Pathogenesis Of Benign Prostatic Hyperplasia Research Articles

Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis.

Despite the high prevalence of benign prostatic hyperplasia (BPH) in the aging male, little is known regarding the etiology of this disease. A better understanding of the molecular etiology of BPH would be facilitated by a comprehensive analysis of gene expression patterns that are characteristic of benign growth in the prostate gland. Since genes differentially expressed between BPH and normal prostate tissues are likely to reflect underlying pathogenic mechanisms involved in the development of BPH, we performed comparative gene expression analysis using cDNA microarray technology to identify candidate genes associated with BPH. Total RNA was extracted from a set of 9 BPH specimens from men with extensive hyperplasia and a set of 12 histologically normal prostate tissues excised from radical prostatectomy specimens. Each of these 21 RNA samples was labeled with Cy3 in a reverse transcription reaction and cohybridized with a Cy5 labeled common reference sample to a cDNA microarray containing 6,500 human genes. Normalized fluorescent intensity ratios from each hybridization experiment were extracted to represent the relative mRNA abundance for each gene in each sample. Weighted gene and random permutation analyses were performed to generate a subset of genes with statistically significant differences in expression between BPH and normal prostate tissues. Semi-quantitative PCR analysis was performed to validate differential expression. A subset of 76 genes involved in a wide range of cellular functions was identified to be differentially expressed between BPH and normal prostate tissues. Semi-quantitative PCR was performed on 10 genes and 8 were validated. Genes consistently upregulated in BPH when compared to normal prostate tissues included: a restricted set of growth factors and their binding proteins (e.g. IGF-1 and -2, TGF-beta3, BMP5, latent TGF-beta binding protein 1 and -2); hydrolases, proteases, and protease inhibitors (e.g. neuropathy target esterase, MMP2, alpha-2-macroglobulin); stress response enzymes (e.g. COX2, GSTM5); and extracellular matrix molecules (e.g. laminin alpha 4 and beta 1, chondroitin sulfate proteoglycan 2, lumican). Genes consistently expressing less mRNA in BPH than in normal prostate tissues were less commonly observed and included the transcription factor KLF4, thrombospondin 4, nitric oxide synthase 2A, transglutaminase 3, and gastrin releasing peptide. We identified a diverse set of genes that are potentially related to benign prostatic hyperplasia, including genes both previously implicated in BPH pathogenesis as well as others not previously linked to this disease. Further targeted validation and investigations of these genes at the DNA, mRNA, and protein levels are warranted to determine the clinical relevance and possible therapeutic utility of these genes.

Alterations in gap junction protein expression in human benign prostatic hyperplasia and prostate cancer.

Gap junctions composed of connexin proteins have an essential role in intercellular communication and differentiation. Dysregulation of connexin expression is believed to have a role in carcinogenesis. The human prostate has been reported to express connexin 32 and 43. However, the expression pattern in prostate cancer is controversial, while to our knowledge connexin expression has not been reported in benign prostatic hyperplasia (BPH). To understand the potential involvement in prostate disease connexin 32 and 43 expression was evaluated in a series of normal prostate, BPH and prostate cancer specimens that were surgically removed due to bladder outlet obstruction. Frozen sections of 23 normal, 43 BPH and 40 cancer involved prostates were evaluated for the presence, staining intensity and pattern of connexin 32 and 43 by immunocytochemical testing. In all specimens examined connexin 43 stain was punctate along the borders of the basal epithelial cells, whereas connexin 32 immunolocalized to luminal epithelial cells. In normal prostate connexin 43 and 32 were present in 87% and 65% of specimens, respectively, at low to moderate stain intensity. Importantly none of the normal samples were negative foreach connexin. In BPH specimens there was a marked increase in the incidence and intensity of connexin 43 and 32 immunostaining within epithelial cells. In addition, 23% of BPH samples showed strong connexin 43 expression in stromal cells. In contrast, connexin was decreased in prostate cancer specimens, of which 65% and 38% were negative for connexin 43 and 32, respectively, and 28% were negative for each type. In poorly differentiated tumors connexin 43 and 32 were present in only 10% and 40% of tumors, respectively, at low immunostaining intensity. In normal human prostate basal cells communicate via connexin 43 gap junctions, whereas luminal cells communicate via connexin 32 gap junctions. In BPH gap junctional intercellular communication is increased in epithelial and stromal cells, which may have a role in BPH pathogenesis. In prostate cancer gap junctional intercellular communication is decreased, is as indicated by decreased expression of connexin 43 and 32 with severe loss in poorly differentiated prostate cancer. These alterations in connexin expression may have a role in dedifferentiation and tumor progression.

5 α-reductase activity in the prostate

The prostate gland depends on androgen stimulation for its development and growth. However, testosterone is not the major androgen responsible for growth of the prostate. Testosterone is converted to dihydrotestosterone (DHT) by the enzyme Δ 4, 3 ketosteroid, 5α-reductase in prostatic stromal and basal cells. DHT is primarily responsible for prostate development and the pathogenesis of benign prostatic hyperplasia (BPH). Inhibitors of 5α-reductase reduce prostate size by 20% to 30%. This reduction in glandular tissue is achieved by the induction of apoptosis, which is histologically manifested by ductal atrophy. Inhibition also diminishes the number of blood vessels in the prostate because of a reduction in vascular-derived endothelial growth factor. 5α-Reductase occurs as 2 isozymes, type 1 and type 2, with the prostate expressing predominantly the type-2 isozyme, and the liver and skin expressing primarily the type-1 isozyme. Patients have been identified with deficiencies in the type-2 5α-reductase, but not type 1. Knockout mice with the type-2 5α-reductase demonstrate a phenotype similar to that seen in men with 5α-reductase deficiency. Type-1 5α-reductase knockout male mice are phenotypically normal. Enzymatic activity for 5α-reductase or immunohistochemical detection has been noted in other genitourinary tissues, such as the epididymis, testes, gubernaculum, and corporal cavernosal tissue. Preputial skin predominately expresses the type-1 5α-reductase, whereas stromal cells in the seminal vesicle also express type-2 isozyme. However, epithelial cells in the epididymis, but not surrounding stroma, express type-1 5α-reductase. In addition to influencing prostatic growth, 5α-reductase also influences the expression of neuronal nitric-oxide synthase in the corpus cavernosum. The contribution of DHT in the serum, which is partially derived from type-1 5α-reductase in the liver and the small amount of type-1 5α-reductase in the prostate, may play a role in maintaining prostatic enlargement. Thus, in an effort to increase efficacy of treatment for BPH, clinical trials are under way using new drugs, such as GI-198745 (Glaxo-Wellcome, Research Triangle Park, NC), PNU 157706 (Pharmacia & Upjohn, Peapack, NJ), FR146687 (Fujisawa, Osaka, Japan), and LY 320236 (Lilly, Indianapolis, IN), which inhibit both the type-1 and type-2 5α-reductase.

ALTERATIONS IN GAP JUNCTION PROTEIN EXPRESSION IN HUMAN BENIGN PROSTATIC HYPERPLASIA AND PROSTATE CANCER

Gap junctions composed of connexin proteins have an essential role in intercellular communication and differentiation. Dysregulation of connexin expression is believed to have a role in carcinogenesis. The human prostate has been reported to express connexin 32 and 43. However, the expression pattern in prostate cancer is controversial, while to our knowledge connexin expression has not been reported in benign prostatic hyperplasia (BPH). To understand the potential involvement in prostate disease connexin 32 and 43 expression was evaluated in a series of normal prostate, BPH and prostate cancer specimens that were surgically removed due to bladder outlet obstruction. Frozen sections of 23 normal, 43 BPH and 40 cancer involved prostates were evaluated for the presence, staining intensity and pattern of connexin 32 and 43 by immunocytochemical testing. In all specimens examined connexin 43 stain was punctate along the borders of the basal epithelial cells, whereas connexin 32 immunolocalized to luminal epithelial cells. In normal prostate connexin 43 and 32 were present in 87% and 65% of specimens, respectively, at low to moderate stain intensity. Importantly none of the normal samples were negative foreach connexin. In BPH specimens there was a marked increase in the incidence and intensity of connexin 43 and 32 immunostaining within epithelial cells. In addition, 23% of BPH samples showed strong connexin 43 expression in stromal cells. In contrast, connexin was decreased in prostate cancer specimens, of which 65% and 38% were negative for connexin 43 and 32, respectively, and 28% were negative for each type. In poorly differentiated tumors connexin 43 and 32 were present in only 10% and 40% of tumors, respectively, at low immunostaining intensity. In normal human prostate basal cells communicate via connexin 43 gap junctions, whereas luminal cells communicate via connexin 32 gap junctions. In BPH gap junctional intercellular communication is increased in epithelial and stromal cells, which may have a role in BPH pathogenesis. In prostate cancer gap junctional intercellular communication is decreased, is as indicated by decreased expression of connexin 43 and 32 with severe loss in poorly differentiated prostate cancer. These alterations in connexin expression may have a role in dedifferentiation and tumor progression.

Localization of angiotensin-converting enzyme in the human prostate: pathological expression in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common hyperplastic disease in man and it is characterized by increased cellular growth (stromal and epithelial hyperplasia) and enhanced local sympathetic tone, both of which are known to be augmented by activation of the renin-angiotensin system (RAS) in other tissues. Angiotensin-converting enzyme (ACE) is an integral component of the RAS that is responsible for the production of the active peptide angiotensin II from the inactive precursor angiotensin I. The present study was undertaken to map the anatomical localization of ACE protein and messenger ribonucleic acid (mRNA) in the normal human prostate and to establish whether their expression is pathologically altered in BPH. Human prostate samples were obtained at post-mortem and histologically defined as normal or hyperplastic. ACE protein binding/expression was determined by in vitro autoradiography and immunohistochemistry using the ACE-specific radioligand [125I]-MK351A and a mouse anti-ACE polyclonal antibody, respectively, whereas the spatiotemporal distribution of ACE mRNA was determined by in situ hybridization using 35S-labelled oligonucleotide probes. ACE protein was localized to the glandular epithelium in the human prostate. ACE binding and immunostaining were increased in BPH compared with normal (non-hyperplastic) prostate specimens [X-ray film autoradiography: normal 873+/-48 dpm/mm2 (n=8) vs. BPH 1631+/-274 dpm/mm2 (n=6), p<0.05; emulsion autoradiography: normal 3.1+/-0.5 grains/mm2 (n=6) vs. BPH 32.8+/-8.6 grains/mm2 (n=5), p<0.01]. ACE mRNA was also localized to glandular epithelial cells in the human prostate with a significant increase in ACE mRNA expression in BPH compared with the normal prostate [normal 11.04+/-2.03 grains/cell (n=220 cells total) vs. BPH 22.29+/-1.34 grains/cell (n=198 cells total), p<0.05]. The findings of the present study suggest that ACE is localized to the glandular epithelium of the human prostate and that its expression, at both protein and mRNA level, is aberrantly increased in BPH. These data support the concept that hyperactivity of the local RAS in the prostate may be involved in the pathogenesis of BPH.

Interleukin-8 Is a Paracrine Inducer of Fibroblast Growth Factor 2, a Stromal and Epithelial Growth Factor in Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men in which there is benign overgrowth of the prostatic transition zone, leading to obstruction of urine outflow. Fibroblast growth factor (FGF) 2, a potent growth factor for prostatic stromal and epithelial cells, is increased twofold in BPH and its concentration is correlated with stromal proliferation in this condition. Immunohistochemistry of normal and hyperplastic prostate revealed that FGF2-expressing stromal cells were present in higher numbers near the epithelial acini, implying that epithelial cells may express a factor that induces FGF2 expression by stromal cells. Conditioned medium from primary cultures of prostatic epithelial cells was capable of inducing increased expression of FGF2 by primary stromal cultures. Blocking experiments with neutralizing anti-interleukin (IL)-8 antibodies and pretreatment with lipopolysaccharide, which down-regulates the IL-8 receptor, show that this inducing activity is because of the presence of IL-8 in the epithelial-conditioned medium. Analysis of normal prostatic peripheral zone and BPH tissue by enzyme-linked immunosorbent assay reveals that IL-8 is present at increased levels in hyperplastic prostate. Therefore IL-8 produced by prostatic epithelial cells can induce FGF2, a potent stromal and epithelial growth factor, and in this manner promote the abnormal proliferation of the prostatic transition zone that is critical in the pathogenesis of BPH.

Association of vitamin D receptor and 17 hydroxylase gene polymorphisms with benign prostatic hyperplasia and benign prostatic enlargement

Objectives. To determine whether polymorphisms in 17 hydroxylase (CYP17) and vitamin D receptor (VDR) genes have an association to prostate volume/histology and endocrine patterns in elderly men with lower urinary tract symptoms (LUTS). Methods. Elderly men with LUTS underwent the following investigations: International Prostate Symptom Score (IPSS), uroflowmetry, serum prostate-specific antigen (PSA) assessment of prostate volume, and an endocrine study. Polymorphisms of CYP17 (T→C substitution in the 5′ promoter region) and VDR (T1055C) genes were detected by polymerase chain reaction followed by restriction-length polymorphism analysis, using DNA from peripheral white blood cells. Clinical and endocrine parameters and the prostate stroma/epithelial ratio were correlated to CYP17 and VDR genotypes. Results. A total of 148 (mean ± SD, 67.0 ± 9.7 years) patients were analyzed. IPSS (17.8 ± 7.0), prostate volume (41.9 ± 17.9 cc), maximum flow rate (10.9 ± 5.8 mL/s), and PSA (4.7 ± 4.7 ng/mL) indicate a typical LUTS population. Mean endocrine levels were consistently within age-specific reference values. Neither CYP17 nor VDR gene polymorphisms revealed an association to prostate size, PSA, clinical parameters, and endocrine parameters. Men who had the A1/A1 CYP17 genotype had on average a greater stromal/epithelial ratio than men with the A1/A2 or A2/A2 genotypes, yet after adjusting for multiple testing, this significance disappeared. Conclusions. Gene polymorphisms of CYP17 and VDR have no association to prostate volume, clinical parameters, and endocrine parameters in elderly men. The association of CYP17 polymorphism and prostate histology warrants further studies. Assessment of gene polymorphisms might provide new insights into the pathogenesis of benign prostatic hyperplasia and benign prostate enlargement and may hold promise as genetic biomarkers of this disease.

Alpha-blockade downregulates myosin heavy chain gene expression in human benign prostatic hyperplasia

Objectives. Smooth muscle (SM), a major component of prostate stroma, plays an important role in the pathogenesis of benign prostatic hyperplasia. In many muscle systems, steroid hormones and alpha 1-adrenergic neurotransmitters tightly regulate expression of contractile proteins. In this study, SM content and the expression of myosin heavy chain (MHC) in tissues from patients with benign prostatic hyperplasia treated with androgen ablation or alpha-blockade were compared with untreated controls. Methods. Prostatic periurethral tissue specimens from patients receiving luteinizing hormone-releasing hormone analogues (n = 12), alpha-blocking agents (n = 12), and no treatment (n = 13) were examined. The samples were analyzed for SM MHC mRNA expression using competitive reverse transcription-polymerase chain reaction. SM content was measured by morphometric analysis of trichrome-stained sections. Results. Stromal SM constituted 45.4% ± 8.6%, 48.1% ± 18.4%, and 45.9% ± 10.8% of the total tissue in androgen ablated, alpha-blocked, and untreated tissues, respectively. No significant difference was observed among these three groups ( P = 0.84, analysis of variance). However, SM MHC mRNA expression was markedly decreased in the alpha-blockade group (0.15 ± 0.02 attomole/mg tissue) compared with the androgen-ablated (0.58 ± 0.15 attomole/mg tissue) or control (0.44 ± 0.10 attomole/mg tissue) groups. The relationship between SM content and expression of SM MHC significantly differed among the groups ( P = 0.02, analysis of variance). Conclusions. Androgen ablation and alpha-blockade do not appear to alter the histologic characteristics of prostate stroma in men with symptomatic benign prostatic hyperplasia. However, contractile protein gene expression in stromal SM cells is significantly altered after alpha-blockade. These data suggest that, in addition to the simple relaxation of muscle tone, alpha-blocking agents may affect the phenotypic expression of contractile proteins in prostate SM cells.

Interleukin-1α Is a Paracrine Inducer of FGF7, a Key Epithelial Growth Factor in Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men in which there is benign overgrowth of the prostatic transition zone, leading to obstruction of urine outflow. FGF7, a potent growth factor for prostatic epithelial cells, is increased by threefold in BPH and is correlated with increased epithelial proliferation in this condition. Immunohistochemistry of normal and hyperplastic prostate revealed that FGF7-expressing fibroblastic cells were present in higher numbers near the epithelial acini, implying that epithelial cells may express a factor that induces FGF7 expression by stromal cells. Conditioned medium (CM) from primary cultures of prostatic epithelial cells was capable of inducing a two- to sixfold increase in the expression of FGF7 by primary stromal cultures. Blocking experiments with neutralizing anti-interleukin-1alpha (Il-1alpha) antibodies and IL-1Ra, an Il-1alpha receptor antagonist, show that this inducing activity was due to the presence of Il-1alpha in the epithelial CM. Analysis of normal prostatic peripheral zone and BPH tissue by enzyme-linked immunoabsorption assay reveal that Il-1alpha is present at increased levels in hyperplastic prostate and that levels of Il-1alpha correlate strongly with tissue FGF7 concentration in BPH. Therefore Il-1alpha 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-1alpha 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.

Ornithine decarboxylase activity and its gene expression are increased in benign hyperplastic prostate.

Ornithine decarboxylase (ODC) is the first key enzyme in the polyamine biosynthesis pathway. Polyamine is believed to participate in cellular proliferation and differentiation. To study the relationship between ODC and the pathogenesis of benign prostatic hyperplasia (BPH), the polyamine levels, ODC activities, and expression of ODC mRNA in benign hyperplastic and normal human prostates were assayed. Polyamine contents and ODC activities in tissue extracts were determined by reverse-phase high-performance liquid chromatography and spectrophotometric procedures, respectively. The ODC mRNA levels were assayed by Northern blot analysis. The contents of putrescine, spermidine, and spermine in BPH tissues were 2.2, 3.4, and 6.0 times higher than those in normal tissues, respectively; the ODC activity of BPH tissue was about 3.2 times higher than in normal tissue; the expression level of ODC mRNA in the BPH tissues was greater than that of normal tissues. The findings imply that 1) the increased ODC activity and polyamine content in prostatic tissue may correlate with the pathogenesis of BPH, and 2) the high level of ODC activity is induced by the overexpression of ODC mRNA.

Transforming growth factor-beta induces growth inhibition and IGF-binding protein-3 production in prostatic stromal cells: abnormalities in cells cultured from benign prostatic hyperplasia tissues.

The IGF axis has been implicated in the pathogenesis of benign prostatic hyperplasia (BPH) via the paracrine action of IGFs and IGF-binding proteins (IGFBPs). In this study, we examined the regulation of cell growth and IGFBP-3 secretion by transforming growth factor-beta (TGF-beta) in prostatic stromal cell (PC-S) cultures from histologically normal tissues and tissues from BPH. PC-S cultures were treated with varying doses of TGF-beta1. Forty-eight hour conditioned media (CM) from these cultures were subjected to Western immunoblotting and ligand blotting for detection and quantification of IGFBPs. IGFBPs-2, -3 and -4 were detected in the CM from normal PC-S cultures. In CM from BPH PC-S, IGFBP-3 levels were 2-fold lower at baseline than in the normal PC-S CM, in addition to the differences in IGFBPs-2 and -5 which we have previously reported. In response to TGF-beta1, a 15-fold increase in the levels of IGFBP-3 was observed in normal PC-S CM, while a mere 2-fold increase was observed in BPH PC-S CM (P<0.001). These findings were confirmed by specific immunoblotting and immunocytochemistry. IGFBP-3 mRNA levels detected by Northern blotting of total RNA extracted from similar cultures showed the induction of IGFBP-3 expression by TGF-beta1 in normal PC-S and its lack of induction in BPH PC-S. Cell growth inhibition in response to TGF-beta1 correlated with the IGFBP-3 concentrations found in CM. Normal PC-S showed a 60% decrease in cell number after 10 days in media with 1 ng/ml TGF-beta1, compared with the untreated control. The decrease in proliferation observed in comparably treated BPH cells was only 20% (P<0.001). In conclusion, BPH PC-S had a reduced IGFBP-3 response to TGF-beta1 and demonstrated decreased TGF-beta1-induced growth inhibition relative to normal PC-S. We hypothesize that in normal PC-S, TGF-beta exerts its anti-proliferative effects by stimulating the production of IGFBP-3, which acts as an inhibitory factor, either by inhibiting IGFs or directly by interacting with cells, and that this process is altered in BPH PC-S.

Myosin heavy chain gene expression in normal and hyperplastic human prostate tissue.

Benign prostatic hyperplasia (BPH) is common among aging men. Over 80% of males 50-60 years and older have various degrees of bladder outlet obstruction secondary to BPH. Despite the tremendous medical impact of BPH, its molecular pathophysiology remains unclear. Current BPH research focuses on steroid hormonal effects, stromal-epithelial cell interaction, and oncogenes and growth factors. But little is known about the potential prostatic smooth muscle (SM) alterations that may occur during stromal hyperplasia. To study SM phenotypic modulation in hyperplastic prostatic growth, we isolated and characterized the 3' end of human SM myosin heavy chain (SMMHC) cDNA as a molecular probe. Expression of SMMHC and nonmuscle myosin heavy chain (NMMHC) in human prostates was analyzed using Western blot, Northern blot, and in situ hybridization to determine if BPH tissue expresses significantly less SMMHC and more NMMHC than a normal prostate. In addition, a competitive, reverse transcription (RT) polymerase chain reaction (PCR) method was adapted to quantify SMMHC and NMMHC mRNA expression at the sensitivity level of 10(-21) mole per mg of wet tissue. Western blot, Northern blot, and in situ hybridization results reveal that both SMMHC and NMMHC are expressed in the human prostate, while SMMHC is the predominant form found in normal prostate stroma. Results from competitive RT-PCR analysis indicate that NMMHC mRNA expression is approximately 10(-20) mole/mg of tissue. The SMMHC mRNA expressed is approximately 10(-18) mole/mg. No significant difference was found when NMMHC mRNA expression was compared between normal and BPH periurethral tissues. However, SMMHC expression was reduced almost fivefold in BPH compared to normal prostate, despite an increase in prostatic stromal mass. Our results suggest the pathogenesis of BPH is associated with a unique type of SM proliferation. Such proliferation is characterized by downregulation of SMMHC mRNA expression but without upregulation of NMMHC mRNA expression, the pattern seen in proliferating SM cells in culture and in other pathologic forms of SM hyperplasia (e.g., atherosclerosis). These findings support a model of BPH typified by active smooth muscle proliferation early in the disease process, and supports clinical observations that suggest ongoing prostate growth of the prostate is minimal in older men. Therapeutic strategies to prevent disease progression should therefore focus on early phases of prostatic growth.

Nitric oxide synthases in normal and benign hyperplastic human prostate: immunohistochemistry and molecular biology.

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The inducible NOS (iNOS or NOS-2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co-expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium.

Modulation of the differentiation status of cultured prostatic smooth muscle cells by an alpha1-adrenergic receptor antagonist.

Prostatic stromal cells are believed to be a key factor in the pathogenesis of benign prostatic hyperplasia (BPH). The effect of phenylephrine, an alpha1-adrenergic receptor agonist, and doxazosin, an alpha1-adrenergic receptor-specific antagonist, on the expression of smooth muscle myosin-heavy-chain isotypes SM-1 and SM-2 was tested in an in vitro model of prostatic smooth muscle cells (SMC). Primary prostatic stromal cells, grown in SMC-specific medium, were treated with 10 microM of phenylephrine or 1 microM of doxazosin or a combination of both. SM-2 to SM-1 mRNA ratios and expression of alpha1-adrenergic receptor subtypes were determined by means of reverse transcriptase polymerase chain reaction (RT-PCR) techniques. Cell growth was measured by a cell viability assay. SM-1 mRNA and only very low levels of SM-2 mRNA were detected in prostatic SMC cultures grown for 4 days in a serum-free base medium. After 6 days of treatment, SM-2 expression increased, highest in the doxazosin-treated cultures. In comparison to unstimulated cells, a statistically significant 10-fold increase of the SM-2:SM-1 ratio was measured in doxazosin-treated cultures. Analysis of alpha1-adrenergic receptor subtype expression revealed the presence of mRNAs of subtypes 1d and 1b mRNAs. Subtype 1a was not expressed. Phenylephrine and doxazosin showed no significant effect on cell proliferation and on alpha1d-adrenergic receptor expression. SMC can differentiate from a proliferative to a contractile phenotype, which is accompanied by increased expression of isotope 2 of smooth muscle myosin heavy chain. Our results suggest that doxazosin seems to have a long-term effect on the differentiation of prostatic stromal cells, indicating that alpha1-adrenergic receptor antagonists do not act solely on SMC contractility.

Localization of oxytocin receptors in the human and macaque monkey male reproductive tracts: evidence for a physiological role of oxytocin in the male.

The peptide oxytocin is present in tissues of the male reproductive tract from a variety of mammalian species. In the human, specific mRNA for oxytocin and the peptide itself have been identified in the testis, epididymis and prostate. The peptide has been shown to modulate both steroidogenesis and contractility in the male reproductive tract and may be involved in the pathogenesis of benign prostatic hyperplasia. We have performed Western blots and immunohistochemistry using a specific antibody to the human oxytocin receptor (OTR) to investigate the distribution and localization of the receptor in the human and macaque monkey (Macaca fasicularis). An immunoreactive band of approximately 55 kDa was detected in human and monkey uterine, testicular and prostatic tissues and in preparations of monkey caput and cauda epididymis. A second, less intense, band of 60 kDa was also seen in testicular and uterine tissue samples. No specific bands were detected in monkey muscle or in any tissue following incubation with mouse immunoglobulin (Ig)M. In the human and monkey testis staining for the OTR was present in the interstitial tissue and in Sertoli cells. Localization of the OTRs varied throughout the epididymis being expressed by epithelial cells proximally but confined to cells at the base of the epididymal ducts and to the surrounding smooth muscle layers distally. In the prostate OTR were localized to the stromal tissue surrounding the ducts. These findings correlate with sites of local production of the peptide and the observed biological actions of oxytocin, and thus support the evidence that oxytocin may play a physiological role in the male reproductive tract.

Morphogenesis of benign prostatic hyperplasia and prostatic carcinoma

Enlargement of the prostate is an age-related, physiological process that is unique in human tissue. The prostate gland is the most common site of neoplastic disorders in men. Despite the growing impact of the various prostate diseases in terms of morbidity and mortality, the pathogenesis of benign prostate hyperplasia (BPH) and prostate cancer remains poorly understood. This reflects the complex composition of the gland with different anatomic, cellular and functional compartments that are differentially involved in benign and malignant disease processes. The present review summarizes new concepts on the morphogenesis of normal and abnormal growth in the human prostate. There is increasing evidence that prostatic stem cells are located in the basal cell layer that is basically involved in normal growth and the development of glandular hyperplasia and prostate cancer. High-grade prostatic intraepithelial neoplasia is considered the most likely precursor of clinically important cancer of the peripheral zone. Severe differentiation and proliferation abnormalities occur during malignant transformation of the prostatic epithelium. These premalignant changes are associated with abnormal expression of growth factor receptors, oncogene and suppressor gene products and genetic instability. During the process of stromal invasion the transformed cells lose their basal cell phenotype and produce basement membrane-like matrices. Common prostate cancer is mainly composed of exocrine cell types that remain androgen-responsive even in hormone-independent disease. The frequent occurrence of neuroendocrine differentiation in common prostate cancer reflects the differentiation potency of its stem cells. The endocrine phenotype derives from exocrine tumor cells via intermediate (amphicrine) cell types. Neuroendocrine tumor cells consistently lack the nuclear androgen receptor and represent an androgen-insensitive cell population in prostate cancer.

ENHANCED EXPRESSION OF THE MESENCHYMAL MARKER, VIMENTIN, IN HYPERPLASTIC VERSUS NORMAL HUMAN PROSTATIC EPITHELIUM

A total of 20 benign prostatic hyperplasia (BPH) tissue samples from 13 patients were evaluated for the coexpression of cytokeratins and vimentin intermediate filaments in the glandular epithelium. Vimentin expression was significantly increased (p <0.001) in BPH epithelium compared to adjacent “normal” epithelium. The data imply that epithelial/mesenchymal (E/M) transformations may play a role in the pathogenesis of BPH.

Inhibitory effects of Serenoa repens on the kinetic of pig prostatic microsomal 5alpha-reductase activity.

The pathogenesis of benign prostatic hyperplasia is linked to the accumulation of dihydrotestosterone (DHT), the active form of testosterone (T), in prostatic tissue. We have defined characteristics of 5alpha-reductase enzyme which catalyzes the conversion of T into DHT in prostatic microsomes of growing pigs. Peaks for the 5alpha-reductase activity were found at pH 5.5 and 8.0, which indicates the presence of both type 1 and type 2 isozymes. Kinetic parameters of porcine 5alpha-reductase in the presence of Serenoa repens extracts revealed uncompetitive, noncompetitive, and mixed types of inhibitions. Our results show the inhibitory action of S. repens on prostate porcine microsomal 5alpha-reductase activity.

Sex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway.

Estradiol (E2) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3alpha-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (R(SHBG)) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3alpha-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3alpha-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3alpha-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3alpha-diol can amplify or substitute for androgens, with regard to activation of the AR via the R(SHBG) by a signal transduction pathway involving cAMP. Because both E2 and 3alpha-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.

Differential expression of acidic and basic fibroblast growth factors in benign prostatic hyperplasia identified by immunohistochemistry.

To detect the expression and to determine the relative cellular locations of the two peptide growth factors, acidic fibroblast growth factor (a-FGF) and basic (b)-FGF in tissues from human benign prostatic hyperplasia (BPH). A series of 50 sequential and unselected cases of human BPH tissues, obtained after transurethral prostatectomy, was examined. Adjacent sections of formalin-fixed and paraffin wax-embedded tissues were stained immunohistochemically for expression of a-FGF and b-FGF using well-characterized and commercially available antibodies. The stained tissue sections were assessed for the cellular distribution of immunohistochemical products and analysed according to the relative intensity of staining as well as the spatial relationships of positively stained cells. Acidic-FGF was weakly expressed with a pancytoplasmic distribution within luminal glandular epithelial cells in regions of prostatic intra-epithelial neoplasia (both PIN I and II) but not by non-dysplastic normal or hyperplastic tissues. No expression of a-FGF was detected in basal epithelial cells or in the stromal compartment of any tissue examined. In contrast, b-FGF was strongly expressed within the cytoplasm of all basal epithelial cells, but not by luminal epithelial cells, in morphologically normal regions of all cases examined. Basal expression of b-FGF was diminished, or absent, in regions of mild epithelial dysplasia, particularly those strongly expressing a-FGF. Extensive nuclear and cytoplasmic expression of b-FGF occurred predominantly in smooth muscle-type stromal cells but not in all types of stromal cells. This study confirmed both a differential and a reciprocal expression of a- and b-FGF in non-dysplastic prostatic hyperplasia and in mildly dysplastic regions of prostatic tissues. While only small amounts of a-FGF were expressed in BPH, exclusively in the luminal epithelial compartment, its consistent appearance in PIN I and II suggests that it might contribute to the early stages of PIN. Conversely, b-FGF may be an important mediator of stromal-epithelial interaction during the pathogenesis of BPH. These results provide new information about the relative expression of these growth factors, particularly in the architectural relationships between different cell-types within normal and non-malignant prostatic tissues.