Purification of a novel protein (ps20) from urogenital sinus mesenchymal cells with growth inhibitory properties in vitro.

Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interactions in the prostate gland.

The induction of epithelial cell growth and differentiation patterns by adjacent mesenchymal cells is a common feature in the organogenesis of many tissues. Such interactions occur in reproductive tissues including seminal vesicle development (1,2), in the androgen-induced regression of male mammary gland (3), and in estrogen-induced proliferation of mammary gland epithelial (4), vaginal epithelial, and uterine epithelial cells (5). Stromal-epithelial interactions have been particularly well studied in the morphogenesis of fetal urogenital sinus (UGS) 1 to mature prostate gland (6,7). UGS epithelial cells progress through a specific morphogenesis pattern (prostatic glandular acini) only when recombined with UGS mesenchymal cells (8)(9)(10). Similar studies addressing the development of differ-entiated epithelium in skin (11), gut (12), and lung (13) have shown a likewise potent inductive nature of stromal cells in directing tissue-specific epithelial growth and differentiation patterns.
In postnatal and fully differentiated adult tissues, stromalepithelial interactions likely maintain histological architecture and differentiated phenotype relative to ongoing modeling and remodeling processes (6). Fully differentiated adult epithelial cells are capable of responding to a heterotypical (different) stroma. In this regard, adult bladder transitional epithelium can be induced by urogenital sinus mesenchyme to change to a prostate-specific epithelial cell phenotype including the expression of prostate-specific proteins, including androgen receptor (14,15). In addition, abnormal patterns of epithelial cell proliferation and differentiation in neoplastic disease progression are also affected by the origin and type of adjacent stroma. The growth of prostatic Dunning tumor adenocarcinoma was inhibited by 7-fold, and the morphology was altered to a more normal phenotype when recombined with UGS mesenchyme and grown in vivo (16). Similarly, the recombination of normal UGS mesenchyme with bladder transitional cell carcinoma resulted in a change of tumor histopathology to an adenocarcinoma phenotype (17). Moreover, the implantation of UGS mesenchyme directly into the adult mouse prostate gland resulted in an induced hyperplastic phenotype typical of benign prostatic hyperplasia (18). These studies together support the suggestion that stromal-epithelial interactions are likely to be central in modulating the progression and histopathology of epithelial cells in proliferation-and differentiation-related diseases such as cancer and benign hyperplasia (19).
Although stromal cell induction of epithelium is critical for organogenesis and growth control in adult tissues, little is understood regarding paracrine effector molecules, fundamental molecular mechanisms of stromal-epithelial interactions, and the basic biology of UGS mesenchymal cells. Indeed, there is currently no well defined set of parameters or markers to clearly identify the stages of mesenchymal cell differentiation to adult stromal cells (fibroblasts, myofibroblasts, or smooth muscle), or the specific roles of these cell types in continued stromal-epithelial interactions. To identify putative mediators and markers of UGS mesenchymal cell action, we have reported previously the development of organ cultures and mesenchymal cell lines from fetal rat urogential sinus (20)(21)(22). Urogential sinus mesenchymal cell lines (U4F and U4F1) were adapted into chemically defined medium and characterized for stromal marker proteins (21,22). Initial studies identified in U4F cell-conditioned medium, a growth inhibitory activity to PC-3 prostatic carcinoma epithelial cells, NBT-II bladder epithelial cells, and Mv-Lu-1 mink lung epithelial cells in vitro (20,21). This activity did not cross-react with a battery of neutralizing antibodies to cytokines (including transforming growth factor-βs, interferons, and interleukins) and was provisionally termed urogenital sinus-derived growth inhibitory factor (UGIF) pending identification of biologically active components (21). Crude UGIF activity acted to inhibit epithelial cell proliferation in a linear and saturable manner, stimulate synthesis of secretory proteins, and alter epithelial phenotypic morphology in vitro, and it was nontoxic/reversible in mechanisms of action (20,21). Gel filtration chromatography analyses of crude UGIF activity showed a consistent elution pattern (peak of activity) assigned to the 18-22-kDa calculated size range (21). Based on additional physicochemical, biological, and immunological properties, UGIF activity could not be ascribed to a previously identified protein or factor (20,21). The present study was conducted to characterize, identify, and purify the protein(s) primarily responsible for the previously described UGIF activity and characterize biological activity in vitro.
We report here the identification and purification of a 20-21-kDa protein secreted from U4F mesenchymal cells in chemically defined conditioned medium, having biological properties identical to the previously characterized UGIF activity. Based on the cell type of origin and the biological activity in vitro, this protein may be relevant to growth and differentiation mechanisms of stromal-epithelial interactions and may serve as a useful marker for the study of mesenchymal cell ontogeny. cutoff = 3,500) from Spectrapore (Los Angeles, CA); polyvinylidene difluoride (PVDF) Immobilon-P membranes from Millipore (Bedford, MA); and Pro-Blott membranes from Promega (Madison, WI).

Materials
Cell Culture -The U4F urogenital sinus mesenchymal cell line, derived originally from UGS organ explants (20), was cultured in medium Bfs (90% DMEM, 5% fetal bovine serum, 5% Nu-Serum, 5 µg/ml insulin, 0.5 fig/ml testosterone, 100 units/ml penicillin, and 100 µg/ml streptomycin), which was replaced every 48 h as previously reported (21). Upon confluence, U4F cultures typically formed multicellular spheroids (multicellular, spherical domes) after 20-30 days in culture, with UGIF activity first detectable in conditioned medium from spheroid-containing cultures (21). To generate sufficient quantities of conditioned medium for analysis of UGIF activity, 75-cm 2 culture flasks were seeded with U4F cells yielding 100-200 spheroids per flask by 30 days in culture. U4F spheroid cultures were maintained for approximately 3-6 months in this manner with conditioned media collected every 48 h, pooled, quick frozen, and used as starting material for analysis of UGIF activity. For preparative purification of UGIF protein(s), spheroid cultures were switched to serum-free, chemically defined medium M 2 (MCDB-110 basal medium plus 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 0.1 µg/ml epidermal growth factor, and 0.5 µg/ml testosterone), formulated empirically to support U4F proliferation and secretion of UGIF activity as reported elsewhere (21). Conditioned medium was collected every 48 h, clarified by centrifugation, and either used immediately, or stored at -20 °C until subsequent use.
Cell Proliferation Assays -Cell proliferation was determined by direct cell counting of viable cells and with a [ 3 H]thymidine incorporation assay following minor modifications of procedures we reported previously (20)(21)(22)(23). For the [ 3 H]thymidine incorporation assay, PC-3 or Mv-Lu-1 target epithelial cells were seeded at 8.1 X 10 3 cells/148 µl/well in 96-well plates and allowed to attach for 24 h. Wells received a 52-µl aliquot of sample to be tested (previously vacuum dried and resolubilized in growth medium in sterile conditions) (200 µl/well final total volume) and were allowed to incubate for an additional 24 h. Cultures were pulsed with [ 3 H]thymidine (2 µCi/ml) during the final 3 h. of incubation. The assay was terminated by fixing cell monolayers in situ with methanol:acetic acid (3:1) (200 µl/well) for 10 min (22 °C), washing with 100% methanol (5 min, 22 °C) followed by a wash with 5% trichloroacetic acid (5 min, 22 °C), and a three times sequential wash with methanol (22 °C, 200 µl/well). Plates were then allowed to air dry for 5-10 min under a heat lamp. Plates could either be stored indefinitely at 22 °C or processed immediately. For processing, monolayers were hydrolyzed by 1 N NaOH (200 µl/well, 5 min, 22 °C), and aliquots (180 µl) were added to scintillation vials containing 180 µl of 1 N HC1 to neutralize pH. Radioactivity was determined by scintillation counting. Results are presented as the reciprocal of incorporated counts/min (1/cpm) where indicated in the figure legends, to reflect inhibition of [ 3 H]thymidine incorporation as a peak of activity. Results from all comparative assays are presented as the mean of n 3 tests ± S.E. (error bars), and results were analyzed for significance using Student's t test.
For direct cell counting, PC-3 cells were seeded at 4.0 X 10 3 cells/148 µl/well in 96-well plates and allowed to attach for 24 h. Wells received 52-µl aliquots of test sample and were allowed to incubate for 5 days. Cultures received fresh medium plus/minus test sample every 48 h. At each 24-h interval, cells were released by exposure to 0.25% trypsin, 0.025% EDTA in calcium, magnesium-free Hanks' solution (30 µl/well) for 4 min. To each well were added 70 µl of PC-3 cell growth medium plus 7% v/v fetal bovine serum, 40 µl of the cell suspension were incubated with trypan blue (4 min, 22 °C), and total and viable cells were counted as described previously (20).
Protein Synthesis Assay -Protein synthesis was assayed by incorporation of [ 35 S]methionine according to procedures published previously (20). PC-3 cells were seeded and processed identically to the cell counting procedure. On day 2-4 of exposure to test samples, wells received [ 36 S]methionine (10 µCi/ml) for 24 h. The medium (200 µl) was harvested and added to an equal volume of 20% trichloroacetic acid for 1 h at 2 °C to precipitate proteins. Aliquots (350 µl) from each sample were added to glass fiber filters (934-AH, 2.4 cm, Whatman) fitted to a filter vacuum manifold (Millipore). Filters were washed sequentially three times with 2 ml of EtOH (-20 °C) to remove unincorporated label, filters were added to scintillation vials, and incorporated radioactivity was determined by scintillation counting. Cell number was counted from each corresponding set of wells using the direct cell counting method, and results were expressed as disintegrations/min [ 35 S]methionine incorporation/10 4 cells to standardize results to cell number.
Ion Exchange Chromatography -Samples were dialyzed (Spectrapore no. 3, M r cutoff = 3,500) with 20 mM ammonium carbonate buffer (pH 8.85, 4 °C) overnight, and applied directly to a DE-52 anion exchange column (1.5 X 9.5 cm, hydrostatic pressure = 30 cm) equilibrated in the same buffer. The column was washed with 3 bed volumes of the same buffer and eluted with a linear gradient of ammonium carbonate (20-300 mM, pH 8.89, 150 ml) and 5 ml/fraction collected. Absorbance at 280 nm (A 280 ) and conductivity were determined for each fraction. Aliquots (25 µl) of each fraction were directly vacuum-dried in sterile microcentrifuge tubes and resolubilized in sterile growth medium Bfs (65 µl), and 52-µl aliquots/well were assayed directly for PC-3 cell growth activity using the [ 3 H]thymidine incorporation assay.
Gel Filtration Chromatography -For analytical studies to assign a protein species to the peak of biological activity, 50-200 ml of U4F conditioned medium Bfs (serum-containing) were dialyzed against 1 M acetic acid (pH 2.25) and lyophilized. Lyophilized proteins were resolubilized in 1 M acetic acid (1-3 ml) and chromatographed through a Bio-Gel P-200 or P-100 gel filtration column (2.5 X 70 cm), equilibrated in 1 M acetic acid, by gravity flow (45 cm), and 3-ml fractions were collected. Aliquots (100 µl) were vacuum-dried and resolubilized in 65 µl of Bfs growth medium, and 52-µl aliquots were added to PC-3 cells for [ 3 H]thymidine incorporation assay. For additional analytical studies and for scaled-up preparative purification of ps20, samples derived from serum-free, chemically defined conditioned medium M 2 were processed through the ion exchange chromatography step as described. Pooled fractions from the biologically active peak were prepared by dialysis (Spectrapore no. 3 tubing, 3,500 M r cutoff) against 4 liters of 1 M acetic acid (pH 2.25) overnight at 4 °C. Dialyzed samples were frozen and lyophilized and either used immediately or stored at -20 °C until use. Lyophilized samples were solubilized in 1 M acetic acid (1 ml) and applied to either a Bio-Gel P-100 column or P-30 column (1.4 X 70 cm) equilibrated in 1 M acetic acid (pH 2.25). Proteins were eluted with a hydrostatic pressure of 55 cm, and 1.4-ml fractions were collected. Aliquots (100 µl) from each fraction were vacuum-dried in sterile microcentrifuge tubes and resolubilized in 65 µl of medium Bfs, and 52-µl aliquots were added to PC-3 cultures for [ 3 H]thymidine incorporation assay. Reverse Phase High Performance Liquid Chromatography -All samples for HPLC were first chromatographed through either ion exchange chromatography and/or gel filtration chromatography prior to HPLC analysis. Samples (pooled fractions from bioactivity peak) were either vacuum dried or lyophilized and solubilized in 50% formic acid (0.5 ml). Samples were applied (three consecutive 150-µl applications) to a Waters C-18 reverse phase column fitted to an HPLC system composed of a Waters 712 WISP automatic injector, and Beckman 412 controller, 110A pumps, and 165 variable wavelength detector. Proteins were eluted with a 1.0 ml/min/fraction flow rate with 0.1% trifluoroacetic acid in a linear acetonitrile gradient as indicated in the text. Aliquots (100 µl) from each fraction were vacuum-dried, resolubilized in 65 µl of medium Bfs, and 52-µl aliquots added to either PC-3 cells or Mv-Lu-1 cultures for the [ 3 H]thymidine incorporation assay.
SDS-PAGE Electrophoresis -Samples were vacuum-dried, resolubilized in Laemmli sample buffer (24) and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) following the procedures of Laemmli (24). Samples were heated (5-10 min, 95 °C), 60-100-µl sample aliquots loaded per lane, and electrophoresed through the stacking gel at 40 mAmp and through the running gel (15% acrylamide) at 70-mAmp continuous current. Proteins were visualized using silver staining procedures as described previously (22). Protein determinations of highly purified samples below detectable range with Bradford analysis (<1 µg) were determined by comparative analysis of sample lanes with sets of protein standards 50-500 ng/lane in SDS-PAGE gels and an assignment was made based on relative staining density after silver staining. For renaturation of activity after SDS-PAGE, gels were washed in double-distilled water, protein standards position was determined by incubation in 1 M KC1 at 4 °C for visualization of opaque bands, and protein bands of interest were precisely excised in 1-2-mm slices. Excised proteins were renatured following minor modifications of the guanidine-HCl method (25) or were directly eluted from gels with 1 M acetic acid overnight (agitation, 22 °C), vacuum-dried, and processed for biological assay as described.
Protein Sequencing -Substantially purified ps20 protein samples were electrophoresed through SDS-PAGE gels as described, except 100 mM thioglycolic acid was added to the cathode buffer, and gels were preelectrophoresed for 20 min at 50 V to reduce amino-terminal blockage. The gel was incubated in transfer buffer (25 mM Tris-base, 192 mM glycine, pH 8.5) for 5 min and assembled with Immobilon-P or Pro-Blott PVDF membranes in a Bio-Rad Trans-Blot cell apparatus according to the manufacturer's recommendations. Proteins were electroblotted using 30 V for 18 h at 4 °C. Proteins were visualized by Coomassie staining, the ps20 protein bands were excised, and the sequence was determined using Applied Biosystems 477A and 773A protein sequencers.

RESULTS
Assignment of UGIF Activity to a Protein Species -Initial studies were conducted to determine whether a putative protein species or set of proteins could be assigned to the peak or peaks of UGIF biological activity in order to assess feasibility of purification and focus on a particular protein for preparative purification. Accordingly, gel filtration chromatography and reverse phase HPLC were used to analyze UGIF activity, and eluted proteins were analyzed with SDS-PAGE. UGIF activity was harvested from U4F mesenchymal cell-conditioned medium following conditions reported previously (20,21). UGIF activity in conditioned medium was first detectable from confluent U4F cultures having small multicellular spheroids, which formed at approximately 25 days of culture, and activity continued to increase with the development of larger U4F cell spheroids as reported previously (21). Identical to previous reports, UGIF activity in CM collected from these long term cultures did not cross-react with neutralizing antibodies to a variety of cytokines including transforming growth factor-βs, interleukin-6, and interferons (21).
Ammonium sulfate precipitation of proteins from conditioned medium was used as a first step to concentrate samples. Approximately 90% of growth inhibitory activity precipitated within the 20-40% saturation range of ammonium sulfate (data not shown), which was then used for subsequent procedures. For analysis of UGIF bioactive proteins, all chromatography buffers were formulated to be both volatile (no salt residues upon vacuum drying) and bacteriostatic (sterile, nonsupportive of bacterial growth) so that aliquots from gel filtration or HPLC columns could be vacuum-dried in sterile vials and used directly for biological assay (addition to target epithelial cells in culture) without additional steps of dialysis and sterilization of sample. This strategy allowed for the biological assay of hundreds of fractions per day. For initial analysis of UGIF biological activity, precipitated proteins from conditioned Bfs medium (conditioned by U4F cells for 48 h) and unconditioned control Bfs medium were resolubilized in ammonium carbonate buffer. The initial chromatography step to analyze biological activity utilized ion exchange chromatography. Samples were dialyzed against 20 mM ammonium carbonate (pH 8.85), applied to and eluted from a DE-52 anion-exchange column according to the methods under "Experimental Procedures." Fig. 1 shows the biological activity and A 280 protein elution profiles. The elution of a major growth inhibitory peak was observed from the 48-h conditioned medium preparations. In comparison, the control (fresh medium) sample did not produce a growth inhibitory peak and exhibited an otherwise similar baseline activity and A 280 elution pattern. These studies indicated the UGIF activity peak was produced by U4F cells and was not a constitutive component of fresh Bfs medium.
Fractions from ion exchange representing the major peak of eluted biological activity (delineated by the bar underlining fractions in Fig. 1) were collected and pooled. The pooled sample was dialyzed against 1 M acetic acid (pH 2.5, 4 liters) overnight at 4 °C. Dialyzed samples were quick frozen, lyophilized, and used either directly or stored at -20 °C. UGIF activity prepared in this manner was further analyzed by gel filtration chromatography for assignment of size using a variety of buffer conditions including 1 M acetic acid, ammonium carbonate, and ammonium acetate. Of these conditions, gel filtration chromatography in 1 M acetic acid reduced the interaction with column matrix optimally and allowed for a reproducible recovery of an activity peak as shown in Fig. 2. Biological activity was detected as a single major peak, eluting consistently in the calculated 18-20-kDa size range (position of molecular mass markers is shown across the top of graph, Fig.  2). SDS-PAGE analysis of eluted fractions (Fig. 2, lower panel) showed the elution pattern of a 20-21-kDa protein to be directly correlated with the elution peak of biological activity (fraction 56, arrow). To further establish the correlation of this protein species with peak activity, additional samples were pooled, chromatographed through C18 reverse phase HPLC columns, and eluted with a linear gradient of acetonitrile as shown in Fig. 3. In direct agreement with gel filtration, the To facilitate the scale up of preparations to allow for the purification of the 20-21-kDa species, a chemically defined (serum-free) growth medium (medium M 2 ) was developed empirically to lower protein complexity in the starting conditioned medium. The M 2 medium supported U4F spheroid growth and production of UGIF activity as described under "Experimental Procedures." To determine whether the 20-21-kDa protein was responsible for UGIF activity in conditioned M 2 medium, increased volumes (up to 600 ml) of conditioned media were prepared, chromatographed through ion exchange chromatography, and analyzed with reverse-phase HPLC as shown in Fig. 4. Biological activity eluted as a major consistent peak and a minor inconsistent peak under these conditions. SDS-PAGE analysis (Fig. 4, lower panel) showed the elution of a 20-21-kDa protein species in a pattern correlating directly with the major peak of biological activity (peak fraction 89, arrow), in agreement with previous results in serum-containing medium. The minor peak of activity was heterogeneous in elution pattern and a protein species could not be assigned. Subsequent analyses focused exclusively on the 20-21-kDa protein.
Purification of ps20 -Subsequent purification procedures utilized conditioned M 2 (chemically defined) medium exclusively and were scaled up to specifically purify the 20-21-kDa protein species. For preparative purification using increased volumes of M 2 conditioned medium ( 600 ml), the initial two steps of ammonium sulfate precipitation and ion exchange chromatography were used (as described in Fig. 1) which pro- duced identical elution patterns of activity. For the third purification step, pooled peak lyophilized material from the ion exchange step was resolubilized in 1 M acetic acid (1 ml) and applied to either a P-100 or P-30 (Bio-Rad) gel filtration column (1.4 X 70 cm) equilibrated in 1 M acetic acid, and each fraction was processed and assayed for biological activity as described under "Experimental Procedures." As observed previously with HPLC of conditioned M 2 medium, biological activity eluted as two separate peak regions when analyzed with either P-100 or P-30 gel filtration chromatography. Shown in Fig. 5 is the elution profile from a scaled-up preparation. The earlier eluting (front) peak was consistently observed in the 20-23-kDa size range (molecular size markers across top of graph), and a second peak of highly variable activity levels corresponded to a lower 9-14-kDa size region. Further SDS-PAGE analysis of eluted fractions (Fig. 5, lower panel) showed the front peak of biological activity consistently (n > 15) corresponded to the elution pattern of the 20-21-kDa protein (arrow, fraction 53). A set of lower molecular mass proteins (8-17 kDa) eluted in patterns correlating to the second, highly variable peak of activity (Fig. 5, fractions 57-63). This variable, secondary peak region of activity was inconsistent from preparation to preparation and was observed only in scaled-up purification procedures from gel filtration and HPLC steps. Proteins or frag- The fourth purification step utilized reverse phase HPLC owing to the utility of HPLC in separating proteins of similar size based on hydrophobic properties. Pooled fractions from gel filtration chromatography (front consistent peak, Fig. 5, fractions 52-55) were vacuum-dried, resolubilized in 50% formic acid, and analyzed with reverse phase HPLC as described under "Experimental Procedures." The column was eluted with a shallow gradient of acetonitrile to produce optimal separation of major peak versus inconsistent minor peak proteins. Fig. 6 shows the biological activity elution profiles and the corresponding SDS-PAGE analysis associated with peak activity. Biological activity eluted as a well defined and consistently observed front peak (fraction 20-23, peak = fraction 22), which was directly correlated with the elution pattern of the 20-21-kDa protein species (Fig. 6, lower panel), purified to near homogeneity as determined by SDS-PAGE analysis and silver staining. The purification procedure as described yielded approximately 600-650 ng of the 20-21-kDa protein from 600 ml of conditioned medium. Subsequent analyses showed a consistent purification to near homogeneity and indicated the 20-21-kDa protein was monomeric in structure as analyzed in either reducing or nonreducing SDS-PAGE conditions. Similar to observations from the previous gel filtration step, a second broad peak or set of peaks (fractions 25-34) of less well defined and highly variable elution patterns and activity levels, eluted at higher acetonitrile concentrations. These inconsistent peaks likely represent variable degrees of 20-21-kDa protein breakdown products, as they were derived from previous step 20- FIG. 4. Identification of the 20-21-kDa protein in serum-free conditioned medium. Upper panel, preparative volumes (>600 ml) of serum-free, chemically defined conditioned medium M2 (U4F cells, 48 h) were processed as described in Fig. 1, chromatographed with a C-18 reverse phase column, and eluted with acetonitrile as described in the text. Aliquots were vacuum-dried and assayed with PC-3 cells as in described in Figs. 1-3. Biological activity eluted as a major peak associated with fraction 89 and a minor, variable peak associated with fraction 96. For microsequence analysis, the 20-21-kDa protein (approximately 700 ng to 1 µg) pooled from multiple purification preparations was electrophoresed through SDS-PAGE gels, blotted to PVDF membranes, and analyzed for amino-terminal sequence as described under "Experimental Procedures." A single sequence was detected with unambiguous assignments made for positions 1-14 and 19-28 as follows: NH2-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-Xaa-Xaa-Xaa-Xaa-Val-Ala-Ala-Thr-Gly-Xaa-Arg-Gln-Pro-His. Analysis with PDB, SwissProt, PIR, SPUpdate, GenPept, and GPUpdate data bases indicated no regions of direct match or homology with previously characterized proteins. The 20-21-kDa protein was hereafter referred to as ps20 (20-kDa prostate stromal protein).
Biological Activity of ps20 -To confirm growth inhibitory activity was endogenous to the ps20 protein, renaturation experiments were conducted with purified ps20 protein extracted from SDS-PAGE gels of samples generated from HPLC steps. Samples purified following the above steps were electrophoresed through an SDS-PAGE gel. The ps20 protein band was precisely excised (1-mm width gel band) as well as control gel bands containing either no loaded protein or protein controls (11-kDa range). The samples were either electroeluted from the gel slices, dialyzed, and renatured following modifications of the guanidine-HCl method (25), or were passively eluted from crushed gel slices in 1 M acetic acid and dialyzed following procedures described under "Experimental Procedures." The ps20 protein renatured with either of these procedures produced significant (p < 0.01) growth inhibitory activity relative FIG. 5. Preparative gel filtration chromatography. Upper panel, the eluted peak of biological activity from ion exchange chromatography using serum-free conditioned medium (48 h, medium M2, preparative volumes, 600 ml) were chromatographed through a P-30 gel filtration column and aliquots assayed for biological activity with PC-3 cells as described in the text and under "Experimental Procedures." Activity eluted in two peaks. An early front peak consistently eluted at fraction 53, in the 18-21-kDa size region (molecular size markers shown across top, as in Fig. 2). A set of secondary, variable and inconsistent peaks eluted in fractions 58-63 as shown. Lower panel, fractions (bar region, upper panel) were analyzed by SDS-PAGE and silver staining. Note the elution pattern of the 20-21-kDa protein (fraction 53, arrow) directly correlated with the front activity peak as shown in the upper panel. The front peak material was pooled (fractions 52-55) and used for reverse phase HPLC (Fig. 6). Molecular size markers are shown in lane 1 and as indicated in Fig. 2. to controls as shown in Fig. 7. Renaturation of ps20 extracted with 1 M acetic acid produced a 15.4% inhibition of [3H]thymidine incorporation over control (Fig. 7A). Guanidine-HCl renaturation of ps20 (Fig. 7B) produced up to a 30.3% inhibition relative to controls. With either procedure in any particular experiment, renaturation efficiency after SDS-denaturation was maximally 15-20% relative to activity levels of samples prior to SDS-PAGE, similar to expected efficiencies of renaturation for proteins extracted and processed from denaturing SDS-PAGE gels (25).
To determine biologically active concentrations, purified ps20 was used for dose-response assays with target PC-3 cells, and exhibited linear and saturable dose-response curve as shown in Fig. 8. Half-maximal activity was observed at 55 ng/ml (2.62 nM) under these conditions. Maximal (saturable) inhibition was observed at 6. Biological activity eluted with a consistently observed front peak (fraction 22) and highly variable secondary peak region (fractions [25][26][27][28][29][30][31][32][33][34]. Lower panel shows the SDS-PAGE analysis and silver staining of proteins in entire eluted fractions (bar region). The elution pattern of the 20-21-kDa protein correlated directly with the initial peak (fractions [20][21][22][23] and was purified to near homogeneity. Molecular size markers (shown in lane 1) are as indicated in Fig. 2. attained confluence at lower cell densities (days 3-5), due to a change in cell shape to a larger, more spread-out phenotype as discussed below. The ratios of viable to nonviable cells were identical between control and ps20 treated cultures indicating the results were not due to increased cell death or toxicity, in agreement with our previous reports using crude UGIF activity (21). The inhibition of cell proliferation was associated with a stimulation of protein synthesis. As shown in Fig. 9B, purified ps20 (7.2 nM) stimulated the synthesis of secreted proteins from PC-3 cells by 210.4% relative to control on a per cell basis. In addition, the ps20-treated (7.2 nM) PC-3 cells assumed a more spread-out cell shape with increased pseudopodia and filopodia cell extensions compared to control cultures (data not shown). The effects of purified ps20 on growth inhibition, stimulation of protein synthesis, and alteration in morphology are each consistent with previous reports on UGIF activity in crude conditioned medium (20,21). Based on these data, the activity ascribed to ps20 protein likely accounts for the previously observed UGIF activity in crude conditioned medium. DISCUSSION Data presented in this study shows the characterization and purification of a novel 20-21-kDa protein (termed ps20) derived from fetal urogenital sinus mesenchymal cells and exhibiting growth inhibitory and protein synthesis stimulatory activities in vitro in a dose-dependent and saturable manner, suggesting responses are mediated through saturable pathways. Ammo-terminal sequence information revealed a unique sequence with no matches to proteins in data bases, indicating ps20 represents a novel protein species. Additional studies beyond the scope of the present report are required to generate probes and determine the specific pattern of ps20 expression in mesenchymal cell ontogeny, the molecular mechanisms of action in stromal-epithelial interactions, and the extent of target FIG. 7. Renaturation of ps20 activity from SDS-PAGE. The ps20 protein isolated by HPLC was electrophoresed through SDS-PAGE, and the ps20 protein band was excised and renatured by the guanidine HC1 method or the gel crushed and proteins extracted with 1 M acetic acid as described in the text and under "Experimental Procedures." Shown in each panel is the inhibition of [3H]thymidine incorporation (1/cpm) in PC-3 cells induced by renatured ps20 (column C) relative to controls (columns A, B, and D) as described below. Panel A, gel extracted in 1 M acetic acid. A, media control; B, extracted gel (no protein) control; C, extracted ps20 protein; D, acetic acid control. Panel B, gel-extracted proteins were renatured with the guanidine HC1 method. A, media control; B, extracted gel control (no protein); C, extracted ps20 protein; D, extracted gel control (arbitrary 11-kDa protein band). Values are mean of n = 3 separate determinations ± S.E. Column C is statistically significant (p < 0.01) in each experiment.
cell-type specificity. Results here report the activity of ps20 with PC-3 (human prostatic carcinoma) and Mv-Lu-1 (mink lung epithelial) assay target cells. Our previous studies with crude preparations showed identical activities with NBT-II (rat bladder epithelial cells) and Y-79 cells (human retinoblastoma cells), in addition to PC-3 and Mv-Lu-1 cells (20,21). The full range of specific cell types responsive to ps20 activities is not yet known and will require greater yields of native ps20 or recombinant protein to address fully.
The in vitro responses elicited by the ps20 protein are consistent with a potential function in stromal-epithelial interactions involving tissue growth and differentiation control in the prostate gland. Extracellular matrices produced by stromal cells, direct cell-cell contact, and secretion of paracrine-acting effectors have each been shown to induce and/or facilitate tissue-specific gene expression and epithelial cell phenotype in a wide variety of tissues. Accordingly, the combined influences of paracrine factors, matrix molecules, and direct cell-cell communication each are likely to contribute to the overall mechanisms of stromal induction of epithelial phenotype. The actions of ps20 may be a component of any such mechanism. Although   FIG. 8. Dose-response of purified ps20 activity. The ps20 protein was purified as described in Figs. 1-6, and final protein yield was determined as described under "Experimental Procedures." Increasing concentrations of purified ps20 were added to PC-3 cells, and cells were assayed for [3H]thymidine incorporation as described under "Experimental Procedures." Incorporated [3H]thymidine (cpm) was plotted as a function of ps20 concentration. One-half maximal activity was determined at 2.62 nM with maximal activity at 6. UGS mesenchyme initially stimulates UGS epithelial cell proliferation during ductal morphogenesis, an epithelial differentiation to mature glandular acini, typified by epithelial cell quiescence (low cell mitosis-turnover) and increased secretory activity (columnar secretory cells) is subsequently induced by the mesenchyme stroma (6,7). Hence, a growth-inhibitory paracrine effector protein which also stimulates protein synthesis concurrent with inhibiting epithelial cell proliferation, as is suggested with ps20, may play a role in this secondary induction to differentiation.
The possible role of ps20 in vivo and the mechanisms of ps20 action are fully unknown. No possible action or role in vivo can be assigned or ruled out based on studies to date. To define ps20 further, the cloning of cDNA encoding ps20 protein is required to determine primary structure and assess likely biological actions. In addition, the characterization of ps20 expression patterns and regulatory pathways are required to define molecular regulatory mechanisms and assess possible functions in stromal cell biology. The ps20 protein may or may not represent a paracrine effector growth regulatory protein involved in receptor-mediated pathways. Alternatively, the ps20 protein may function as a matrix component laid down by mesenchyme or as an external cell membrane protein involved in cell-cell or cell-matrix adhesion, thereby affecting proliferation/differentiation. However, it should be cautioned that the present report addresses biological properties in vitro only, and no data yet exist to indicate a similar activity or properties of ps20 in the fetal urogenital sinus or normal prostate gland in vivo.
Progress to understand both prostate organogenesis and prostate disease progression requires the elucidation of genes and proteins involved in stromal cell biology and the mechanisms of stromal-epithelial interactions. The ps20 protein may be of significance to the initiation and progression of prostatic diseases, typified by alterations in epithelial cell proliferation, differentiation, and patterns of stromal cell histology. The observation that ps20 growth inhibits the PC-3 carcinoma cell line derived from a human prostatic adenocarcinoma points to the possibility that ps20 actions may affect prostatic carcinoma progression. Understanding the expression patterns of ps20 in prostatic disease (benign prostatic hyperplasia and prostatic carcinoma) and defining molecular mechanisms of action will be of importance in assessing any potential role of this protein in prostatic disorders.
Of additional interest is the possibility that ps20 may represent a new marker for stromal cell ontogeny and functional differentiation. Our preliminary studies to date2 indicate that significant ps20 expression is limited to mesenchymal cells and adult stromal cells expressing smooth muscle differentiation marker proteins and is not observed in either fibroblast or epithelial cells. Recent studies indicate that prostate smooth muscle represents the major androgen-regulated stromal cell type in the postnatal prostate gland (26,27). Prostate smooth muscle cells evolve from androgen receptor-positive mesenchymal cells, such as U4F cells, in the immediate proximity to developing pockets of epithelial islands (28). Accordingly, ps20 expression may correlate with ontogeny of mesenchymal differentiation to smooth muscle and may provide insight to mechanisms of mesenchymal/smooth muscle actions.
This report serves as the initial characterization of the ps20 protein and associated biological properties. The full extent of ps20 activity and relevance to prostate gland biology awaits more extensive characterization of cDNA encoding this protein and determination of specific biological activity in vivo.