(Received for publication, June 19, 1995; and in revised form, July 11, 1995)
From the
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) ()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 differentiated 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, stromal-epithelial 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-s, 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.
The PC-3 cell line (ATCC CRL 1435, prostatic carcinoma epithelial) and Mv-Lu-1 cell line (ATCC CCL 64, mink lung epithelial) were received from American Type Culture Collection (Rockville, MD). PC-3 cells were cultured in 93% DMEM/Ham's F-12 medium (1:1), supplemented with 7% fetal bovine serum, and Mv-Lu-1 cells in 90% DMEM, supplemented with 10% fetal bovine serum, with each containing penicillin (25 units/ml) and streptomycin (25 µg/ml). Medium was replaced every 2-3 days. Cultures were passaged at confluence by brief exposure to trypsin-EDTA (0.25% trypsin, 0.025% EDTA in calcium, magnesium-free Hanks' salt solution). Cell viability was established by trypan blue dye exclusion and cells counted with an improved Neubauer type hemocytometer. All cell lines were routinely tested for mycoplasma contamination (MycoTect Kit, Life Technologies, Inc.).
For direct
cell counting, PC-3 cells were seeded at 4.0 10
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) .
For additional analytical studies and for scaled-up preparative
purification of ps20, samples derived from serum-free, chemically
defined conditioned medium M 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
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
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
[
H]thymidine incorporation assay.
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. 1shows the
biological activity and A 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
elution pattern. These studies indicated the
UGIF activity peak was produced by U4F cells and was not a constitutive
component of fresh Bfs medium.
Figure 1:
Ion exchange
chromatography. Proteins from U4F cell 48-h conditioned medium
(serum-containing media Bfs) or volume-matched fresh Bfs (as control)
were precipitated with (NH)
SO
and
chromatographed through a DE-52 anion exchange column. Aliquots (100
µl) from each fraction were vacuum-dried and assayed for inhibition
of [
H]thymidine incorporation in PC-3 cells as
described under ``Experimental Procedures.'' Activity was
plotted as reciprocal of incorporated counts/min (1/cpm) to illustrate
inhibition of [
H]thymidine incorporation as a
peak of activity. With conditioned medium samples, activity eluted as a
single peak and fractions were pooled and processed for analytical gel
filtration chromatography and HPLC as described in Fig. 2Fig. 3. Fresh medium preparations were negative for
biological activity. Bottom panel shows the corresponding A
pattern of total
protein.
Figure 2:
Analytical gel filtration chromatography.
Proteins from U4F mesenchymal cell conditioned medium (Bfs medium) were
precipitated with (NH)
SO
,
chromatographed through DE-52 as described in Fig. 1, and
analyzed with gel filtration chromatography, and fractions were assayed
for activity with PC-3 cells as described in Fig. 1and under
``Experimental Procedures.'' Upper panel, elution
profile of biological activity from P-100 gel filtration column.
Biological activity eluted as a single peak (maximum activity at fraction 56) associated with the 18-20-kDa size region.
The elution position of molecular size markers are shown across the top of the graph: bovine serum albumin (66 kDa)
chymotrypsinogen A (27.5 kDa) soybean trypsin inhibitor (20.1 kDa), and
lysozyme (14.3 kDa). Lower panel, SDS-PAGE analysis of eluted
fractions. Fractions (underlined by bar, top
panel) from gel filtration were vacuum dried, electrophoresed
through a 15% acrylamide gel, and stained with the silver method as
described. The elution pattern of a 20-21-kDa species (arrow, fraction 56, bottom panel)
correlated directly the position of eluted bioactivity peak. Molecular
size markers are shown in lanes 1 and 10: myoglobin
fragment I (8.16 kDa), lysozyme (14.3 kDa), myoglobin (16.9), soybean
trypsin inhibitor (20.1 kDa), carbonic anhydrase (29 kDa), ovalbumin
(43 kDa), and bovine serum albumin (66
kDa).
Figure 3:
Analytical reverse phase HPLC. Upper
panel, proteins were chromatographed with a C-18 reverse phase
column as described in the text and under ``Experimental
Procedures,'' and aliquots were vacuum-dried and assayed for
inhibition of [H]thymidine incorporation in
Mv-Lu-1 cells as described under ``Experimental Procedures.''
Biological activity eluted as a major peak associated with fraction 93. Lower panel, SDS-PAGE analysis of eluted fractions from HPLC (bar region, upper panel) shows the elution pattern
of the a 20-21-kDa protein species correlating directly to the
peak of biological activity. Molecular size markers are as described in Fig. 2.
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 major peak of UGIF activity from HPLC was associated with a protein of approximately 20-21 kDa as analyzed by SDS-PAGE (Fig. 3, lower panel, arrow, peak fraction 93).
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) was developed empirically to lower
protein complexity in the starting conditioned medium. The M
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
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.
Figure 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 M (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 Fig. 1Fig. 2Fig. 3. Biological activity eluted as
a major peak associated with fraction 89 and a minor, variable peak
associated with fraction 96. Lower panel, SDS-PAGE analysis of
eluted fractions (bar region, upper panel) shows the
elution pattern of the a 20-21-kDa species (fraction 89, arrow) directly correlated with the peak pattern of biological
activity as shown in the upper panel. Molecular size markers
are shown in lane 1 and are as described in Fig. 2.
Figure 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 M, 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.
Figure 6: Preparative HPLC chromatography. Upper panel, fractions from gel filtration chromatography as shown in Fig. 5were pooled (fractions 52-55) and used for reverse phase HPLC as described in the text and under ``Experimental Procedures.'' Shown is the elution profile of biological activity with PC-3 cells and acetonitrile elution gradient. Biological activity eluted with a consistently observed front peak (fraction 22) and highly variable secondary peak region (fractions 25-34). 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 20-23) and was purified to near homogeneity. Molecular size markers (shown in lane 1) are as indicated in Fig. 2.
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. 6shows 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 20-21-kDa protein peak fractions and were observed only in scaled-up preparations in later stages of purification.
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:
NH-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).
Figure 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 HCl 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 [H]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 HCl 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.
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
6.3-8 nM. To correlate inhibition of
[H]thymidine incorporation with inhibition of
cell proliferation, PC-3 cells were incubated with 7.2 nM ps20
or vehicle control for 5 days, and cells were counted every 24 h as
shown in Fig. 9A. Under these conditions, PC-3 cell
proliferation was inhibited by 52-60% relative to control at any
particular time point, in general agreement with the maximal percent
inhibition of [
H]thymidine incorporation
(approximately 70%) at 7.2 nM shown in Fig. 8. Under
these conditions, the population doubling time of subconfluent PC-3
cells (days 1-3) was increased from an average of 19.8 h in
control cultures to 25.8 h in ps20-treated cultures. In addition,
ps20-treated cultures 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.
Figure 8:
Dose-response of purified ps20 activity.
The ps20 protein was purified as described in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 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
[H]thymidine incorporation as described under
``Experimental Procedures.'' Incorporated
[
H]thymidine (cpm) was plotted as a function of
ps20 concentration. One-half maximal activity was determined at 2.62
nM with maximal activity at 6.3-8 nM. Values
are mean of n = 3 separate determinations,
±S.E.
Figure 9:
Inhibition of PC-3 cell proliferation and
stimulation of protein synthesis by purified ps20. Panel A,
PC-3 cells were incubated with either 7.2 nM purified ps20
(maximal active concentration) or vehicle control for 5 days and
counted each 24 h as described under ``Experimental
Procedures.'' Purified ps20 produced a 52-60% decrease in
PC-3 cell proliferation, increased population doubling time from 19.8
to 25.8 h (days 1-3), and achieved confluence at a lower cell
density. Panel B, PC-3 cells were incubated with 7.2 nM purified ps20 or vehicle control for 3 days and pulsed with
[S]methionine (10 µCi/ml) the final 24 h,
cells were counted, and proteins from the medium were assayed for
[
S]methionine incorporation as described under
``Experimental Procedures.'' Presented are
disintegrations/min of incorporated
[
S]methionine/10
cells. Values are
mean of n = 3 separate determinations, ±
S.E.
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. Amino-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 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 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 date ()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.