From the § Department of Cell Biology and the
Cell and Molecular Biology Program, Baylor College of
Medicine, Houston, Texas 77030
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ABSTRACT |
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We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.
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INTRODUCTION |
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Signaling between stromal and epithelial cells directs specific
growth and differentiation patterns in many tissues, including the lung
(1, 2), gastrointestinal tract (3), liver (4), skin (5), the mammary
gland (6), and the prostate gland (7-10). Growth factors comprise a
key component of stromal-epithelial interactions. Growth factors
implicated in prostate gland biology include: the fibroblast growth
factor family (11, 12), the transforming growth factor- family (13),
transforming growth factor-
(14), the insulin-like growth factor
family (15), epidermal growth factor (16, 17), heparin-binding
epidermal growth factor-like growth factor (18), tumor necrosis
factor-
(19), and heparin growth factor-scatter factor (20). Each of
these growth factors are bound to the extracellular matrix, cell
membrane, or other binding proteins in an inactive or sequestered form
and their activity regulated by secreted proteases and protease inhibitors. The actions of proteases and protease inhibitors are fundamental to tissue homeostasis through the regulation of both growth
factor bioavailability and cell interaction with extracellular matrix.
Alterations in protease and protease inhibitor actions affect tissue
development patterns, angiogenesis, cell motility, and tumor invasion
(21-24).
In previous studies to identify stromally derived paracrine-acting factors which regulate prostatic epithelial cell proliferation and differentiation, we identified a urogenital sinus mesenchymal secreted growth-inhibitory activity (UGIF1 activity), which altered epithelial cell proliferation, morphology, and protein secretion in several epithelial cell lines (25, 26). A novel mesenchymally secreted protein, termed ps20 (20-kDa prostate stromal protein), responsible for UGIF activity was purified to homogeneity and its amino acid sequence determined (27). Purified ps20 inhibited PC-3 prostatic epithelial carcinoma cell proliferation in a dose-dependent and saturable manner, altered phenotypic morphology, and stimulated protein synthesis and secretion in vitro (27). Specific mechanisms of ps20 action have not yet been determined.
We report here the cloning of full-length cDNA encoding ps20, expression of biologically active recombinant ps20, development of a ps20 antibody, and analysis of in vivo expression patterns. The ps20 protein is a novel member of the whey acidic protein (WAP)-type "four-disulfide core" domain protein family (28, 29). This family is composed of small, secreted serine protease inhibitors, which exhibit a variety of growth- and differentiation-regulatory functions and affect extracellular matrix remodeling and carcinoma progression. We demonstrate here that recombinant ps20 inhibits the proliferation of prostate carcinoma PC-3 cells and COS-7 cells. In addition, we show that ps20 is expressed specifically in the smooth cell type of the prostate gland and other tissues including vascular smooth muscle. These studies suggest that ps20 may function as a mediator of local growth and differentiation mechanisms.
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EXPERIMENTAL PROCEDURES |
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Preparation of the PS-1 (Adult Rat Prostate Smooth Muscle Cell
Line) ZAP Express (PS-1_BfsZAP) cDNA Library--
An oligo(dT)
reverse transcribed cDNA library of 109 to
1012 plaque-forming units/ml was prepared using the ZAP
Express cDNA synthesis kit (Stratagene, La Jolla, CA). PS-1 cells
(adult rat prostate smooth muscle-like cell line) (30), were grown to
post-confluence in Bfs medium (90% Dulbecco's modified Eagle's
medium (DMEM), 5% fetal calf serum, 5% NuSerum (Becton Dickinson,
Bedford, MA), 5 µg/ml insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin) supplemented with androgens (0.5 µg/ml
testosterone) (26). RNA was prepared using the RNAgents total RNA
isolation system and poly(A) RNA selected with the
PolyATtract® system (both from Promega, Madison, WI). RNA
was size-fractionated on a Sephacryl column prior to ligation with
vector arms.
ps20 Cloning-- The amino-terminal amino acid sequence of ps20 was determined and previously reported (27): N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-X-X-X-X-Val-Ala-Ala-(Thr)-Gly-X-Arg-Gln-Pro-(His)-C. Degenerate ps20 primers were designed from this sequence: primer P34 (256-fold degenerate, amino acids 1-7), primer P36 (16-fold degenerate, amino acids 1-7), primer P37 (64-fold degenerate, amino acids 7-13), and primer P38 (16, 384-fold degenerate, amino acids 27-19). Inosines were incorporated at sites of complete degeneracy (31) in primers P34 and P38, whereas optimal mammalian codon usage was assumed in the design of primers P36 and P37. The ps20-specific primers P40 and P42 were designed from clone 3438-1pCRII sequence and ps20-specific primers P45 and P46 from clone 42T7pCRII sequence. Primer sequences (IUPAC nomenclature) are identified in terms of rat ps20 full-length cDNA sequence (Fig. 2): P34 (nt 130-150), 5'-ACI TGG GAR GCI ATG YTI CC-3'; P36 (nt 130-150), 5'-ACM TGG GAR GCY ATG CTS CC-3'; P37 (nt 148-168), 5'-CCY GTS MGR CTG GCY GAR AA-3'; P38 (nt 209-183), 5'-GGC TGY CKI IIS CCK GTK GCR GCN AC-3'; P40 (nt 169-151), 5'-TCA GCT TGG GAC TTC TCA G-3'; P42 (nt 153-171), 5'-GAG AAG TCC CAA GCT GAA G-3'; P45 (nt 425-444), 5'-GGA GGA AGT GTT ACA AGC AG-3'; and P46 (nt 1011-992), 5'-GGT GGG CAG ATT TAT TCG GG-3. Primers P34-P42 were synthesized and purified using high performance liquid chromatography by Keystone Labs, Inc. (Menlo Park, CA). Primers P45 and P46 were synthesized by Genosys (The Woodlands, TX).
Primary PCR amplifications were performed using a ps20 degenerate primer and a vector primer (T3 or T7) with 1 µl of the PS-1_BfsSequencing-- Multiple pass DNA sequencing was performed on an Applied Biosystems model 377 Sequencer version 2.1.1 (University of Texas Houston Molecular Genetics Core Facility) using Ampli-Taq polymerase, 3 pmol of primer (pBKT3-1-, pBKT7-1-, M13-20-, and ps20-specific primers), and 1 µg of double-stranded DNA per reaction, or by manual sequencing with Sequenase version 2.0 DNA sequencing kit (U. S. Biochemica Corp./Amersham Life Science) (33). Sequences were assembled using MacVector version 4.1 and AssemblyLign version 1.0 (Kodak).
Sequence Analysis-- Nucleotide sequence searches were performed on all available data bases using the BLASTN and TBLASTN (blast enhanced alignment utility) algorithms (34). Secondary structure predictions of deduced amino acid sequence were made by SOPMA (Self Optimized Prediction Method from Alignment) (35) and SBASE (36) and confirmed by BLASTX and TBLASTX (34) searches of the available data bases (37). Secretory peptide prediction was confirmed by PSORT version 6.3.
Northern Analysis--
Total RNA was isolated from adult male
Sprague-Dawley rat tissues (intact, castrate 2 weeks, and castrate 6 weeks), adult female rat tissues (kindly provided by J. S. Richards, Baylor College of Medicine) and from U4F cells grown to
post-confluence in Bfs medium. Total RNA was prepared using RNA Stat-60
total RNA isolation reagent (Tel-Test "B," Inc., Friendswood, TX).
RNA was electrophoresed through 1% agarose gels (containing 1 × MOPS and 5.1% of a 37% formaldehyde solution), which were transferred to neutral charged nylon membranes (Schleicher & Schuell) by downward capillary blotting. Inserts from T340pCRII and 42T7pCRII were excised,
gel-purified, labeled by random priming with
[-32P]dCTP, purified, and denatured as described
above. Blots were UV-cross-linked and exposed to probe at 1-5 × 106 counts/ml, overnight, at 42 °C; washed with multiple
20 min washes at 60 °C in 0.1% SDS containing 2 to 0.1 × SSC;
and exposed to Kodak XAR film for 24 h to 2 weeks, as
necessary.
Preparation of ps20 Antisera and Affinity Purification-- The amino-terminal sequence of native ps20 was determined as described previously (27). A unique 14-amino acid synthetic peptide, ps20 peptide-(1-14), corresponding to the amino terminus of purified ps20 (see Fig. 2), was synthesized on an Applied Biosystems model 430A peptide synthesizer: N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-C (Baylor College of Medicine Protein Core Facility). For initial immunization, ps20 peptide was solubilized in sterile, tissue culture grade H2O (400 µg/ml), mixed with Freund's complete adjuvant (1:1 ratio), and injected in 500-µl (100-µg) aliquots at multiple subcutaneous and intramuscular sites of three female New Zealand rabbits. At 3 weeks following immunization, sera samples were analyzed by solid phase enzyme-linked immunoabsorbent assay (ELISA). Each rabbit received a booster of 100 µg of peptide in 500 µl of Freund's incomplete adjuvant injected subcutaneously and intramuscularly at 2 weeks and 5 weeks. Serum samples were tested for ps20-specific antibody every 2 weeks and immunoglobulin subtype determined by ELISA analysis. High titer antiserum (reactive at 1:1 × 106 dilution) was pooled, and the IgG class was precipitated by ammonium sulfate (50% saturation), resolubilized in 1 × PBS, and dialyzed overnight against 1 × PBS at 4 °C. This antibody preparation is referred to as ps20ab-1.
ps20ab-1 was affinity-purified by peptide column chromatography. ps20 peptide-(1-14) (10 mg) was coupled to 1 g of cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech Inc.) (38) and poured into a 2-ml Poly-Prep column (Bio-Rad). ps20ab-1 was diluted in BBS buffer (200 mM sodium borate, 160 mM sodium chloride, pH 8.0) and chromatographed through the column at 4 °C. The column was washed extensively in BBS (10-15 column volumes) and bound antibodies eluted with glycine-Cl buffer (0.05 M glycine, 0.15 M NaCl, pH 2.28). Fractions (2 ml) were eluted and collected directly in tubes containing 0.5 ml of neutralizing buffer (0.5 M phosphate, pH 7.7). Fractions were assayed for protein content, and peak fractions were pooled and assayed for specific immunoreactivity by ELISA.Cell Culture-- The U4F urogenital sinus mesenchymal cell line (26) was derived from a urogenital sinus organ explant (25) and the PS-1 cell line (30) derived from an adult rat prostate organ explant. U4F cells and PS-1 cells were maintained in medium Bfs as described previously (26, 27, 30). PC-3 cells (ATCC CRL-1435, human prostate adenocarcinoma) were maintained as recommended by the ATCC and as described previously (27). The COS-7 cell line (ATCC CRL-1651, SV40 transformed African green monkey kidney fibroblast-like) was maintained in 90% DMEM, 10% fetal calf serum (DMEM-10) (BioWhittaker, Walkersville, MD), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were tested for mycoplasma every 6 months.
COS-7 Cell Transient Transfection-- COS-7 cells were seeded at 1.7 × 106 cells/ml in 100-mm culture dishes in 90% DMEM, 10% fetal calf serum (DMEM-10) with no antibiotics 24 h prior to transfection. Cells were transfected at 80% confluence with 4.1 µg of ps20-2bpBKCMV-lac DNA or pBKCMV-lac control plasmid DNA (prepared by Qiagen tip-500) and 41.0 µl of LipofectAMINE reagent (Life Technologies, Inc.), following recommendations of the manufacturer.
COS-7 Cell Stable Transfectant Cell Lines-- To select for stably transfected COS-7 cell lines, 48 h following transfection with either ps20-2bpBKCMV-lac DNA or pBKCMV-lac control plasmid DNA cells were split 1:15 and replated in COS-7 cell medium containing 600 µg/ml G418 (Sigma). Cells were grown in G418-containing medium for 3 weeks when pools of G418-resistant colonies were collected. Clonal cell lines were obtained by serial dilution in 96-well plates and assayed for ps20 expression by Western analysis of the conditioned medium. Once established, stable cell lines were maintained in 400 µg/ml G418-containing medium (DMEM-10/G418).
Crystal Violet Staining-- Cells were grown to desired density and then incubated with a solution of crystal violet (0.5% (w/v) crystal violet, 3.2% formaldehyde, 0.17% (w/v) NaCl, 22% ethanol overnight, washed three times with H2O, allowed to dry, and photographed.
[3H]Thymidine Incorporation Assays--
Cells were
seeded in a 150-µl volume in 96-well plates at a density of 5.0 × 104 cells/ml (PC-3) and 2.5 × 104
cells/ml (COS-7-derived cell lines), and allowed to attach overnight. Sample was added in triplicate in a 50-µl volume (if applicable) and
allowed to incubate an additional 24 h. Cultures were pulsed with
10 µl of 2 µCi/ml [3H]thymidine (Amersham) during the
final 3 h of incubation, fixed, and processed as described
previously (27). To assay for ps20 activity in the conditioned medium
(CM) of transfectant lines, cultures were seeded in a 5-ml volume in
25-mm2 flasks at 1 × 105 cells/ml in
standard medium, CM collected after 24 or 48 h, and 100 or 200 µl of CM added to pBKCMV-4 cells and cultures assayed as described
above. All assays were performed in triplicate and each experiment
repeated multiple times (n 3). Data are presented as
mean ± S.E. (error bars) and significance determined by
Student's t test.
Immunoprecipitation-- 75-mm2 flasks of PS-1 and U4F cells, grown in Bfs medium, were allowed to reach post-confluence. Cells were then incubated in M medium for 6 h then for 24 h in 4 ml of medium M made with DMEM lacking Cys, Met, and glutamine (ICN, Costa Mesa, CA), 2 mM L-glutamine, and 0.6 mCi of 35S (ICN, Boston, MA). 300 µl of 35S-labeled medium was incubated overnight with an equal amount of RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) and 10 µl of ps20ab-1 or 50 µl preimmune sera followed by a 1-h incubation with 50 µl of Protein A-Sepharose (Pharmacia). Beads were washed four times in 500 µl of RIPA buffer, resuspended in SDS sample buffer with or without 100 mM dithiothreitol, and resolved by 12% SDS-PAGE. The gel was fixed for 30 min in 30% methanol with 10% acetic acid, incubated for 20 min in Enlightning (NEN Life Science Products), dried, and exposed to X-OMAT AR film (Kodak) for 12-48 h.
Expression and Purification of rps20-His--
PCR primers, P69
and P60, were used to synthesize a modified 585-nt fragment of the ps20
coding region for cloning into the bacterial expression vector, pET23c
(Novagen, Madison, WI), a vector that allows incorporation of a His tag
(six consecutive histidine residues) at the carboxyl terminus. At the
amino terminus, the secretory peptide (amino acids 1-26) was replaced
with a Met-Ala and the fifth codon was modified to optimize for
bacterial expression. Primer P69 (nt 117-153; 5'-C TCT cat
atg GCT ACT TGG GAA GCA ATG TTG CCG
GTC-3') includes an NdeI site (lowercase) containing an internal ATG start site. Nucleotides modified from ps20 cDNA
sequence are underlined. Primer P60 (nt 672-697; 5'-C TCC
gaa ttc TGG AAA GTG CCT CTG T-3'), contains an
EcoRI site (lowercase) immediately following the final amino
acid for cloning into pET23c. Because the EcoRI site in the
pET23c vector is out of frame with the His tag, the vector was modified
by deleting the four-nucleotide SacI 3' overhang downstream
of the EcoRI site; the entire construct was digested with
SacI, digested with T4 polymerase in the presence of 100 µg/ml dNTPs at 37 for 10 min, re-ligated, and confirmed by
sequencing. The ps20 cDNA clone ps20-2bpBKCMV (1 ng) was used as
template using standard PCR procedures with primers P69 and P60 to
amplify a PCR product that was cloned into the T-tailed vector, pCRII.
The insert from one clone, rps20pCRII, confirmed by automated
sequencing, was excised by a NdeI/EcoRI double
digest and ligated with NcoI/EcoRI-digested
pET23c. An insert-containing clone, rps20-1pET23c, was confirmed by
sequencing and transformed into the BL21(DE3)pLysS bacterial expression
cell line (Novagen). 500-ml cultures were grown to OD600 = 0.6, protein expression induced by 0.4 mM
isopropyl-1-thio--D-galactopyranoside for 3 h, and
soluble cell extracts generated by sonication in SB buffer (50 mM phosphate, 300 mM NaCl, pH 8.0) on ice
followed by centrifugation at 15,000 × g for 20 min.
Soluble recombinant rat ps20 His-tagged protein (rps20-His) was
purified on a nickel-charged resin (Ni+-NTA, Qiagen). The
column was washed and eluted with an imidazole gradient, as recommended
by the manufacturer. Fractions containing rps20-His with apparent
>95% purity were pooled and dialyzed to remove imidazole.
Cloning, Expression, and Purification of GST-rps20-- The rps20pCRII clone was modified by cloning the oligonucleotide 5'-CATAcGGATCCCTCGAGCATATG-3' into the NdeI site providing an artificial ATG prior to nucleotide 129 (amino acid 26) of the rps20 cDNA sequence. The lowercase nucleotide represents a mutation in the 5' NdeI site, and the underlined nucleotides represent BamHI and XhoI sites, respectively. This clone, rps20BXpCRII, was digested with BamHI and EcoRI and the 647-base pair fragment purified on a 0.8% agarose gel by the following procedure. The 647-base pair fragment was run onto DE-81 paper (Whatman) and incubated overnight at 25 °C in 500 µl of 0.5 × TBE, 1.25 M NaCl. The paper was then filtered through a 0.45-µm filter, and the DNA was ethanol-precipitated and resuspended in dH2O. This rps20 fragment was then cloned into the BamHI and EcoRI sites in the pGEX2TK vector (Pharmacia) (39), containing a thrombin cleavage and cAMP-dependent protein kinase phosphorylation site, creating the vector rps20pGEX2TK.
To purify GST-rps20 or GST protein preparations, a 1-ml overnight bacterial culture of rps20pGEX2TK or pGEX2TK in XA90 bacteria was used to inoculate 100 ml of fresh LB plus ampicillin (50 µg/ml) at 37 °C. When the culture reached an OD600 = 0.5 M, 1 mM isopropyl-1-thio-Western Analysis--
CM from transfected COS-7 cells and from
U4F cells were prepared identically for Western analysis. U4F cells
were seeded in Bfs medium and COS-7 stable transfectants seeded in
DMEM-10/G418. Both were switched to serum-free, fully defined medium M
(MCDB-110, ITS, 0.5 µg/ml testosterone, 0.1 µg/ml epidermal growth
factor, 100 units/ml penicillin, and 100 µg/ml streptomycin) (40).
CM-M was collected every 48-72 h, clarified by centrifugation, and stored at 20 °C until use. Proteins from CM were precipitated with
ammonium sulfate (0-45% saturation) for 3 h at 4 °C,
centrifuged at 9.5 × g for 20 min at 4 °C, and
pellets resuspended in 0.5 ml of 1 M acetic acid. Samples
were dialyzed 12-16 h against 1 M acetic acid in
Spectra/Por Mr 3500 dialysis tubing (Spectrum Medical Industries, Inc., Houston, TX), dried by Speed Vac (Savant Instruments), and resolubilized in 1 × SDS Laemmli sample buffer containing (700 mM)
-mercaptoethanol at 37 °C.
Bacterial cell extracts and purified protein were mixed with 3 × SDS-sample buffer with or without 700 mM
-mercaptoethanol or 100 mM dithiothreitol. Samples were
boiled at 95 °C for 10 min, and resolved by 0.75-mm or 1.5-mm 15%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) following the
procedures of Laemmli (41), as described previously (27).
Immunohistochemistry--
Two-month-old and 6-month-old male
Sprague-Dawley rats were sacrificed (intact and 2-week and 8-week
castrates), and tissues removed and fixed in 10% neutral buffered
formalin overnight. Fixed tissues were dehydrated in a graded series of
ethanol washes, embedded in paraffin, cut into 5-µm-thick sections,
applied to poly-L-lysine-coated slides, and baked at
37 °C prior to staining. Tissue sections were deparaffinized by
immersion in Hemo D (Fisher) twice for 10 min each; rehydrated by 5-min
graded washes in 100%, 95%, and 70% ethanol; permeabilized by
immersion in 1 × PBS, 0.1% Triton X-100 for 5 min; and treated 5 min with 3% hydrogen peroxide (H2O2) to
neutralize endogenous peroxidase activity. Conditions for primary
antibody incubations were determined empirically. Affinity-purified
ps20ab-1 was incubated with tissue sections at a 1:4 dilution overnight
at room temperature in a humid chamber. Negative controls included
affinity-purified ps20ab-1 preabsorbed with peptide, preimmune sera
(1:18.9), and secondary antibody alone. Mouse monoclonal antibody
against the smooth muscle-specific isoform of -actin (clone asm-1,
Boehringer Mannheim) was incubated at a 1:4 dilution for 1 h at
room temperature. Immunoreactivity was visualized by a 45-min
incubation with either biotinylated goat anti-rabbit or goat anti-mouse
secondary antibodies (Sigma), diluted 1:15; followed by a 30-min
incubation with ExtrAvidin-conjugated peroxidase (Sigma), diluted 1:15;
and concluding with a 7-min incubation with diaminobenzadine and
mounting on a glass slide with Gel Mount.
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RESULTS |
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Cloning of ps20 cDNA--
The amino-terminal amino acid
sequence of ps20 was previously determined and reported (27) (see
"Experimental Procedures"). Degenerate ps20 primers, P34
(corresponding to amino acids 1-9) and P38 (corresponding to amino
acids 19-27) (primer sequences given under "Experimental
Procedures"), were designed from this amino acid sequence and used in
a nested PCR strategy in combination with vector primers to amplify a
fragment of ps20 cDNA from the PS-1_BfsZAP cDNA library, a
cDNA library developed from the PS-1 adult rat prostate smooth
muscle cell line (30). Degenerate ps20 primer P38 was used in a primary
PCR amplification with vector primer T7 to amplify a product from PS-1
cDNA library phage lysate. This primary amplification reaction was
used as template for a secondary PCR amplification with primer P38 and
degenerate ps20 primer P34 to yield a single 81-bp reaction product
(Fig. 1A). To confirm that the
81-bp fragment was derived from ps20 sequence, two additional ps20
degenerate primers, P36 (amino acids 1-7) and P37 (amino acids 7-13),
were generated and used independently in combination with P38 to
amplify expected 81-bp and 63-bp fragments, respectively, in analogous
nested PCR reactions. The 81-bp fragment produced by P34 and P38 was
cloned into the pCRII T-tailed vector (clone 3438pCRII), and sequenced
(Fig. 1A). Clone 3438pCRII sequence (primer sequence
omitted) was: 5'-GGTCAGGCTGGCTGAGAAGTCCCAAGCTGAAGAG-3'. The
deduced amino acid sequence of clone 3438pCRII matched directly with
native ps20 amino acid sequence (27). The clone 3438pCRII was therefore
assumed to be a fragment of the DNA sequence encoding the amino
terminus of mature ps20 protein.
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Analysis of Endogenous ps20 Secreted by U4F and PS-1 Cells-- Prior to expression of recombinant ps20 protein, CM from U4F cells (the cell line from which ps20 was identified) (25-27) was analyzed by Western analysis using ps20ab-1. CM from U4F cells was collected, partially purified (as described under "Experimental Procedures"), and analyzed by Western analysis under reducing conditions with ps20ab-1. Two specific bands of apparent molecular mass 21 and 27 kDa were specifically recognized by ps20ab-1 (Fig. 5A, lane 1), relative to preabsorbed control (Fig. 5A, lane 2).
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Expression of Recombinant ps20 Protein-- To further clarify the molecular forms of ps20, the ps20 open reading frame without signal peptide was subcloned into a bacterial expression vector and expressed as a carboxyl-terminal histidine (His)-tagged fusion protein (rps20-His) in bacteria. A protein of 29 kDa was observed by SDS-PAGE under reducing conditions in induced cells (Fig. 5C, lane 2). Western analysis with ps20ab-1 (ps20, amino-terminal) and anti-His antibody (His tag, carboxyl-terminal), demonstrated specific immunoreactivity of both antibodies with the 29-kDa protein (Fig. 5C, lanes 3 and 4, respectively), confirming intact amino and carboxyl termini. rps20-His protein was purified by nickel affinity chromatography, resolved by 15% SDS-PAGE under both reducing and non-reducing conditions, and visualized by silver staining. Consistent with immunoprecipitation of endogenous protein (Fig. 5A), rps20-His migrated at an apparent molecular mass of 29 kDa under reducing conditions (Fig. 5C, lane 5) and at 22-24 kDa (Fig. 5C, lane 6) under non-reducing conditions. These data confirm the apparent molecular mass of ps20 resolved by SDS-PAGE is sensitive to reducing agents. In addition, these data suggest that the bacterial and mammalian expressed forms of ps20 are equivalent in their structural properties as resolved by SDS-PAGE. Given the presence of the WAP four-disulfide core domain, it is not surprising that ps20 in its folded, non-reduced (21-23 kDa) form exhibits a more rapid migration in SDS-PAGE as compared with the reduced (27-29 kDa) form.
To demonstrate the expression of the full-length ps20 cDNA clone in eukaryotic cells and secretion of the recombinant protein, COS-7 cells were transfected with ps20 cDNA or vector control. Both transiently transfected cultures and stable transfectant clonal lines were established. CM from both transiently and stably transfected COS-7 cells was collected, processed, resolved by reducing SDS-PAGE, and analyzed by Western analysis with ps20ab-1. As expected under reducing conditions, the 27-kDa protein was detected in the CM of COS-7 cells transfected with ps20 cDNA (Fig. 5D, lane 2) but not in CM of mock-transfected COS-7 cells (Fig. 5D, lane 1). All ps20 stable transfectant line CM was screened for ps20 secretion with identical results (data not shown). These data confirm that COS-7 cells transfected with ps20 cDNA express and secrete an immunoreactive protein of molecular mass consistent with endogenous ps20 secreted by U4F and PS-1 cells (Fig. 5, A and B).Biological Activity of Recombinant ps20 Protein-- As reported previously for ps20/UGIF-treated cells (25-27), growth inhibition of the ps20 transfectant cell lines was measured using [3H]thymidine incorporation assays. Four ps20 stable transfectant COS-7 cell lines (rps20-1, rps20-3, rps20-4, and rps20-6pBKCMV COS-7), one mock-transfected cell line (pBKCMV-4 COS-7), and the parent cell line (COS-7) were seeded at identical densities, and the rate of [3H]thymidine incorporation by each cell line measured daily over a 4-day time course (Fig. 6A). The rate of [3H]thymidine incorporation by the ps20 transfectant lines was significantly reduced (p < 0.01) relative to the mock transfectant line and the COS-7 parent line at each time point assayed. Consistent with previous data indicating that ps20-induced inhibition of [3H]thymidine incorporation correlated directly with cell number (27), direct cell counting of these cultures indicated 14-45% fewer cells in cultures of the ps20 transfectant cell lines relative to the mock transfectant line when counted 24-96 h after seeding (data not shown). To demonstrate that the growth inhibition of the ps20 transfectant COS-7 cell lines was due to an activity secreted into the growth medium, mock transfectant cells were incubated with CM from ps20 transfectant or mock cell lines and assayed for [3H]thymidine incorporation. Incorporation of [3H]thymidine in cultures incubated with CM from ps20 transfectant cell lines was reduced (p < 0.01) relative to cultures incubated with CM from mock transfectant cells (Fig. 6B).
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Localization of ps20 Protein in Vivo--
Immunohistochemical
analysis with affinity-purified ps20ab-1 and adult rat tissues was
performed to assess ps20 protein expression patterns in specific cell
types. In adult rat prostate, specific immunoreactivity was apparent
only in the periacinar smooth muscle cells of the stroma (Fig.
8c), as determined by
comparison of a serial section probed with a monoclonal antibody
recognizing the smooth muscle-specific isoform of -actin (SM
-actin), a smooth muscle cell marker (Fig. 8d). The
smooth muscle staining pattern was eliminated by preabsorption of
ps20ab-1 with ps20 peptide-(1-14) (Fig. 8b). No
immunoreactivity was observed in the absence of primary antibody, and
only nonspecific background reactivity was observed with preimmune sera
(data not shown). No immunoreactivity was observed in the stromal
fibroblast population. A weak immunoreactivity, noted in some
epithelial cells, was eliminated by preabsorption with ps20 peptide.
ps20 was detected in smooth muscle of both normal and castrate animals,
with an apparent moderate increase of immunoreactivity noted in the
smooth muscle of castrate animals (data not shown).
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DISCUSSION |
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We report here the cloning of full-length cDNA encoding ps20, expression of biologically active recombinant ps20 protein, and characterization of ps20 mRNA and protein expression patterns in vivo. The cloning of ps20 from the PS-1 (prostate smooth muscle-like cell line) cDNA library and immunolocalization of ps20 expression in smooth muscle in vivo is consistent with the previous characterization of ps20 secretion by prostatic mesenchymal and smooth muscle cell lines in culture (27). We have demonstrated here that both bacterial and mammalian expressed recombinant ps20 protein exhibit growth-inhibitory effects on mammalian cells in culture, consistent with properties reported for UGIF/native ps20 protein (25-27). The ps20 amino acid sequence contains a whey acidic protein (WAP)-type four-disulfide core motif (29), which places ps20 in this family of small secreted proteins.
Biological activity of ps20 is consistent with the biological activity of other WAP family proteins, the majority of which have protease inhibitor activity. Protease inhibitors have been reported previously to have growth-inhibitory effects both in vitro and in vivo. Pefabloc, a synthetic serine protease inhibitor, inhibited prostatic PC-3 carcinoma cell proliferation through inhibition of an IGFBP-3 protease that regulated availability of active insulin-like growth factor-II (12). Other serine protease inhibitors, including maspin (50), plasminogen activator inhibitors (51), and tissue inhibitors of matrix metalloproteinases (52), have exhibited growth-inhibitory activities. The WAP family member elafin inhibited abnormal vascular smooth muscle proliferation in an in vivo rabbit arteriopathy model of coronary neoinitimization (12). In vitro, the mechanism for elafin-induced inhibition of pulmonary artery smooth muscle cell proliferation involves inhibition of elastase-mediated proteolytic release of mitogenic biologically active FGF-2 from extracellular matrix (12).
Members of the WAP signature motif family exhibit fundamental roles in growth control, differentiation, and tissue remodeling in development and maintenance of homeostasis in adult tissue. The Kallmann syndrome protein is a peripheral membrane protein, which is proteolytically released in soluble form and may be a neuronal axon chemoattractant (53). Mutations or deletions in the Kallmann syndrome protein lead to defective embryonic migration and differentiation of olfactory and gonadotrophin-releasing hormone-synthesizing neurons in development, resulting in the hypogonadotropic hypogonadism and anosmia of Kallmann syndrome (49). Expression of elafin is down-regulated in poorly differentiated squamous cell carcinoma (48) and in mammary carcinoma cells (54), and its loss of expression has been postulated to facilitate tumor progression in these tissues. WDNM1 was identified in a subtractive hybridization screen to identify potential tumor suppressor genes in mammary cancer (47, 55) as a transcript down-regulated in metastatic breast cancer cell lines. Although the significance of ps20 expression in vivo is not yet known, alterations in the expression of other WAP family members clearly influence growth control in vivo.
We initially identified ps20 protein as the major protein component of the mesenchymally produced UGIF activity, which has a growth-inhibitory effect on epithelial cell lines in vitro (25-27). We now report that ps20 localizes to the periacinar ring of smooth muscle in immediate contact with the secretory epithelial acini in the prostate gland. Whether or not the weak immunoreactivity observed in epithelial cells represents a low level of expression by these cells or stromally produced ps20 incorporated by these cells is not yet clear. The in vivo localization of ps20 in smooth muscle supports a putative role for ps20 in stromal-epithelial interactions in the prostate gland.
The significance of the apparent differential expression of ps20 by different types of smooth muscle cells is unknown, but is evident at both the mRNA and protein levels. Expression of ps20 message and protein was relatively low in many organs containing visceral smooth muscle, including the stomach and small and large intestines, which have thick walls of smooth muscle layers. Of interest, the staining intensity of ps20 in vascular smooth muscle, particularly in both larger elastic arteries and small muscular arteries, was consistently high and similar to or higher than in prostate smooth muscle. The elevated mRNA expression observed in heart tissue by Northern analysis is not likely due to expression by cardiac muscle cells, which were not immunoreactive, but is likely due to high expression by coronary artery and large elastic artery smooth muscle cells, which were highly immunoreactive with ps20ab-1. Vascular diseases are typified by medial hypertrophy and neointimal proliferation. The localization of ps20 in vascular smooth muscle suggests ps20 may play an autocrine role in regulation of smooth muscle proliferation and maintenance of homeostasis in the vasculature.
The specific mechanisms of ps20 biological action and functional role in vivo are not yet known. ps20 could potentially inhibit cell proliferation through alterations of growth factor bioavailability, extracellular matrix remodeling, or alterations of cell adhesion. Based on our data to date, no specific ps20 mechanism can be ruled in or ruled out. Definitive characterization of ps20 mechanisms of biological action awaits a more complete functional characterization of activity in vitro and in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeff Rosen for helpful discussions and Liz Hopkins for excellent technical assistance in preparation of tissue sections for immunohistochemical analysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA58093, DK45909, and SPORE CA58204 and by a grant from Sheffield Medical Technologies.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) AF037272.
¶ To whom requests for reprints should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6220; Fax: 713-790-1275; E-mail: drowley{at}bcm.tmc.edu.
1 The abbreviations used are: UGIF, urogenital sinus mesenchymal secreted growth-inhibitory factor; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CM, conditioned medium; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; SM, smooth muscle; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.
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