antibody to a C-terminal
peptide (amino acids 238–254) of rat 5-R type 1 (24).
Immunoblotting with the peptide antibody detected a single component in
rat liver macrophages of the appropriate size for 5-R type 1 (24). A
biotin-streptavidin-conjugated peroxidase staining system was used
(Zymed, San Francisco, CA). The sections were counterstained with
hematoxylin. To demonstrate specific immunoreactivity of the 5-R
type 1 antibody, immunostaining was repeated after preincubation of the
antibody with the peptide antigen.
DNA probes
Rat 5-R type 1 complementary DNA (cDNA) is an 834-bp
fragment inserted into the transcription vector pBluescript SK at the
EcoRI site of the polylinker (24). Rat 5-R type 2 cDNA
was obtained from Dr. D. W. Russell (University of Texas, Dallas,
TX) as a 1786-bp fragment inserted in the EcoRI site of the
polylinker of pBluescript SK. Glyceraldehyde-3-phosphate-dehydrogenase
(GAPDH) cDNA consisted of a 1270-bp fragment inserted into the
PstI site in the polylinker of the transcription vector pGEM
3Z (25). The plasmids containing the 5-R type 1 and 2 were digested
with EcoRI, and the 834-bp fragment of 5-R type 1 and the
1786-bp fragment of type 2 were isolated from agarose gels. A 500-bp
fragment of GAPDH was similarly isolated after digestion with
BglI. Random labeled DNA probes were constructed using
random hexanucleotides and the Klenow fragment of DNA polymerase I in
the presence of 32P-labeled deoxycytidine triphosphate and
three other nucleotide triphosphates (26).
Riboprobes
The vectors containing the 5-R cDNAs were cut with PST I and
re-ligated. The 216-bp riboprobe for type 1 and the 199-bp riboprobe
for type 2 were made by linearizing the respective plasmids with the
restriction enzyme HindIII and then using T3 RNA polymerase
in the presence of -32P-labeled uridine triphosphate
(UTP) and three other nucleotide triphosphates (26). A 109-bp internal
control probe was created by linearizing pT7 18S ribosomal RNA (rRNA)
(Ambion, Austin, TX) and transcribing with T7 RNA polymerase as
above.
Northern blot analysis
Total RNA (10 µg per sample) isolated by the guanidinium
thiocyanate method (23) was separated on a formaldehyde-containing
agarose gel, transferred to a Duralon UV membrane (Stratagene, La
Jolla, CA ) by capillary elution, prehybridized, and then hybridized
with random labeled DNA probes at 42 C for 16–20 h. The filter was
subsequently washed and exposed to x-ray film (26). GAPDH was used as
an internal loading control.
RNAse protection assay
Total RNA (10 µg) was hybridized to 32P-labeled
antisense RNA probes specific for 5-R type 1 and 2. An antisense RNA
probe to 18S rRNA was used to normalize data for variations in the
amount of RNA in each reaction. Unprotected RNA was digested with RNase
A and T1 for 30 min at 37 C. The protected RNA hybrids were
subsequently separated on a 5% polyacrylamide/8 M urea gel
(27). The gel was dried and exposed to x-ray film, and the bands were
quantified using an AMBIS (San Diego, CA) image acquisition and
analysis system.
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Results
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Immunohistochemistry demonstrated the type 1 iso-zyme in
the basal immature cells of the preputial gland, as well as in the most
mature sebocytes in the center of the preputial acini. 5-R
expression was found predominantly in the cytoplasm of the preputial
cells, suggesting this as the primary site of DHT formation (Fig. 1A
). Immunocytochemistry of dispersed
single cell suspensions and primary cell culture likewise demonstrated
expression of 5-R type 1 mainly in the cytoplasm of both preputial
and epidermal cells, with little if any expression in the nuclei (Fig. 1, B and 1C). Incubation with a nonspecific control antibody showed no
staining (Fig. 1, A–C). Preincubation of the type 1 antibody with the
peptide antigen completely abolished the staining (results not shown,
but similar to control antibody).
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Figure 1. A, Immunohistochemistry of a sectioned preputial
gland using either a rabbit antirat polyclonal antibody to a C-terminal
peptide of 5-R type 1 (left panel), or a control
antibody (right panel), in a biotin-streptavidin-
conjugated peroxidase staining system, whereby the bound antibody
stains (dark). 5-R is present in preputial acinar cells, mainly in
the cytoplasm, at all stages of differentiation. B, Immunocytochemistry
of dispersed single cell suspensions of preputial cells (left
panel), and of epidermal cells (right panel).
5-R staining is marked in the preputial and epidermal cell
cytoplasm, with little if any staining in the nucleus. The
center panel shows preputial cells incubated with a
control antibody. C, Immunocytochemistry of preputial (left
panel) and epidermal (right panel) cells in
primary culture. Both cell types show expression of the type 1 isozyme
within the cytoplasm. Preputial cells incubated with a control antibody
(center panel) showed no staining.
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Northern blots of total RNA isolated from homogenized tissues showed
type 1 mRNA in preputial RNA from both male and female rats, as well as
in skin at a lower level of expression than in preputial sebocytes. The
type 1 isozyme was also expressed in liver, ovary, and prostate. The
type 2 mRNA was detected in prostate and epididymis. A faint band at
2.4 kb may represent a low amount of type 2 mRNA expression in
preputial sebocytes or nonspecific binding (Fig. 2).
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Figure 2. Northern blot analysis. Autoradiograph of 5-R
type 1 and type 2 in male (m) and female (f) preputial gland and skin,
epididymis (epid), ovary (ov), liver (liv), and prostate (pr). Type 1
mRNA (2.4 kb) was detected in preputial RNA from both males and females
and was more abundant in preputial than in skin RNA, second only to
liver. It was detected to some extent in all tissues. Type 2 mRNA (2.4
kb and 4.4 kb) was detected in epididymis and prostate. The faint band
in preputial RNA may represent a low amount of expression or
nonspecific binding.
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RNase protection assays demonstrated only the type 1 isozyme in
preputial sebocytes and skin (Fig. 3).
Freshly isolated preputial cells expressed amounts of the type 1
isozyme similar to homogenized preputial gland (Fig. 4A) and 2-fold more than skin
(P < 0.01; Table 1) (Figs. 3 and 4A).
Type 1 mRNA abundance averaged 7-fold greater in freshly isolated
preputial cells than in cultured preputial cells (P <
0.01; Table 1) (Fig. 4B). In contrast, both cultured and freshly
isolated epidermal cells expressed similar amounts of type 1 mRNA
(Table 1 and Fig. 4B).
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Figure 4. RNase protection assay/autoradiographs of 5-R
type 1. A, Expression of type I mRNA is similar in homogenized (h) and
freshly isolated cells (f). Liver RNA served as a positive control for
preputial gland (Pp), dorsal skin (sk), prostate (pr), liver (liv), and
epididymis (epi). B, Type 1 mRNA expression was greater in freshly
isolated preputial cells than in cultured (cx) preputial cells. Freshly
isolated skin and cultured skin expressed similar levels of the type 1
isozyme and approximately twice that of cultured preputial cells.
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Discussion
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We have demonstrated by immunostaining, Northern analysis, and
RNase protection assay that the type 1 isozyme is the predominant form
of 5-R present in rat preputial sebocytes and skin. We also found
the type 1 isozyme to be expressed in the ovary and liver of the female
rat as has been previously reported (11, 28). This high amount of type
1 enzyme expression present in the liver likely plays a catabolic role
in terms of inactivating testosterone and other steroids. The presence
of the type 1 isozyme in the ovary may also serve a catabolic role
(19). We demonstrated the type 2 isozyme in prostate on both Northern
blot and RNase protection assay, although at low levels; previous
studies have augmented the expression of this isozyme in the
regenerating prostate, after castration and pharmacological
testosterone treatment (11, 29).
These data signify that sebocyte 5-R isozyme expression is
programmed by a skin-specific signal rather than a genital tract
signal, which is consistent with the embryologic origin of sebaceous
glands as down-growths from embryonic skin epithelium (30, 31). These
results are important because the preputial gland is an androgen target
gland in the midst of the genital tract where the type 2 isozyme
otherwise predominates. Although we only found expression of the type 1
isozyme, it is possible that the type 2 isozyme is expressed at a
different time point in development, as has been found in human skin at
various ages (20).
Our results indicate that the expression of 5-R mRNA is less in
cultured preputial cells than in freshly isolated cells. Since cells
grown in this monolayer culture system do not mature to the point of
developing fused lipid droplets (10), our results suggest that type 1
5-R expression, like androgen receptor gene expression (9), is low
before the stage at which lipogenesis occurs and thus imply a role for
5-R and DHT in augmenting lipogenesis in the later stages of
sebocyte maturation.
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Acknowledgments
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We thank Richard A. Hiipakka and John Kokontis for their
excellent technical advice.
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Footnotes
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1 This research was supported in part by USPHS Grants HD-06308 (to
R.L.R.), DK-41670 (to S.L.), and T32 DK-07011 (to D.D.), and a grant
from Eli Lilly Pharmaceuticals (to D.D.). 
Received March 7, 1997.
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Abstract
Introduction
