Cloning and Characterization of a Novel Member of the Transforming Growth Factor-beta /Bone Morphogenetic Protein Family*

Vishwas M. ParalkarDagger §, Amy L. VailDagger , William A. GrasserDagger , Thomas A. Brown, Hong Xu, Slobodan Vukicevicparallel , Hua Zhu KeDagger , Hong QiDagger , Thomas A. OwenDagger , and David D. ThompsonDagger

From the Dagger  Department of Metabolic Diseases and the  Department of Molecular Sciences, Central Research Division, Pfizer, Inc., Groton, Connecticut 06340 and the parallel  School of Medicine, University of Zagreb, 10000 Zagreb, Croatia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Members of the transforming growth factor-beta (TGF-beta ) superfamily of growth and differentiation factors have been identified in a wide variety of organisms, ranging from invertebrates to mammals. Bone morphogenetic proteins (BMPs) constitute a subgroup of proteins belonging to the TGF-beta superfamily. BMPs were initially identified by their ability to induce endochondral bone formation at ectopic sites, suggesting a critical role for this family in development and regeneration of the skeleton. They are also expressed at a variety of nonskeletal sites during development, suggesting possible extraskeletal roles for these proteins. We cloned a novel member of the BMP family that is expressed at high levels in the placenta and the prostate and that we have designated as prostate-derived factor (PDF). Based on cDNA sequence analysis, the predicted PDF protein contains two cysteines in addition to the seven conserved cysteines that are the hallmark of the members of the TGF-beta superfamily. In addition, Northern blot hybridization to poly(A)+ RNA showed low levels of expression in the kidney and pancreas. We further characterized the expression of this member of the BMP family by in situ hybridization and immunohistochemistry. These results show high expression in the terminal villae of the placenta. The expression of the protein as visualized by immunohistochemistry shows an expression pattern identical to that of the message in the terminal villae of the placenta. In day 18 rat embryos, protein expression was also seen in the skin and in the cartilaginous tissue of developing skeleton. Orchidectomy and dihydrotestosterone treatment of rats revealed that PDF expression is regulated by androgens in the prostate. In addition, subcutaneous implantation of recombinant PDF induced cartilage formation and the early stages of endochondral bone formation. These data indicate that PDF has a functional relationship to the BMPs.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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-beta (TGF-beta )1 superfamily. This superfamily includes bone morphogenetic proteins (BMPs), cartilage-derived morphogenetic proteins (CDMPs), Müllerrian inhibiting substance, activins, inhibins, TGF-beta , 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta /BMP superfamily, designated prostate-derived factor (PDF).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Cloning and Sequencing the Human PDF cDNA-- In order to identify potentially novel family members of the TGF-beta /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% beta -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-beta 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-beta 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-beta 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 5alpha 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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Characterization and Cloning of PDF-- To identify new members of the TGF-beta /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-beta superfamily (Fig. 2A). Based on the presence of nine cysteine residues in the mature protein, PDF is structurally more related to the TGF-beta s and the beta -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-beta 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).

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.

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.

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 5alpha 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 5alpha 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 ± 5alpha 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 5alpha 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.

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-beta /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-beta , 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-beta superfamily.


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Fig. 8.   Activation of TGF-beta -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 beta 1. The transcriptional response induced by either PDF or by TGF beta  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.

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.

We report here the cloning of a new member of the TGF-beta /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-beta 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-beta /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-beta superfamily (31, 32). So far, a number of studies have shown the regulation of expression of TGF-beta 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 beta -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-beta /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-beta superfamily members in prostate development and maintenance.

    ACKNOWLEDGEMENT

We thank the DNA sequencing laboratory of Pfizer Central Research for their excellent help and support during this project.

    FOOTNOTES

* 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 GenBankTM/EMBL Data Bank with accession number(s) AF003934.

§ To whom correspondence should be addressed: Department of Cardiovascular and Metabolic Diseases, Pfizer Central Research, Groton, CT 06340. E-mail: Vishwas_M_Paralkar{at}groton.pfizer.com.

1 The abbreviations used are: TGF-beta , transforming growth factor-beta ; BMP, bone morphogenetic protein; CDMP, cartilage-derived morphogenetic protein; DPP, Drosophila decapentaplegic; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TEN, 200 mM Tris-HCl, pH 7.5, 0.1 M EDTA, 1 M NaCl; CMF, calcium-magnesium-free; CHO, Chinese hamster ovary; 5alpha DHT, 5alpha -dihydrotestosterone.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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