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Is a Paracrine Inducer of FGF7, a Key Epithelial Growth Factor in Benign Prostatic Hyperplasia
From the Department of Pathology, Baylor College of Medicine; and the Houston Department of Veterans Affairs Medical Center, Houston, Texas
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(Il-1) antibodies and
IL-1Ra, an Il-1 receptor antagonist, show that this
inducing activity was due to the presence of Il-1 in the epithelial
CM. Analysis of normal prostatic peripheral zone and BPH tissue by
enzyme-linked immunoabsorption assay reveal that Il-1 is present at
increased levels in hyperplastic prostate and that levels of Il-1
correlate strongly with tissue FGF7 concentration in BPH. Therefore
Il-1 is produced by prostatic epithelial cells and can induce
FGF7, a potent epithelial growth factor, which can in
turn lead to further epithelial growth and increased Il-1
secretion, thus establishing a double paracrine loop that is
functionally equivalent to an autocrine growth loop. This double
paracrine loop may play a key role in the abnormal proliferation of the
transition zone, which is critical to the pathogenesis of
BPH.
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Introduction |
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Prostate growth is controlled by a variety of polypeptide growth factors, including members of the fibroblast growth factor (FGF) gene family.3,4 FGF2, FGF7, and FGF9 are all present in high concentrations in normal human prostate.5-7 All of these growth factors can stimulate proliferation of primary prostatic epithelial cells in culture. These FGFs are produced by prostatic stromal cells, and appropriate receptors are present on prostatic epithelial cells, so that these FGFs can act as paracrine growth factors for prostatic epithelial cells in vivo.
The concentrations of both FGF7 and FGF2 are significantly increased in hyperplastic prostate in comparison to normal peripheral and transition zone tissue, and this overexpression is not due to alterations in the percentage of stroma in the tissue.5 Interestingly, the normal transition zone tissues had higher levels of both of these FGFs than the normal peripheral zone, indicating a fundamental difference in the biology of the two prostatic zones, and is consistent with the idea that increased expression of FGF2 and FGF7 in the transition zone may be important in the eventual development of BPH. FGF7 is a potent mitogen for prostatic epithelial cells, and quantitative analysis of cellular proliferation by Ki67 immunohistochemistry of frozen sections of the tissue taken before protein extraction revealed a strong correlation of epithelial proliferation with FGF7 content in BPH tissue, consistent with a key role for this growth factor in driving the abnormal epithelial proliferation in BPH. This correlation of the FGF7 content with epithelial proliferation is the first demonstration that alterations of growth factor concentration in BPH in vivo can be linked directly to increased proliferation.
Given that FGF7 protein is increased in BPH and that this
overexpression has functional consequences for the proliferation of
prostatic epithelial cells, we sought to determine what factors control
FGF7 expression in the human prostate. We report here that
interleukin-1 (IL-1) is produced by prostatic epithelial cells
and can act as a paracrine inducer of FGF7 production by prostatic
stromal cells in vitro. In addition, we have found that
IL-1 is present in substantial quantities in human prostate in
vivo and that IL-1 concentration correlates with FGF7
concentration in BPH tissue. Thus epithelial proliferation in BPH is
controlled, at least in part, by a double paracrine loop in which
epithelial cells produce IL-1, which stimulates FGF7 production by
stromal cells, which in turn induces epithelial proliferation and
further production of IL-1. This double paracrine loop ultimately
leads to increased tissue mass in the prostatic transition zone, which
is critical in the pathogenesis of BPH.
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Materials and Methods |
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Samples of the benign peripheral zone of the prostate or hyperplastic transition zone were taken from radical prostatectomies. Tissues were received fresh, and portions were snap-frozen in liquid nitrogen or used to establish primary cell cultures (see below). The frozen tissues were then analyzed by frozen section to confirm the absence of carcinoma or high-grade prostatic intraepithelial neoplasia. Samples of hyperplastic transition zone were also harvested from suprapubic prostatectomies performed for the treatment of severe BPH.
Cell Culture, Production of Epithelial Conditioned Medium, and Assay for FGF7 Induction
Primary epithelial and stromal cell cultures were established from prostatic tissue samples from the peripheral zone as described previously.8 To prepare conditioned medium (CM), primary epithelial cells were plated in 10-cm tissue culture dishes. When the cells were subconfluent, epithelial growth medium was replaced with 8 ml of MCDB 153 medium containing insulin, transferrin, selenium, bovine serum albumin (BSA), and oleic acid (1% ITS+2; Sigma Chemical, St. Louis, MO). Conditioned medium (CM) was collected after 72 hours. The epithelial cells tolerated this treatment well and appeared to be healthy after this period. To assay for FGF7 induction, 1 x 106 primary stromal cells were plated in 10-cm tissue culture dishes. The following day cells were placed in 7.2 ml RPMI 1640 with 1% ITS+2. Cells were then treated with either 800 µl of epithelial conditioned medium or MCDB153 with 1% ITS+2 as a control. At 24-hour intervals aliquots were removed for the analysis of FGF7 content by enzyme-linked immunoabsorption assay (ELISA) (see below).
To determine whether IL-1 could induce FGF7 release, stromal
cultures were plated as above and treated with either 2.5 or 6 pg/ml of
recombinant IL-1 (R&D Systems, Minneapolis, MN) in RPMI 1640 with
1% ITS+2, and the FGF7 content was determined by ELISA on aliquots
removed at 24-hour intervals. To block IL-1 activity, 800 µl of CM
was preincubated with 5 µg of neutralizing anti-IL-1 antibody
(MAB200; R&D Systems) for 2 hours at 37°C. The negative control
antibody was 5 µg of anti-VEGF antibody (AF-293-NA; R&D Systems). A
second approach to blocking IL-1 activity was to use recombinant
IL-1Ra, a naturally occurring antagonist of IL-1 activity. Before the
addition of CM, stromal cultures were pretreated with 400 ng of
recombinant human IL-1Ra (R&D Systems) and incubated for 2 hours. For
both blocking experiments FGF7 concentration was determined by ELISA of
stromal medium removed after 48 hours of treatment.
Immunohistochemistry
Frozen tissue sections were fixed in acetone for 10 minutes and stored at -80°C. All sections were treated with Autoblocker (R&D Systems) to inhibit endogenous peroxidase and avidin/biotin (Vector Laboratories, Burlingame, CA) to block endogenous biotin. The sections were incubated with 2.5 ng/ml of anti-FGF7 mouse monoclonal antibody (R&D Systems) at 4°C for 12 hours. After liberal washing with phosphate-buffered saline (pH 7.4) (PBS), sections were then incubated with biotinylated horse anti-mouse antibody at 7.5 µg/ml for 45 minutes at room temperature (Vector Laboratories). Sections were then washed with PBS containing 0.1% Tween 20 and incubated with avidin-biotin complex (Vectastain Elite, Vector Laboratories) for 15 minutes. The antigen-antibody reaction was demonstrated, using diaminobenzidine as the substrate, and the sections then counterstained with hematoxylin.
Enumeration of FGF7-Expressing Cells
To determine the number of FGF7 cells that were located near epithelial acini or in stromal areas without adjacent epithelium, a point-counting protocol was used. After immunohistochemistry with anti-FGF7 antibody, random fields in each slide were examined at 200x, using a Nikon Labphot-2 microscope with an ocular micrometer. If epithelium was present in the field, then the center of the ocular micrometer was placed approximately 50 µm from the nearest epithelial unit and aligned parallel to its base. Any positively staining cell that intersected the ocular micrometer was counted. A total of 10 such periacinar fields were counted, and the result was summed. Fields without epithelium were counted directly in a manner similar to that of periacinar fields, and a total of 10 stromal fields were counted and summed. The ocular micrometer corresponded to a distance of approximately 400 µm at 200x. A total of six slides from the normal peripheral zone and 11 from hyperplastic transition zone were examined.
Enzyme-Linked Immunoabsorption Assay
Cell extracts were prepared from snap-frozen tissues as described
previously.7
Determination of the FGF7 and IL-1
concentrations in the media and cell extracts was carried out by ELISA,
based on a quantitative sandwich immunoassay technique. Typically,
96-well enzyme immunoassay plates (Nunc, Rochester, NY) were coated
with 1 µg/ml of capture antibody (MAB 251 for FGF7 and MAB 200 for
IL-1; R&D Systems) for either 12 hours (FGF7) or 24 hours (IL-1)
at room temperature. The plates were washed with PBS with 0.05% Tween
20 (wash buffer) to remove unbound antibodies. Plates were then blocked
with PBS containing 1% BSA, 5% sucrose, and 0.05% sodium azide for 1
hour at room temperature. This was followed by the addition of 100 µl
of culture medium or 25 µl of tissue extract (prepared as described
previously)7
made to 100 µl with diluent (0.1% BSA,
0.05% Tween 20, 10 mM Tris, and 150 mmol/L sodium chloride, pH 7.3)
per well and incubation at room temperature for 2 hours. Recombinant
FGF7 or IL-1 (R&D Systems) was used as the standard. After multiple
washes with wash buffer, the plates were incubated with either
biotinylated anti-human FGF7 (BAF251, R&D Systems) at 100 ng/ml or
biotinylated anti-human IL-1 (BAF200, R&D Systems) at 50 ng/ml for 2
hours at room temperature. Plates were washed liberally with wash
buffer, and 100 µl of diluted peroxidase conjugated streptavidin
(1:4000; Zymed Laboratories, San Francisco, CA) was aliquoted per well
and incubated for 30 minutes at room temperature. The plates were then
washed with wash buffer and incubated with 100 µl per well of
substrate consisting of a 1:1 mixture of peroxidase solution and
tetramethyl benzidine substrate (KPL, Gaithersburg, MD) for 20 minutes
at room temperature. The enzymatic activity was stopped with 5 N
sulfuric acid, and the optical density was determined at 450 nm in a
microplate reader. The FGF7 ELISA was linear between 60 and 2000 pg/ml,
whereas the IL-1 ELISA was linear between 2 and 200 pg/ml.
Statistical Analysis
Data were analyzed by paired or unpaired t-tests for comparison of two groups as appropriate. ANOVA was used for the comparison of multiple groups. Calculations were performed using SigmaPlot and SigmaStat programs.
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Results |
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To determine the localization of the increased FGF7 in BPH we
examined tissue sections from normal peripheral zone and BPH tissues
after immunohistochemistry with anti-FGF7 monoclonal antibodies. FGF7
was expressed in stromal cells with fibroblastic morphology. In these
sections we noted a definite tendency for the FGF7-positive cells to be
located near the epithelial acini. Examples of such concentrations of
FGF7-positive cells near epithelial acini are shown in Figure 1
, as is a control photomicrograph of an
adjacent stromal area. It should be noted that this distribution was
not uniform, in that some epithelial acini had large numbers of
adjacent positive cells, whereas others had relatively few. To try and
quantitate this phenomenon, a simple point-counting protocol was used
to enumerate the number of cells staining positively on FGF7 IHC in
periacinar and stromal areas. At x200 magnification, an ocular
micrometer was aligned parallel to the base epithelial acini chosen at
random, and all positively staining cells crossing the ocular
micrometer were counted for a total of 10 measurements per slide. The
same measurement was then performed with the ocular micrometer placed
in a stromal area without adjacent epithelium. Six slides from normal
peripheral zone and 11 BPH tissues were evaluated in this manner
without knowledge of their origin. The results are shown in Figure 2
. For each type of tissue the number of
FGF7-positive cells was significantly higher in the periacinar area
(paired t-test, P < 0.03). In addition,
higher cell counts were present in the periacinar areas of the BPH
tissues compared with the peripheral zone tissue, although this
difference is not statistically significant (P =
0.31, t-test). The increase in cell number in the periacinar
tissue of BPH (one- to threefold) is not proportional to the increase
in FGF7 protein content in BPH (threefold). The most likely explanation
for this discrepancy is that in BPH there are both increased numbers of
FGF7-expressing stromal cells and increased expression of FGF7 per cell
in BPH. We could not identify any obvious association of these areas of
increased FGF7-positive cells with atrophy, metaplasia, or inflammatory
infiltrate
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Given that the FGF7-positive cells are more common closer to the
epithelial acini, we wished to determine whether there was a paracrine
factor or factors secreted by epithelial cells that induced FGF7
production by prostatic stromal cells. Such paracrine factors could
play a role in normal growth if they are secreted into the stroma or
might be released into the surrounding tissue if the epithelium is
disrupted during inflammation. To determine whether such a factor(s) is
secreted in vitro we collected conditioned medium (CM) from
primary cultures of prostatic epithelial cells after 72 hours of
incubation. The collection medium contained insulin as the only added
growth factor, to avoid the effect of EGF and/or FGFs, which are
normally present in the epithelial media, on the stromal cells. The CM
was then added at a 1:10 dilution to primary cultures of prostatic
stromal cells in RPMI 1640 containing insulin, and aliquots were
removed at 24-hour intervals over 96 hours. The aliquots were then
assayed by ELISA for FGF7 content. Results of several such experiments
are shown in Figure 3
. FGF7 accumulates
in the medium of the untreated stromal cells over the four day
incubation, and the diluted CM induces a two- to fourfold increase in
the FGF7 content relative to untreated controls after 4 days. We have
repeated this experiment with multiple batches of conditioned medium
and have found a consistent, two- to sixfold stimulatory effect of the
conditioned medium on FGF7 production. To determine whether the
increased concentration of FGF7 in the conditioned medium was due to an
increase in cell number or increased production of FGF7 per cell, the
number of prostatic stromal cells was determined after 5 days of
incubation with or without treatment with diluted conditioned medium.
There was no significant difference in cell number between the treated
and untreated stromal cells (data not shown). We have repeated these
experiments with both a low-passage and high-passage number of stromal
cells and have found a similar response, although the low-passage cells
consistently have a higher basal (unstimulated) FGF7 expression, so
that the fold increase in FGF7 production for any given CM was altered
by the passage number of the stromal cells used for testing (data not
shown). It should be noted that the induction seen (two- to sixfold) is
on the same order of magnitude as the increase in FGF7 in BPH (relative
to normal PZ) that was correlated with increased epithelial
proliferation.
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Is the Paracrine Factor Secreted by Prostatic Epithelial
Cells That Stimulates FGF7 Secretion
To determine the identity of the factor responsible for the FGF7
accumulation, we assayed the epithelial CM for Il-1, because it is
known to induce FGF7 transcription.9-12
Il-1 was
present at an average concentration of 33.9 ± 6.9 pg/ml (SEM,
n = 12) in the CMs from the prostatic epithelial cells.
Using recombinant Il-1 at 2.5 or 6 pg/ml, roughly equivalent to the
concentration after a 1:10 dilution of the CM, we found a two- to
threefold increase in accumulation of FGF7 production by prostatic
stromal cells (Figure 4)
, similar to the
level of induction by the CM. When compared to CM containing an equal
amount of Il-1, the pure Il-1 induced 60–98% as much FGF7,
indicating that the majority of FGF7-inducing activity in the CM can be
attributed to Il-1. To verify that the FGF7-inducing activity of the
conditioned medium was due to Il-1, we pretreated the CM with
anti-Il-1-neutralizing antibody. As can be seen in Figure 5A
, this pretreatment essentially
eliminated the response of the stromal cells to the CM. As expected,
similar treatment also completely blocks the response to added
recombinant Il-1. Treatment with control anti-VEGF antibody had no
effect. Il-1Ra is a secreted Il-1 antagonist that blocks Il-1 and
Il-1ß activity.13
When cells were pretreated with
recombinant Il-1Ra before the addition of the CM, the FGF7-inducing
activity was again completely abrogated (Figure 5B)
. IL-1ß, which is
also blocked by IL-1Ra, was almost undetectable in the epithelial CM by
ELISA (less than 1 pg/ml; data not shown). It should also be noted that
no Il-1 was detected in medium collected from stromal cells,
indicating that little or no Il-1 is produced by these cells. Thus
Il-1 is the major paracrine inducer of FGF7 production present in
epithelial CM.
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in Human Prostate in Vivo
Having established that IL-1 can act as an epithelial-derived
paracrine inducer of FGF7 production by stromal cells in
vitro, we sought evidence that this paracrine stimulation occurs
in vivo. We therefore assayed tissue extracts from normal
peripheral zone and BPH tissue for IL-1 content by ELISA. The tissue
content of IL-1 was quite variable, ranging from undetectable (<5
pg/g wet weight) to 954 pg/g. The mean IL-1 content of the
peripheral zone was 200 ± 108 pg/g wet weight (SEM,
n = 9), whereas BPH tissue contained 451 ± 99
pg/g (SEM, n = 26). Although this difference is not
statistically significant (P = 0.12,
t-test), the two- to fivefold higher Il-1 concentration
in BPH tissue is similar to the threefold higher level of FGF7 in BPH.
When the IL-1 and FGF7 contents were determined in the same BPH
tissue extract, we found a strong correlation between IL-1 and FGF7
content, as shown in Figure 6A
. The
correlation coefficient by linear regression is 0.73, which is a very
good correlation, particularly when dealing with human tissue samples
from a histologically heterogeneous tissue. When the tissue samples are
divided into four groups based on their IL-1 content (<200,
200–400, 400–600, and >600 pg/g), there is a clear correlation of
increased IL-1 and FGF7 content (Figure 6B)
. The difference between
these four groups is highly significant statistically
(P = 0.001; ANOVA). In summary, IL-1 is
expressed at relatively high levels in prostate tissue, and the tissue
content of IL-1 is strongly correlated with FGF7 content in BPH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that FGF7-producing stromal cells tend to be located near
epithelial acini in the prostate and that epithelial cells release
IL-1, which can induce FGF7 secretion in vitro. At the
present time we do not know the mechanism by which IL-1 increases
FGF7 secretion, but it is likely that it reflects, at least in part,
increased FGF7 transcription, because IL-1 and other cytokines have
been shown to increase FGF7 transcription in other
systems.9-12
IL-1 lacks a signal peptide but is
secreted by a defined mechanism,17
so that it can act as a
paracrine factor in vivo. The strong correlation between
IL-1 and FGF7 levels in vivo argue that this is the case.
This in vivo correlation, in conjunction with our
morphological observations and in vitro data, is consistent
with the idea that IL-1 plays an important role in controlling FGF7
production in BPH. In turn, FGF7 appears to be a major factor in the
increased epithelial proliferation in BPH.
Given the highly variable levels of IL-1 in vivo, it is
likely that control of IL-1 expression in the prostate is complex.
We have shown that IL-1 is produced and secreted by prostatic
epithelial cells. We do not know the extent of variability of IL-1
expression by epithelial cells in vivo. It is highly likely
that inflammatory cells, such as macrophages and T lymphocytes, also
contribute to IL-1 levels in vivo, because IL-1 is
produced by inflammatory cells,13
and it is well known
that normal prostate and BPH tissue are often subject to chronic
inflammation, which can be extensive.18,19
However, we did
not observe any obvious correlation between FGF7-expressing cells
detected by immunohistochemistry and infiltrates of chronic
inflammatory cells, so the extent to which inflammation contributes to
IL-1 and FGF7 expression in vivo is unclear. In addition,
we do not know the extent to which the IL-1 produced by the
epithelial cells is normally released to the stroma and whether
inflammation and atrophy may increase such release. Thus, whereas the
strong correlation of IL-1 and FGF7 levels argues that IL-1 plays
an important role in driving FGF7 production in BPH, the control of
IL-1 expression and the manner in which it comes to interact with
prostatic stromal cells need further investigation.
Although our data indicate that FGF7 secretion is induced by IL-1,
there are almost certainly other factors that also control FGF7
expression. Some of our tissue samples from normal and BPH tissue did
not contain detectable IL-1 and yet had significant, although lower,
FGF7 expression. One factor that may be important is androgens, which
have been shown to induce FGF7 expression by prostatic stromal cells
in vitro.20
More recent observations in
castrated rats indicate that FGF7 expression is not decreased in such
animals.21
Thus the role of androgens in the control of
FGF7 expression in the prostate is controversial. Further
investigations are needed to determine the role of androgens and other
factors in the control of prostatic FGF7 expression and the
interactions of these factors with IL-1.
In this report we have demonstrated that FGF7-expressing stromal cells
tend to be located near epithelial acini and that epithelial cells can
release IL-1, which can induce FGF7 expression. Thus it is possible
that a double paracrine loop can be established in which IL-1 is
secreted by epithelial cells, which then induces FGF7 expression by
stromal cells, which can then induce epithelial proliferation and
further increases in Il-1 expression, which in turn lead to
increased FGF7 expression, and so forth. Our in vivo
observations showing a correlation between IL-1 and FGF7 levels
support the existence of such a double paracrine loop in
vivo. This double paracrine loop is functionally equivalent to an
autocrine loop and can result in increased and poorly controlled
proliferation, which can explain, in part, the increased tissue mass of
the prostatic transition zone that is characteristic of BPH.
Interruption of this double paracrine loop is an attractive target for
therapeutic interventions aimed at preventing BPH and its associated
morbidity in older men.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant R01 DK54170.
Accepted for publication April 3, 2000.
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References |
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 B Kwabi-Addo, M Ozen, and M Ittmann The role of fibroblast growth factors and their receptors in prostate cancer Endocr. Relat. Cancer, December 1, 2004; 11(4): 709 - 724. [Abstract] [Full Text] [PDF]
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, 2.5 pg/ml, Il-1
, 6 pg/ml; 
, CM equivalent to 2.5 pg/ml Il-1
, CM
equivalent to 6 pg/ml Il-1
