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Original Studies |
Department of Fisiopatologia Medica, II Endocrinologia (S.M., R.I., C.M., S.L., P.F., M.P., A.S., F.S., V.T.), and Department of Urologia (F.D.S.), University La Sapienza of Rome, 00161 Rome, Italy
Address all correspondence and requests for reprints to: Dr. Vincenzo Toscano, II Endocrinologia, Dip Fisiopatologia Medica, Università La Sapienza, 00161 Rome, Italy. E-mail: i.s.g.s.h{at}agora.stm.it
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-androstanediol (3Diol) was also
determined to verify their possible androgen dependence.
Prostates were removed by suprapubic prostatectomy from 14 BPH patients
and sectioned in the periurethral, intermediate, and subcapsular
regions. Gene expression of IGF-I, IGF-II, and IGFR1 was evaluated by
semiquantitative RT-PCR, using ß-actin as a control. irIGF-I was
measured by RIA, and irIGF-II was measured by IRMA after acidification
and chromatography on Sep-Pak C18 cartridges. DHT and
3Diol concentrations were evaluated by RIA after extraction and
purification on Celite microcolumns.
IGF-II and IGFR1, but not IGF-I, mRNA was higher in the periurethral
than in the intermediate (P < 0.05) and
subcapsular (P < 0.01) region. Also, prostatic
levels of irIGF-II, expressed as picomoles per g tissue, were higher in
the periurethral (20.84 ± 1.84) than in the intermediate
(14.81 ± 2.11; P < 0.05) and subcapsular
(10.88 ± 1.21; P < 0.001) region. No
significant differences were found in irIGF-I content. Considering
prostatic androgen levels, DHT and 3Diol presented a regional
variation, with the highest concentrations in the periurethral region.
IGF-II mRNA and irIGF-II levels were positively correlated with both
DHT and 3Diol content.
These results demonstrate that in BPH tissue a greater IGF-II activity is present in the periurethral region, the site of origin of BPH. Moreover, we can hypothesize that the tissue androgen content may modulate prostatic production of IGF-II, acting at the transcriptional and probably the posttranscriptional level. Therefore, even though further studies will need to confirm this hypothesis, DHT may increase IGF-II activity, mainly in the periurethral region, which, in turn, induces, through IGFR1, benign proliferation of both epithelial and stromal cells, characteristic of BPH.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The stromal-epithelial interaction plays a critical role in the regulation of the differentiation, growth, and function of the prostate gland (2, 3, 4). In particular, experiments using epithelium/stroma cocultures have shown that 1) the stroma is the inductor of prostatic development during both embryogenesis and puberty; and 2) adult prostatic epithelium, normally quiescent in adulthood, remains responsive to the products arising from the embryonic urogenital sinus stroma cells, which are responsible for growth and changes in morphology and differentiation of the epithelial cells. These findings suggest that stroma-epithelial interaction is important not only during embryogenesis and puberty, but also in adulthood and therefore may be implicated in the pathogenesis of proliferative prostatic diseases. In particular, McNeal (5) suggested that BPH may be caused by reactivation of the inductive activity of the stroma in adulthood, which is responsible for benign proliferation of both stroma and epithelium through autocrine and paracrine effects.
For the development of the normal prostate and its proliferative diseases, adequate androgen stimulation is required, and it is essential (6). At least during prostatic development, the stroma is the mediator of part of the androgenic actions on the epithelium (2). However androgen stimulation alone does not completely explain the development of BPH, probably because androgen action is only in part direct and is mainly indirect through prostatic production of some growth factors (7). These locally produced peptides are considered to be autocrine and/or paracrine mediators of the stromal-epithelial interaction, and abnormal synthesis and secretion of these peptides may be related to the inductive embryonic capacities of the stroma (8).
Insulin-like growth factor I (IGF-I) and IGF-II are two important
growth factors (9). They interact with two specific
membrane receptors: the type 1 (IGFR1) and the type 2 (IGFR2) IGF
receptors (10). However most of the biological actions of
IGFs, in particular mitogenic and antiapoptotic effects, are mediated
via the IGFR1 (11), which is composed of two -subunits,
with ligand-binding sites, and two ß- subunits, with intrinsic
tyrosinic activity (10). In the prostate the most
important source of IGF production is the stroma (12, 13, 14).
However, IGFR1 has been identified mainly in the epithelial
compartment, but it is also present in the stroma
(15, 16, 17).
In vitro studies, using primary cultures of epithelial and
stromal cells, have shown a different protein distribution of IGFs and
their binding proteins in the different regions of the prostate
(14). However, as far as we know, no in vivo
studies have been performed on IGF production and its possible androgen
dependence in the different regions of the human prostate. To clarify
this aspect we determined the expression of IGF-I, IGF-II, and IGFR1
messenger ribonucleic acid (mRNA); the immunoreactive levels of IGF-I
(irIGF-I) and IGF-II (irIGF-II); and dihydrotestosterone (DHT) and
3-androstanediol (3Diol) contents in periurethral, intermediate,
and subcapsular regions of BPH tissue.
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Subjects and Methods |
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The population studied included 14 patients with established BPH (age range, 57–79 yr; mean age, 67.21 ± 6.88 yr). The 14 men had no history of outlet surgery, prostatitis, neurogenic disorders, urinary bacterial infections, or other conditions known to interfere with normal voiding bladder, except BPH. There was no evidence of prostate cancer on digital rectal examination or transrectal ultrasonography. In all cases the clinical diagnosis of BPH was confirmed by histological examination. None of the patients had received any prior treatment. The study was approved by our local ethical committee, and all patients gave written informed consent.
Prostatic tissue
BPH tissues were removed by suprapubic prostatectomy. As previously described, from each enucleated benign hyperplastic prostate gland three different regions were obtained: the periurethral, intermediate, and subcapsular zones (18, 19). Each prostate was sectioned on a frontal plane parallel to the urethra, in front of and behind it; the remaining tissue, represented by the two lateral portions of the gland, was cut along the sagittal plane, parallel to the urethra, into sections of 0.6 cm; the most internal was the periurethral region, the outer one was the subcapsular, and the one included between these two was the intermediate region. Each region weighed about 1 g. Immediately, prostatic samples were put in liquid nitrogen and processed or stored at -80 C for no more than 1 month.
Tissue specimens were analyzed in three ways. The first was a
histological examination to confirm the diagnosis of BPH; to exclude
carcinoma, inflammation, and other possible confounding variables; and
to evaluate morphometric characteristics of the different prostatic
regions by stereological analysis. The second was RT-PCR analysis to
evaluate gene expression of IGF-I, IGF-II, and IGFR1. In the third,
tissue samples were pulverized in liquid nitrogen with a porcelain
mortar and then homogenized in 5 vol TEGM buffer (0.01 mol/L Tris-HCl,
0.001 mol/L ethylenediamine tetraacetate, 0.002 mol/L mercaptoethanol,
0.02 mol/L sodium molybdate, 0.001 mol/L phenylmethylsulfonylfluoride,
0.001 mol/L dithiothreitol, and 10% glycerol, pH 7.4, at 25 C)
(20) to determine irIGF-I, irIGF-II, DHT, and 3Diol by
RIA or immunoradiometric assay (IRMA).
Histological analysis
Prostatic samples were cut in 4-µm-thick sections and stained with hematoxylin and eosin. After histological diagnosis of BPH, prostatic tissues were morphometrically evaluated using a stereological analysis (21) to determine whether the histological compositions of the different prostatic regions influence our results.
RNA extraction and RT-PCR
Total RNA was extracted from prostatic tissues by the acid
guanidinium thiocyanate-phenol-chloroform method (22).
After precipitation, RNA pellets were dissolved in
diethylpyrocarbonate-treated water and quantified
spectrophotometrically at 260 nm; the integrity of the extracted RNA
was verified by agarose gel electrophoresis. Typically, single stranded
complementary DNA (cDNA) was synthesized from total RNA by RT, using
Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI) according to the manufacturer’s
instructions. Briefly, 25 µL of the RT reaction volume, containing 2
µg total RNA, 50 mmol/L Tris-HCl, 75 mmol/L KCl, 3 mmol/L
MgCl2, 10 mmol/L dithiothreitol, 0.5 mmol/L
deoxy-NTPs (dNTPs), 1 µg oligo(deoxythymidine) primer, 25 U
ribonuclease inhibitor, and 200 U Moloney murine leukemia virus reverse
transcriptase, were incubated at 42 C for 60 min. Two microliters of
the RT reaction mixture were then subjected to PCR amplification in a
final volume of 50 µL containing 2 mmol/L Tris-HCl, 10 mmol/L KCl,
0.1 mmol/L dithiothreitol, 2 mmol/L MgCl2, 0,25
µmol/L of each primer, and 1 U Taq DNA polymerase
(Promega Corp.). Primers used for human IGF-II and IGFR1
have been described previously (23), whereas primers used
for human IGF-I were designed by us; each primer pair was localized on
different exons to evaluate the presence of contaminating genomic DNA.
The sequences of oligonucleotide primers are reported in Table 1
. These primers amplify IGF-I, IGF-II,
and IGFR1 sequences of 270, 153, and 300 bp, respectively. The
PCR reactions were carried out on a DNA thermocycler and consisted of
denaturation at 94 C for 30 s, annealing at 65 C for 30 s,
and extension at 72 C for 60 s. The denaturation phase of the
first cycle was prolonged by 5 min, and the extension phase of the last
cycle was prolonged by 9 min. The number of cycles was determined
previously to be within the exponential range of PCR product
amplification necessary for quantitative densitometry and was 27 for
IGF-II, 30 for IGFR1, and 32 for IGF-I. A specific unpublished primer
pair (Table 1) was used for the amplification of human ß-actin, a
housekeeping gene used to control PCR reactions and to normalize
mRNA levels of IGF-I, IGF-II, and IGFR1. PCR amplification of ß-actin
was carried out using the protocol described above for 25 cycles.
Negative controls included the substitution of cDNA templates with an
equal volume of distilled water and were amplified in parallel to check
contamination of the samples. To evaluate the presence of the
contaminating genomic DNA in the RNA-extracted preparation, a PCR
reaction, where the cDNA was replaced with a corresponding quantity of
total RNA extracted, was performed without obtaining amplification. At
least two independent RTs, starting from two different RNA extractions,
and at least six separate PCRs (three for each RT) were performed for
each sample. The PCR-amplified products were analyzed by
electrophoresis on a 2% agarose gel in TAE running buffer (40 mmol/L
Tris base, 20 mmol/L sodium acetate, and 0.5 mmol/L ethylenediamine
tetraacetate) and visualized by ethidium bromide staining under
UV light. The intensities of the bands were quantified by densitometric
analysis and normalized to that of the ß-actin.
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irIGF-I and irIGF-II were measured in tissue homogenate, as previously described (24). The extraction and purification procedures consisted of acidification and filtration on Sep-Pak C18 cartridges (Waters Corp., Milford, MA) (25) to eliminate IGF-binding protein (IGFBP), which interferes with IGF radioligand assays (26). After centrifugation of the homogenate at 800 x g for 10 min, the resulting supernatant was acidified with HCl to a final 0.5-N concentration, stirred at room temperature for 45 min, and centrifuged at 1400 x g for 20 min. A second extraction of the pellet, under the same conditions, was performed, and the supernatants of the two extractions were loaded onto a prewashed Sep-Pak C18 cartridge. The IGFBPs were eliminated by washing with 10 mL 4% acetic acid, and the IGFs were eluted with 5 mL 95% acetonitrile-5% H2O containing 0.1% trifluoroacetic acid. The filtration was repeated for a second time under the same conditions, and the eluate was dried on a Speed-Vac rotor and neutralized with assay buffer. Prostatic irIGF-I and irIGF-II were determined in triplicate at two different dilutions by RIA (Medgenix Diagnostic, Fleurus, Belgium) and IRMA (Diagnostics Systems Laboratories, Inc., Webster, TX), respectively, using commercial kits. The sensitivity of the IGF-I assay was 25 pg/tube, and the inter- and intraassay coefficients of variation were 8.2% and 4.1%, respectively. The sensitivity of the IGF-II assay was 13 pg/tube, and the inter- and intraassay coefficients of variation were 7.9% and 4.2%, respectively.
The mean recoveries of [125I]IGF-I (NEN Life Science Products, DuPont, Boston, MA) and [125I]IGF-II (Diagnostics Systems Laboratories, Inc.), incubated for 24 h at 4 C at the beginning of the extraction procedure to monitor the recovery of endogenous IGFs, were 84 ± 5% and 88 ± 4%, respectively. The recoveries of three different concentrations of unlabeled human IGF-I (3, 9, and 15 ng/mL) and IGF-II (15, 45, and 75 ng/mL) incubated for 24 h at 4 C with two different homogenates were 84%, 90%, and 92% and 87%, 89%, and 91%, respectively, for IGF-I, and 88%, 90%, and 93% and 89%, 90%, and 94%, respectively, for IGF-II.
To evaluate whether binding proteins that may produce artifacts in IGFs assays have been removed, we evaluated in the extracted samples IGF-I and IGF-II concentrations in the presence of an excess of IGF peptide, specifically by adding 100 ng/mL IGF-II in the IGF-I assay and 400 ng/mL IGF-I in the IGF-II assay, saturating the possible residual IGFBPs (27). Comparing the concentrations of IGF-I and IGF-II obtained with and without excess IGFs, we found similar results. Moreover, a second procedure to evaluate whether IGFBPs had been completely removed was performed: an aliquot of the extracted specimens for the assay of IGFs was used to evaluate the presence of IGFBP-1 by IRMA and of IGFBP-2, IGFBP-3, and IGFBP-6 by RIA, using commercial kits (Diagnostics Systems Laboratories, Inc.) (25). No detectable IGFBP was found in any sample. Both of these procedures confirm the validity of our IGFs extraction and purification methods.
Androgen assay
DHT and 3Diol were determined in the homogenate tissue, as
previously described (28). [3H]DHT
and [3H]3Diol (NEN Life Science Products, DuPont; 2000 cpm each) were added to each homogenate
to monitor the recovery of endogenous androgens (DHT and 3Diol)
after extraction and purification methods. The homogenate was extracted
with 5 vol ice-cold acetone, stirred at 4 C for 60 min, and centrifuged
at 1400 x g for 20 min. The supernatant was collected,
and the pellet was extracted a second time as described above. The two
resulting supernatants were united, dried under nitrogen, extracted
twice with 5 vol ether, and redried under nitrogen. The sample was
resuspended in isooctane and loaded on Celite microcolumns. DHT and
3Diol were eluted with a mixture of isooctane-benzene (60:40,
vol/vol, and 40:60, vol/vol, respectively) (29). The
eluates were dried under nitrogen and resuspended in phosphate buffer.
Intraprostatic levels of DHT and 3Diol were determined by RIA in
duplicate at two different dilutions using a commercial kit
(Diagnostics Systems Laboratories, Inc.) and specific
antibody from Sera-Lab Ltd. (Crawley Down, UK), respectively, as
previously described (28). The interassay coefficient of
variation was 4.5 for DHT and 5.1 for 3Diol. The intraassay
coefficients of variation for DHT and for 3Diol were 6.3 and 6.1,
respectively. The recovery (mean ± SD) of
[3H]DHT and
[3H]3Diol added to the homogenate at the
beginning of the extraction was 71 ± 9% for DHT and 74 ±
8% for 3Diol. Accuracy, evaluated by adding known amounts of DHT
and 3Diol to tissue samples of different weigh, showed coefficients
of variation less than 10%.
Statistical analysis
Correlation of degrees of association for any two parameters was
determined by Pearson’s r correlation test and validated by
Spearman’s rank and Kendal 
correlation tests. Regional variations
in irIGF-I, irIGF-II, IGF-I mRNA, IGF-II mRNA, IGFR1 mRNA, DHT, and
3Diol were determined by paired t test. P
< 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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RT-PCR analysis was performed using specific primers for
human IGF-I, IGF-II, and IGFR1; the PCR products were of the expected
sizes of 270, 153, and 300 bp, respectively (Fig. 1). In all samples examined, expression
of these three genes was found. No amplification was observed when cDNA
was replaced with distilled water (negative control) or total RNA; the
latter excludes contamination of genomic DNA, also demonstrated by the
absence of amplification of fragments longer than those expected. Using
the housekeeping gene ß-actin as a control, periurethral,
intermediate, and subcapsular zones had similar levels of IGF-I mRNA
(Fig. 2A). In contrast, a higher
expression of IGF-II and IGFR1 mRNA was found in the periurethral zone
than in the intermediate (P < 0.05) and subcapsular
(P < 0.01) regions (Fig. 2, B and C).
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irIGF values are reported as the mean ± SE
and expressed as picomoles per g tissue. irIGF-I levels were 3.92
± 0.69 in the periurethral, 3.69 ± 0.78 in the intermediate, and
3.11 ± 0.37 in the subcapsular region, but no significant
differences were found in the three zones (Fig. 3A). In contrast, in the periurethral
region irIGF-II concentrations (20.84 ± 1.84) were higher than
those in the intermediate (14.81 ± 2.11; P <
0.05) and subcapsular (10.88 ± 1.21; P < 0.001)
regions (Fig. 3B). In the three prostatic zones, levels of irIGF-II
were always higher than those of irIGF-I (P <
0.0001).
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Diol levels
Tissue androgen levels are reported as the mean ±
SE and expressed as picomoles per g tissue. Prostatic DHT
levels were significantly higher in the periurethral (25.82 ±
1.68) than in the intermediate (16.21 ± 1.44; P
< 0.001) and subcapsular (14.08 ± 1.75; P <
0.001) regions (Fig. 4A). Similarly,
tissue 3Diol concentrations were significantly higher in the
periurethral than in the intermediate (P < 0.01) and
subcapsular (P < 0.001) regions, with values of
6.18 ± 0.61, 3.72 ± 0.61, and 2.76 ± 0.51,
respectively (Fig. 4B).
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A morphometric study was performed to evaluate the percentage of
stroma, epithelium, and glandular lumen in each region of the prostate,
which may influence our data. In fact, it is known that prostatic
stroma is the most important source of IGF production
(12, 13, 14) and is particularly rich in 5-reductase, the
enzyme responsible for DHT production (30, 31). The
periurethral region contained a mean (±SD) of 67 ±
10.9%, 14.1 ± 7.2%, and 18 ± 5.9% stroma, epithelium,
and glandular lumen, respectively. The intermediate region was
characterized by 65.3 ± 10.9% stroma, 13.6 ± 5.8%
epithelium, and 21.1 ± 6.1% glandular lumen. The subcapsular
region was composed of 65.4 ± 10.5% stroma, 14.46 ± 6.3%
epithelium, and 20.2 ± 5.7% glandular lumen. The
stroma/epithelium ratio was 6.7 ± 5.2 in the periurethral,
6.5 ± 4.3 in the intermediate, and 5.9 ± 4.4 in the
subcapsular region. No statistically significant differences were found
between stroma, epithelium, and glandular lumen content and
stroma/epithelium ratio in the three zones analyzed.
The regional variations in IGF-II and IGFR1 mRNA, irIGF-II, DHT, and
3Diol, but not in IGF-I mRNA and protein, were also observed when
the content of each region was compared with the correspondent
histological composition (stroma, epithelium, and glandular lumen
content and stroma/epithelium ratio), excluding an influence of tissue
morphometric characteristics in our result.
Correlation analysis
Correlation of degrees of association between two parameters was
evaluated both separately in each region and considering the three
regions as a whole. Positive linear correlations between the levels of
DHT and IGF-II mRNA, DHT and irIGF-II, DHT and IGFR1 mRNA, 3Diol and
IGF-II mRNA, 3Diol and irIGF-II, and 3Diol and IGFR1 mRNA were
seen in all of the conditions studied (Table 2); moreover, a linear positive
correlation between the levels of DHT and irIGF-I and between 3Diol
and irIGF-I was found only in the periurethral region.
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Diol. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In our study no significant differences in mRNA and protein levels of IGF-I are present in the three regions of BPH tissue, even though in the periurethral region irIGF-I is 20% higher than in the subcapsular region. Therefore, these data do no definitively rule out a role of IGF-I in BPH, because it is possible that with a larger number of prostatic samples this difference could be significant.
IGF-II mRNA and irIGF-II levels present a significant regional variation, with higher values in the periurethral zone, where the disease originates, than in the subcapsular and intermediate regions. These results are in agreement with those of Boudon et al. (14) obtained in vitro using primary cultures of epithelial and stromal prostatic cells grown separately; they have, in fact, demonstrated that IGF-II protein concentrations secreted in stroma cell culture medium from normal prostate were higher in the periurethral zone than in the peripheral zone.
Considering that IGFBPs are important modulators of IGF action, regulating IGFs availability for their receptor (10), it is possible that variations in IGFBP prostatic secretion may occur in BPH tissue, neutralizing the highest concentrations of IGF-II of the periurethral region. However, even though we have no data regarding IGFBPs, our findings suggest that the different levels of IGF-II found in the three regions are associated with different IGF actions. Specifically, the highest levels of IGF-II mRNA and irIGF-II in the periurethral region appear related with an increase in free IGF-II available for IGFR1 interaction, as a corresponding modification of IGFR1 mRNA content is present in this region. Therefore, because antiapoptotic and mitogenic effects of IGF-II are mediated by IGFR1 (11) and because in prostatic tissue it has been reported that high mRNA levels of IGFR1 are associated with high protein content of IGFR1 (15), our results, showing the highest levels of IGF-II mRNA, irIGF-II, and IGFR1 mRNA in the periurethral region of BPH, strongly support the possibility that in this region a greater IGF-II activity is present.
During the fetal period, IGF-II is expressed at high levels in the rapidly growing tissues (34), and in primary cultures of stromal cells the content of IGF-II mRNA is 10-fold higher in those derived from BPH than in those from normal prostate (35). These findings support the hypothesis that BPH represents a return of the prostatic adult stroma to a fetal status (5), with IGF-II considered a mediator of the reawakening of the embryonic capacities of the prostatic stroma. In particular, a greater action of IGF-II in the periurethral region, where the primitive micronodular lesion of the disease originates, may cause BPH growth acting on both epithelial and stromal cells through paracrine and autocrine effects.
In our previous study performed on whole BPH tissue, a linear
positive correlation between irIGF-II and both DHT and 3Diol levels
was observed (24). In this study, where the three regions
are considered, both IGF-II mRNA and irIGF-II were positively
correlated with DHT, suggesting that this hormone may be a modulator of
the prostatic synthesis and secretion of IGF-II, acting at the
transcriptional and probably the posttranscriptional level.
Alternatively, the correlation between both IGF-II mRNA and irIGF-II
and DHT may be a casual event or may be due to the fact that DHT
formation in BPH tissue is IGF-II dependent. This latter hypothesis is
suggested by the observation that 5-reductase activity of human
scrotal skin fibroblasts is increased by IGF-I stimulation through
IGFR-1 activation, but this effect is not reproduced by IGF-II
(36). Therefore, even though the positive correlation
between both IGF-II mRNA and irIGF-II and DHT does not necessarily
prove causality, it suggests that DHT may regulate prostatic IGF-II
production, and then the regional variation of this growth factor may
better reflect its androgen dependence. According to this hypothesis it
has been recently demonstrated that androgen deprivation reduces IGF-II
mRNA levels in prostatic cells (37).
Both IGF-II mRNA and irIGF-II were also correlated with 3Diol.
These data may reflect the origin of this metabolite. In fact, 70% of
3Diol derives from the reduction of DHT (38), as
confirmed by the positive linear correlation between these two
androgens observed in this study. In particular, it may be possible
that intraprostatic 3Diol originates mostly from DHT bound to
nuclear androgen receptor and then is active at the genomic level
(28). From this point of view, 3Diol may be a valuable
marker of prostatic androgenization (28). However, it
cannot be excluded that 3Diol is involved in the control of
prostatic synthesis and secretion of IGF-II, acting either indirectly,
through back-conversion to DHT, or directly, through activation of the
sex hormone-binding globulin/sex hormone-binding globulin receptor
complex (39, 40).
Unlike IGF-II, whose gene and protein expression appears to be
dependent on androgen stimulation of BPH tissue, the intraprostatic
content of IGF-I may be modulated by androgens only in the periurethral
region and exclusively at the posttranscriptional level. In fact, in
the periurethral region only irIGF-I, but not IGF-I mRNA, was
correlated with both DHT and 3Diol, whereas in the intermediate and
subcapsular regions no correlation was present between IGF-I mRNA and
irIGF-I and both DHT and 3Diol.
Our results, then, suggest that in BPH tissue, androgens control IGF-II and only in part IGF-I production. These data are only apparently in disagreement with the recent report showing that the DHT deprivation during finasteride treatment induces a reduction of IGF-I protein content in BPH tissue evaluated by immunostaining (41). This study, in fact, was performed exclusively on the first chips of the periurethral prostatic tissue obtained by transurethral prostatectomy. The samples studied, therefore, approximately correspond to the periurethral region of our study obtained by suprapubic prostatectomy.
It is known that in the prostate the stroma is the major source
of IGFs (12, 13, 14) and is particularly rich in
5-reductase (30, 31), the enzyme responsible for DHT
formation. Therefore, we performed morphometric analysis of the
periurethral, intermediate, and subcapsular regions, excluding that
histological composition may be responsible for the differences
observed. In fact, IGF-II and IGFR1 mRNA, irIGF-II, DHT, and 3Diol
were significantly higher in the periurethral zone than in the
intermediate and subcapsular regions and when their content was
normalized to the stroma, epithelium, and glandular lumen content and
the stroma/epithelium ratio.
The different androgen dependence of IGF-I and IGF-II may be responsible for the different distributions of the two growth factors in BPH tissue. In fact, we can hypothesize that IGF-II presents a significant regional variation, with highest levels in the periurethral region, as its synthesis and secretion correlated to the androgen content in all three regions of BPH analyzed. However, further studies will need to be performed to confirm this hypothesis.
This study confirms our previous investigations (18, 28),
showing a relative hyperandrogenism of the periurethral zone, with the
highest levels of DHT, 3Diol, and nuclear androgen receptor compared
to the subcapsular and intermediate regions. This assumption is
supported by the preferential size reduction of the periurethral region
during DHT deprivation (42).
The most acceptable hypothesis to explain the androgen distribution, having excluded that histological composition may be responsible for the periurethral accumulation of DHT (18), is the transfer of testosterone/DHT from the vas deferens and/or the deferential veins, where these androgens are present at high concentrations, to the prostatic tissue (20, 43, 44). In fact, the vas deferens reachs the urethra passing through the prostate, and the transfer of material from the deferential vein to the prostate has been demonstrated in the dog (45), which has an anatomical situation similar to that in man. This hypothesis may explain the fact that man and dog are the only two species affected by BPH.
In conclusion, the results of this study demonstrate that in BPH
tissue 1) a regional variation in IGF-II, but not IGF-I, is present,
where gene and protein expressions of IGF-II are higher in the
periurethral region than in the intermediate and subcapsular regions;
2) the highest levels of IGF-II mRNA and irIGF-II are associated with
the highest levels of IGFR1 mRNA, the receptor that mediates IGF-II
effects, indicating a greater action of IGF-II in the periurethral
region; 3) IGF-II synthesis and secretion may be androgen-dependent
events, as both IGF-II mRNA and irIGF-II are correlated with tissue DHT
and 3Diol levels, which are higher in the periurethral than in the
intermediate and subcapsular regions. Because a simple correlation does
not prove causality, further studies are needed to confirm this
hypothesis. However, on the basis of our data, it is possible that in
BPH tissue DHT may increase gene and protein expression of IGF-II,
mainly in the periurethral region where the disease originates. IGF-II,
which may be the mediator of reawakening of the embryonic capacities of
the adult prostatic stroma, induces, through IGFR1, a benign
proliferation of both epithelial and stromal cells, which is
characteristic of BPH, particularly in the periurethral region whose
enlargement is responsible for urinary obstruction.
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Footnotes |
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Received August 25, 2000.
Revised December 5, 2000.
Accepted December 6, 2000.
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  G. E. Lim, G. J. Huang, N. Flora, D. LeRoith, C. J. Rhodes, and P. L. Brubaker Insulin Regulates Glucagon-Like Peptide-1 Secretion from the Enteroendocrine L Cell Endocrinology, February 1, 2009; 150(2): 580 - 591. [Abstract] [Full Text] [PDF]
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D. L. Kleinberg, W. Ruan, D. Yee, K. T. Kovacs, and S. Vidal Insulin-Like Growth Factor (IGF)-I Controls Prostate Fibromuscular Development: IGF-I Inhibition Prevents Both Fibromuscular and Glandular Development in Eugonadal Mice Endocrinology, March 1, 2007; 148(3): 1080 - 1088. [Abstract] [Full Text] [PDF]
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 S. T. Page, D. W. Lin, E. A. Mostaghel, D. L. Hess, L. D. True, J. K. Amory, P. S. Nelson, A. M. Matsumoto, and W. J. Bremner Persistent Intraprostatic Androgen Concentrations after Medical Castration in Healthy Men J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3850 - 3856. [Abstract] [Full Text] [PDF]
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C. Crescioli, P. Ferruzzi, A. Caporali, R. Mancina, A. Comerci, M. Muratori, M. Scaltriti, G. B. Vannelli, S. Smiroldo, R. Mariani, et al. Inhibition of Spontaneous and Androgen-Induced Prostate Growth by a Nonhypercalcemic Calcitriol Analog Endocrinology, July 1, 2003; 144(7): 3046 - 3057. [Abstract] [Full Text] [PDF]
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 R. O. Roberts, D. J. Jacobson, C. J. Girman, T. Rhodes, G. G. Klee, M. M. Lieber, and S. J. Jacobsen Insulin-like Growth Factor I, Insulin-like Growth Factor Binding Protein 3, and Urologic Measures of Benign Prostatic Hyperplasia Am. J. Epidemiol., May 1, 2003; 157(9): 784 - 791. [Abstract] [Full Text] [PDF]
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M. W. Elmlinger, I. Mayer, D. Schnabel, B. S. Schuett, D. Diesing, G. Romalo, H. A. Wollmann, W. Weidemann, K.-D. Spindler, M. B. Ranke, et al. Decreased Expression of IGF-II and Its Binding Protein, IGF-Binding Protein-2, in Genital Skin Fibroblasts of Patients with Complete Androgen Insensitivity Syndrome Compared with Normally Virilized Males J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4741 - 4746. [Abstract] [Full Text] [PDF]
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