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INTRODUCTION |
Morphogenetic proteins have been identified that play critical
roles in regulating tissue differentiation and maintenance during
embryogenesis and in the adult organism. These inductive proteins
(morphogens) belong to a family of molecules called the transforming
growth factor-
(TGF-
)1
superfamily. This superfamily includes bone morphogenetic proteins (BMPs), cartilage-derived morphogenetic proteins (CDMPs),
Müllerrian inhibiting substance, activins, inhibins, TGF-
,
growth and differentiation factors, and the Drosophila
decapentaplegic gene complex (DPP) (1-3). These morphogenetic proteins
are synthesized as large precursor molecules that are cleaved at a
dibasic cleavage site (RXXR) to release carboxyl-terminal
domains containing a characteristic motif of seven conserved cysteines.
The biologically active proteins are homo- or heterodimers of the
carboxyl-terminal domains (for review, see Ref. 1).
TGF-
superfamily members have been implicated to perform various
roles during embryogenesis, including dorsal ventral specification (DPP) (4), formation of the axial and apendicular skeleton (various
BMPs and CDMPs) (6), and normal male sex development (Müllerrian
inhibiting substance) (5). Based on in vitro and in
vivo studies, it is clear that these proteins perform important roles during embryogenesis and in the adult animal. Several members of
this family show overlapping patterns of expression during embryonic
development, as well as functional redundancy, indicating the apparent
importance of these proteins in embryogenesis (7-8). In addition to
these redundant activities, recent data generated from identification
of mutations in members of this protein family clearly show specific
functions for different family members. For example, multiple
recombinant BMPs and CDMPs are capable of inducing endochondral bone
formation at ectopic sites (1, 3). However, mutations in CDMP-1 in
humans and growth and differentiation factor-5, the murine homologue of
CDMP-1, leads to specific morphological abnormalities in limbs and
joints (9, 10). Thus, members of this family have specific activities
in addition to the pluripotent functions each member might share with
the rest of the family. These specific activities could be the result
of exclusive expression in a spatial and temporal fashion during
development or due to the expression or lack of expression of
receptors.
TGF-
superfamily proteins interact with and initiate intracellular
effects through specific membrane bound receptors. A family of cell
surface receptors that interacts and activates cell-specific responses
upon binding ligands of the TGF-
superfamily has been identified.
These receptors belong to a family of Ser/Thr kinase receptors and have
been designated as either type I or type II based on their molecular
weights (11-13). Several different type I and type II receptors have
been identified and cloned. Additional evidence indicates that the
formation of a heteromeric complex of type I and type II receptors is
required for signal transduction (14). Members of the TGF-
family
first bind the type II receptor. Ligand binding to the type II receptor
causes phosphorylation and formation of a heteromer with the type I
receptor. It is apparent that different combinations of type I and type
II receptors can signal in response to the same ligand, and thus, the
tissue-specific effect of any of the ligand would be dependent on the
combinations of type I and type II receptors that are expressed by the
different cell types. Receptor activation by ligands of the TGF-
superfamily results in the phosphorylation of downstream signaling
molecules. Recent work has identified members of the mothers against
dpp (MAD) proteins as downstream signaling molecules in the Ser/Thr kinase receptor signaling pathway. Ligand binding and receptor heterodimerization results in phosphorylation of the (MAD) proteins and
their accumulation in the nucleus (15-19). The activation of specific
(MAD) proteins is ligand- and receptor subtype-specific; thus, a
combination of the ligand, the receptor profile, and the profile of the
(MAD) proteins would define the final cellular response to a particular
member of the TGF-
family. Recent work has also shown the ability of
other secreted molecules to interact with and inhibit the activity of
BMPs and other morphogens, such as Wnt, indicating yet another
mechanism by which the activities of these proteins are regulated
in vivo (20, 21).
It is clear that ligands belonging to the TGF-
superfamily are
highly conserved throughout evolution and that related genes play
similar roles in vastly different organisms. The remarkable conservation of these proteins and their mechanisms of action are
illustrated by the ability of Drosophila dpp to initiate
endochondral bone formation in mammals and the ability of the mammalian
homologues of dpp (the BMPs) to replace dpp function in
Drosophila (22, 23). In addition, recent work based on
phenotypic effects identified upon inactivation of members of this
family also illustrates the crucial role for these proteins in tissue
differentiation and interaction in organisms as diverse as
Drosophila and humans (Ref. 9; for review, see Ref. 24).
Many of these proteins are expressed in a variety of tissue and cell
types during organogenesis (7, 8). They seem to play an important role
in mesenchymal-epithelial interactions based on their expression
pattern, as well as on their in vitro and in vivo
effects (3).
However, despite the significant advances in the identification and
characterization of members of the TGF-
superfamily, there still
remain organs and tissues that do not appear to require members of this
family for their differentiation and/or maintenance. It is possible
that these proteins do not play a role in differentiation of some
tissues or that further characterization of existing family members
will show their involvement in as yet unidentified functions. On the
other hand, it is likely that there remain members of this family that
are not yet identified. In order to test the second hypothesis, we
aimed to identify other members of this family that may play important
roles in mammalian development. In this study, we report the cloning
and characterization of a new member of the TGF-
/BMP superfamily,
designated prostate-derived factor (PDF).
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MATERIALS AND METHODS |
Cloning and Sequencing the Human PDF cDNA--
In order to
identify potentially novel family members of the TGF-
/BMP family, we
searched the Integrated Molecular Analysis of Genomes and Their
Expression (IMAGE) Consortium data base of expressed sequence tags
(25). Expressed sequence tag entry R33078 exhibited limited sequence
homology to BMP family members. We designed a nested 5'RACE strategy in
order to obtain more sequence data and confirm the homology. The source
DNA was a human spleen cDNA library, and the 3' oligonucleotide
primers for the first and second round of amplification were DW3
(5'-GGGTCTTTTGAATGAGCACCATTTGGGATT-3') and DW4
(5'-CGCGCCGTAGCACATGGTCACTTGCACCTC-3'). A SP6 primer was used as
the 5' primer in both rounds. The polymerase chain reaction (PCR)
product was sequenced and shown to extend to nucleotide 10, as shown in
Fig. 2. Having confirmed sequence homology to known BMP family members,
we attempted to isolate the full-length cDNA by library screening.
IMAGE clone 140296 (R66917) was ordered from Research Genetics and used
to probe a human placental cDNA library (26)
(CLONTECH). One clone representing the full
sequence except for the 5' three amino acids was isolated. The absence of the initiation codon in this clone was recognized by querying the
IMAGE data base with the 5' sequence of the cDNA clone. Multiple clones within the data base demonstrated that the open reading frame
extended to that shown in Fig. 2. Based on these data, we engineered
the initiation codon by PCR. The PCR product was cloned into pCR
3.1-Uni 3 (Invitrogen, Carlsbad, CA), and the sequence was confirmed by
double stranded DNA sequencing (GenBankTM accession no.
AF003934).
Northern and Southern Blot Hybridization--
Human multiple
tissue Northern blots were obtained from CLONTECH.
These were prehybridized for 4 h at 42 °C in 50% formamide, 6× SSC, 0.5% SDS, 7.5× Denhardt's solution (26). Hybridization was
performed for 18 h at 42 °C in the same buffer. The probe was
the same cDNA probe used for the library screening. The blots were
washed in 1× SSC, 0.1% SDS, three times for 20 min each at room
temperature followed by two washes for 20 min each in 0.3× SSC, 0.1%
SDS at 55 °C and then exposed to Eastman Kodak XAR film. For
Southern blot hybridization, 1 µg of each of genomic human DNA was
digested overnight with EcoRI, XbaI,
HindIII, or BamHI. The digested DNA was separated
by electrophoresis overnight on a 1% agarose gel and transferred to a
Nytran membrane (Schleicher and Schuell). The membrane was then probed
with 32P-labeled PDF probe as described above and subjected
to autoradiography and normalized to 18 S ribosomal RNA.
In Situ Hybridization--
For in situ hybridization
IMAGE clone 140296 was subcloned into PCR 2.0 plasmid (Invitrogen). The
plasmid was linearized by digesting with either XbaI or
SpeI, and the linearized plasmids were then used as a
template to make sense and antisense 35S-labeled PDF
riboprobes using T7 (sense probe using SpeI
linearized plasmid) or SP6 (antisense probe with the
XbaI linearized plasmid) RNA polymerase, respectively.
Sections of human placenta, prostate, and accessory male genital glands
were obtained by Dr. S. Vukicevic, University of Zagreb. Tissues were
fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH
7.2), embedded in paraffin, serially sectioned at 5 mm, and mounted on
silanated slides. Sections were deparaffinated in Histoclear (National
Diagnostics, Atlanta, GA), rehydrated in a descending ethanol series
(100-50%), and washed twice for 5 min each in PBS. Nonspecific sulfur
binding sites were blocked in PBS containing 4 mM
dithiothreitol and protease inhibitors (iodoacetamide and
N-ethymaleimide) for 30 min at 45 °C. Sections were
prehybridized in 50% formamide, 0.6 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 µg/ml
heparin, 10 mM dithiothreitol, 0.5 mg/ml salmon sperm DNA,
0.5 mg/ml tRNA, 10% polyethylene glycol 8000, and 1× Denhardt's
solution for at least 1 h at 50 °C; sections were then
hybridized in the same buffer with 2 × 106
counts/section of either sense or antisense 35S-labeled PDF
riboprobe for 16-18 h at 50 °C in a humid chamber. After
hybridization, sections were rinsed in 2× SSC for 15 min at 50 °C;
in 2× SSC, 50% formamide, 20 mM dithiothreitol for 20 min
at 65 °C; in TEN buffer twice for 10 min each; and in 20 µg/ml RNase A for 30 min at 37 °C. Sections were then washed as described in (27). Slides were dipped in NTB-2 autoradiography emulsion (Kodak)
and exposed for 1 week in the dark at 4 °C. Slides were developed in
Kodak D-19 solution (diluted 1:1 with water) for 4 min at 16 °C,
fixed, rinsed in water, stained with hematoxylin and eosin, and mounted
in Permount (Fisher).
Expression of Recombinant PDF and Western Blot Analysis--
An
antibody was raised in rabbits to a 15-amino acid peptide sequence
(DTGVSLQTYDDLLIA) conjugated to keyhole limpet hemocyanin via an added
carboxyl-terminal cysteine. Anti-PDF antibody was purified using
peptide affinity chromatography and characterized by Western blot
analysis against recombinant PDF expressed in CHO cells following
transfection with a mammalian expression plasmid containing PDF
(pcDNA 3.1). Stable cell clones expressing PDF were selected and
made serum-free for 48 h. The serum-free media were collected and
precipitated with 10% trichloroacetic acid. The resulting protein
pellets were dissolved in 5× Laemmli sample buffer with 2%
-mercaptoethanol and subjected to electrophoresis on a 14%
SDS-polyacrylamide gel (28). The proteins were transferred to a Nytran
membrane and visualized by Western blot analysis using the
affinity-purified antibodies described above and detected by
chemiluminescence (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Immunostaining--
Tissues for immunolocalization were fixed in
4% paraformaldehyde in calcium-magnesium-free phosphate-buffered
saline (CMF/PBS) for 24 to 48 h, rinsed in CMF/PBS and embedded in
paraffin using standard protocols. For immunolocalization of PDF,
sections were deparaffinated with 3× Histoclear. Endogenous peroxidase
activity was blocked by a 10-min incubation in 3%
H2O2 in methanol followed by a 10-min
incubation in CMF/PBS, 0.1% bovine serum albumin, 0.1% Triton X-100
at room temperature. Nonspecific binding was blocked with 1.5% donkey
serum in CMF/PBS for 30 min at room temperature followed by incubation
in 10 µg/ml affinity-purified rabbit anti-PDF diluted in CMF/PBS,
0.1% bovine serum albumin overnight at 4 °C. The next day, sections
were rinsed three times in CMF/PBS, 0.1% bovine serum albumin for 5 min at room temperature and then incubated in a biotinylated donkey
anti-rabbit antibody for 30 min at room temperature and developed
according to the manufacturer's instructions (Vector Laboratories,
Burlingame, CA).
Cell Culture, Transfection and Transcriptional Response
Assay--
Mink lung epithelial cells (Mv1Lu) were obtained from
American Type Culture Collection and cultured in minimum essential
medium (Life Technologies, Inc.) containing 10% fetal bovine serum and antibiotics. R mutant mink lung epithelial cells (clonal Mv1Lu cells
that lack the receptors for TGF-
1) were obtained from
Dr. J. Massague (Memorial Sloan-Kettering Cancer Center) and cultured in minimum essential medium without L-Histidinol, 10%
fetal bovine serum, and antibiotics. The full-length PDF expression
plasmid and a p3TP-Lux promoter-reporter construct (obtained from J. Massague) were transfected into either wild type or R mutant Mv1Lu
cells using Tfx 50 (Promega Inc., Madison, WI) according to
manufacturer's instructions. The p3TP-Lux promoter contains three
consecutive TPA response elements and a portion of the plasminogen
activator inhibitor 1 promoter region and has been shown to be induced
by TGF-
and the various BMPs (14). The transfected cells were trypsinized and then replated into 6-well dishes the next day. Cells
were made serum-free 2 h after replating, and the control wells
were treated with recombinant human TGF-
1 (R&D Systems) at a concentration of 5 ng/ml. The next day, luciferase activity in the
cell lysate was measured using the luciferase assay kit (Promega)
according to manufacturer's direction.
Effect of Androgens on PDF Expression in Vivo--
Seven to
eight-month-old male Charles River rats weighing approximately 600 g were orchidectomized and allowed to recover for 3 weeks. The groups
of animals (n = 3) were treated with 1 mg/kg 5
DHT
(Sigma) and sacrificed at the indicated time points. Poly(A+) RNA was isolated from the ventral prostates and
used (1 µg/lane) for Northern blot analysis of PDF mRNA
expression.
Determination of in Vivo Activity--
Serum-free tissue culture
media obtained from either CHO cells expressing recombinant PDF (650 ng
or 1 µg/pellet of total protein) or nontransfected CHO cells was
reconstituted with 25 mg of guanidine-insoluble collagenous residue of
demineralized rat bone matrix (31). The resulting pellets were
implanted subcutaneously into 1-month-old male Long-Evans rats. The
implants were recovered on day 11 followed by histological analysis for
bone and cartilage induction.
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RESULTS AND DISCUSSION |
Characterization and Cloning of PDF--
To identify new members
of the TGF-
/BMP superfamily we searched the IMAGE Consortium data
base of expressed sequence tag. Northern blot analysis indicated that
one expressed sequence tag was expressed at very high levels in the
placenta and the prostate and had restricted expression in other
tissues (Fig. 1). Other than the placenta
and prostate, only the kidney and pancreas showed detectable levels of
expression among the various tissues tested. Given the expression
pattern, this sequence was designated PDF and used as a radiolabeled
probe to screen a human placental cDNA library. Two cDNA clones
were isolated and sequenced. One of the clones had a 5' deletion in the
open reading frame about 30 amino acids from the start site, and this
clone was not pursued further. Based on a search of the expressed
sequence tag data base, we realized that the second clone lacked the
coding region for the starting methionine and the next two amino acids.
We used a PCR-based strategy to add the 5' missing base pairs and
obtain a full-length PDF cDNA as described under "Materials and
Methods." The open reading frame codes for a protein containing a
hydrophobic leader sequence, a prodomain of approximately 175 amino
acids, a dibasic cleavage site, and a mature domain containing nine
conserved cysteine residues, hallmarks of members of the TGF-
superfamily (Fig. 2A). Based
on the presence of nine cysteine residues in the mature protein, PDF is
structurally more related to the TGF-
s and the
-chain of inhibins
than to other members of the superfamily (Fig. 2B). However,
the mature protein also has sequence similarities to the BMP6,
BMP7/OP-1 subfamily, as well as sequences that are identical to those
present in growth and differentiation factor-7 (not shown).

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Fig. 1.
Human multiple tissue Northern blot analysis
of PDF expression. Northern blot of 2 µg of poly(A+)
RNA from each of the indicated human tissues was probed with a
32P-labeled PDF probe. A single transcript of human PDF of
1.4 kb was observed in the placenta (lane A), prostate
(lane I), kidney (lane E), and pancreas
(lane F). Lanes A-N represent placenta, lung,
liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,
testis, ovary, small intestine, colon, and peripheral blood leukocytes,
respectively. Size markers are indicated on the left.
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Fig. 2.
Nucleotide and amino acid sequence of the
full-length PDF cDNA. A, nucleotide and predicted amino
acid sequence of PDF. One of two dibasic cleavage sites is indicated by
a box. Note that cleavage could also take place one amino
acid carboxyl-terminal of the boxed sequence. The peptide used for
raising the antibody is indicated by underlining.
B, sequence alignments of the mature carboxyl-terminal
domain of PDF with other members of the TGF- superfamily.
Dashed lines indicate gaps introduced for alignment.
Boxes show the conserved residues. C, antibody
characterization and Western blot analysis of recombinant PDF. In order
to characterize the anti-PDF anti-sera, recombinant PDF was expressed
in CHO cells. Western blot analysis of proteins in the serum-free
conditioned media from PDF-transfected cells separated by
SDS-polyacrylamide gel electrophoresis under reducing conditions showed
the presence two specific bands at 42 and 16 kDa (lane A).
Conditioned media from cells transfected with pcDNA 3 alone did not
express these proteins (lane B).
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An anti-peptide antibody to a sequence present in the mature protein
was raised in rabbits. Western blot analysis of media obtained from CHO
cells stably transfected with a PDF expression construct showed that
upon reduction, PDF is detectable as a monomer of 16 kDa. A second band
migrating at 42 kDa probably corresponds to the unprocessed PDF monomer
(Fig. 2C). Both of these bands were absent in medium
obtained from control (pcDNA 3-transfected) cells (Fig. 2C,
lane B). Similar bands, corresponding to the reduced and
unprocessed monomeric forms, have been seen in tissue culture media
when other members of the BMP family have been expressed (30). Southern
blot analysis of human genomic DNA yielded a very simple pattern,
suggesting the presence of a single PDF gene (Fig.
3).

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Fig. 3.
Southern blot analysis shows no evidence of
multiple PDF genes. Southern blot hybridization of genomic DNA
restriction digested with the indicated enzymes revealed a simple
pattern of hybridizing bands, strongly suggesting that PDF is a single
copy gene.
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Localization of PDF--
Northern analysis of human tissues
expressing PDF suggests that it is not widely expressed (Fig. 1). In
order to understand and characterize the precise cell types that
express PDF, we used a combination of in situ hybridization
and immunohistochemistry to localize PDF in the most highly expressing
tissues placenta and prostate. The cell type-specific distribution of
PDF was initially analyzed in the placenta by in situ
hybridization and revealed that PDF mRNA is expressed at very high
levels in the cells of the terminal villi (Fig.
4A). Control hybridizations
done with the probe corresponding to the sense strand showed no
binding, indicating the specificity of the localization obtained with
the antisense probe (Fig. 4B). Immunohistochemistry in
corresponding serial sections using the affinity-purified anti-PDF
polyclonal antibody showed a pattern of expression identical to the one
obtained with in situ hybridization (Fig. 4, C
and D). The expression of PDF in whole embryo sections was
further characterized using serial sections from 18-day-old rat
embryos. In these sections, it can be seen that the protein was also
expressed specifically in the skin and in cartilaginous tissue of the
developing embryo (Fig. 4, E-G). Very little expression of
the protein was detected in other skeletal tissues at this stage of
development. In a human postnatal prostate, in situ
hybridization of PDF revealed that PDF message was expressed in both
the normal and hypertrophic prostate. Expression was seen in the
epithelium of the main prostatic glands (Fig.
5). No specific hybridization was seen in
the fibromuscular stroma. To determine the specificity of PDF
localization, we performed further experiments using other accessory
male genital glands, namely seminal vesicles and bulbourethral glands.
Results indicate that PDF was mainly present in the secretory
epithelium of the prostatic main glands (Fig.
6B), whereas mucosal prostatic
glands close to the urethra in the main lobe, tubulo-alveolar
epithelium of the bulbourethral glands, and the epithelium of the
seminal vesicle did not express PDF protein (Fig. 6, C-E).
These data suggest that main prostatic glands but not other accessory
male genital glands produce PDF.

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Fig. 4.
Expression pattern of PDF. PDF
expression in the placenta. Bright-field images show the specific
localization of PDF visualized by in situ hybridization
(arrows) (A) and immunohistochemistry
(C) in the placenta using an antisense riboprobe and the PDF
anti-peptide antibody. Controls with either the sense probe
(B) or secondary antibody alone (D) showed no
signal. PDF also localized to the cartilaginous tissue and the
epidermal tissue during embryonic development, as shown by
immunohistochemistry using day 18 gestation embryo (E-G).
Magnification, × 125 in A-D; × 25 in E;
and × 250 in F and G.
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Fig. 5.
PDF mRNA expression in human prostate.
A, bright-field image of a normal prostate (36 years of
age). B, dark-field image of the same section with mRNA
localized in the epithelium of the main glands from the left lobe of
the prostate. C, bright-field image of a hypertrophic
prostate surgically removed from a 57-year-old patient. D,
dark-field image of the same section, with a signal in the epithelium
of glandular structures. The control (sense) probe did not show a
specific signal (data not shown). Magnification, × 30.
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Fig. 6.
Immunolocalization of PDF in accessory male
glands. A, control section of the prostate from the area of
main glands from the left lobe (arrow), incubated with only
the primary (PDF) antibody. B, secretory epithelium of the
prostatic main glands from the left lobe showed intensive staining
(arrow) with the PDF antibody. C, a section of
the same prostate from the area of mucosal prostatic glands of the
median lobe close to the urethra did not stain for PDF
(arrowhead). Tubulo-alveolar epithelium of the left
bulbourethral gland (D; arrowhead) and the
epithelium of a section from the left seminal vesicle (E)
stained negatively for PDF. Magnification, × 60 in D and
E; × 100 in A-C.
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Regulation of PDF by Androgens--
The role of androgens in the
control of PDF expression in prostate was investigated by studying the
expression pattern of PDF following orchidectomy. Orchidectomy resulted
in a dramatic decrease in prostate size and a similar decrease in the
expression of PDF compared with sham-operated controls animals (Fig.
7). Treatment of orchidectomized animals
with 5
DHT (1 mg/Kg) resulted in a time-dependent
increase in PDF expression, with maximum increase (2-fold) over control
seen 6 h after treatment (Fig. 7). Although the levels of PDF do
not approach those of sham-operated control animals, it should be noted
that the increase in expression was seen upon a single dose of
5
DHT.

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Fig. 7.
Regulation of PDF by androgens.
Poly(A+) RNA was prepared from ventral prostates of rats
that were either orchidectomized or sham-operated ± 5 DHT at 0, 6, 24 and 48 h. after treatment. A, Northern blot
analysis of the RNA showed a dramatic decrease from sham-treated
(lane A) to orchidectomized (lane B) rat in PDF
expression. Treatment with 5 DHT for 6 h showed an increase in
PDF expression (lane D) over control (lane C),
which dropped back to control levels by 24 h (lane E)
and 48 h (lane F). B, quantitation of the
bands shown in A.
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Signaling Activity of PDF--
The ability of PDF to activate
specific cell surface receptors was investigated using a p3TP-Lux
promoter reporter construct. This construct is regulated by members of
the TGF-
/BMP superfamily in a receptor-specific manner (14). This
reporter construct was transfected into wild type and R mutant Mv1Lu
cells (data not shown). The R mutant cells lack type II receptors for
TGF-
, and although they express the endogenous BMP receptors,
co-transfection of BMP receptors along with the reporter construct
results in a superinduction of luciferase activity upon BMP treatment.
Transfection of PDF along with p3TP-Lux resulted in a 5-fold induction
of luciferase activity over control cells, which were transfected with
pcDNA3 along with p3TP-Lux (Fig. 8).
These results suggest that PDF activates signal transduction along the
same pathway as other members of the TGF-
superfamily.

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Fig. 8.
Activation of TGF- -like signaling by
PDF. A PDF expression vector was co-transfected with the p3TP-Lux
promoter reporter construct into Mv1Lu mink lung epithelial cells.
Control cells were transfected with p3TP-Lux alone. The cells were
split, made serum-free the next day, and cultured an additional 24 h in the presence or absence of 5 ng/ml of TGF 1. The
transcriptional response induced by either PDF or by TGF was
measured by determination of luciferase activity. The values are
relative to control and reflect a minimum of three independent
determinations in each experiment. The experiment was repeated three
times.
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Biological Activity of PDF--
Because PDF is a member of the BMP
superfamily, we next investigated the ability of PDF to induce ectopic
bone formation. Tissue culture medium containing recombinant PDF was
reconstituted and subcutaneously implanted as described previously
(29). Histological examination of the implants revealed the presence of
cells with a typical chondrocytic morphology (Fig.
9, B-D, arrows), as well as
metachromatic staining of cartilage matrix (Fig. 9, B-D),
indicating biological activity in the media. Pellets reconstituted with
tissue culture media from mock-transfected cells did not show any
chondrocytic cells (Fig. 9A). These results were similar to
those observed upon implantation of CDMP-1 (6), when development of a
predominantly cartilagenous tissue was observed following subcutaneous
implantation.

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Fig. 9.
Induction of cascade of endochondral
ossification by PDF. A 50-µl aliquot of concentrated protein was
mixed with rat type I collagen and demineralized bone powder, and the
resulting pellets were subcutaneously implanted in Long-Evans rats.
After 11 days, the implants were removed, fixed in 4% paraformaldehyde
in PBS, paraffin-embedded, sectioned, and stained. A,
control implant without PDF showing intact demineralized bone particles
with fibrous tissue between individual particles. B, a low
dose (650 ng/implant) of PDF induced formation of small cartilage
islands in the center of the implant (arrow). C,
a high dose of PDF (1 µg/implant) induced formation of larger
cartilage islands in the middle of the implanted pellets
(arrows). D, higher magnification of C
showing hypertrophied chondrocytes on day 11 after implantation
indicating early stages of endochondral ossification. Toluidine blue
staining was used. Magnification, × 60 in A and
B; × 125 in C; × 250 in D.
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We report here the cloning of a new member of the TGF-
/BMP
superfamily. In contrast to other members of the family, PDF expression is more tissue restricted, at least at the current level of detection. Identification of additional sites of expression of PDF will help to
better define the in vivo physiological role of PDF. The
activity of proteins of the BMP superfamily is regulated through
specific cell surface receptors. Because the family of TGF-
receptors bind more than one ligand, it is clear that a certain
combination of receptors present on the surface of a cell dictates its
response to a specific member of the TGF-
/BMP family of proteins. At
present, we have shown the ability of PDF to initiate activation of the p3TP-Lux promoter reporter construct indicating receptor activation similar to other members of the superfamily. Clearly, the
identification of specific receptors that interact with PDF and their
expression pattern will be important in understanding the physiological
function of PDF. Extracellular matrix interactions can also regulate
the activity of members of the TGF-
superfamily (31, 32). So far, a
number of studies have shown the regulation of expression of TGF-
family proteins during embryonic development. However, little is known
about the ability of other hormones or morphogens to regulate the
expression of these proteins. The identification of these upstream
signaling molecules will be a major focus of future studies.
In conclusion, PDF is related to the inhibin
-chain based on
structural similarity of the conserved nine cysteine residues, but it
is also related to the BMP6, BMP7/OP-1 subfamily in terms of sequence
identities. Based on biological activity, PDF appears to initiate a
signaling cascade similar to that of other members of the TGF-
/BMP
family. Although PDF is a member of this family of proteins, it is
difficult to place in any one subgroup due to the presence of sequences
and structural motifs that place it in more than one subgroup. Based on
its in vivo biological ability to induce bone, it has
classical BMP-like activity. Based on its expression in the prostate,
regulation by androgens, and ability to induce bone formation, we
believe that future work regarding the identification of its in
vivo functional role in the prostate and its regulation of
expression by other hormones and growth factors will result in
obtaining important insight into the role of TGF-
superfamily
members in prostate development and maintenance.
We thank the DNA sequencing laboratory of
Pfizer Central Research for their excellent help and support during
this project.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF003934.