REVIEwS. Estrogen and androgen signaling in the pathogenesis of BPH. Clement K. M. Ho and Fouad K. Habib

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1 REVIEwS Estrogen and androgen signaling in the pathogenesis of BPH Clement K. M. Ho and Fouad K. Habib Abstract Estrogens and androgens have both been implicated as causes of benign prostatic hyperplasia (BPH). Although epidemiological data on an association between serum androgen concentrations and BPH are inconsistent, it is generally accepted that androgens play a permissive role in BPH pathogenesis. In clinical practice, inhibitors of 5α-reductase (which converts testosterone to the more potent androgen dihydrotestosterone) have proven effective in the management of BPH, confirming an essential role for androgens in BPH pathophysiology. To date, multiple lines of evidence support a role for estrogens in BPH pathogenesis. Studies of the two estrogen receptor (ER) subtypes have shed light on their differential functions in the human prostate; ERα and ERβ have proliferative and antiproliferative effects on prostate cells, respectively. Effects of estrogens on the prostate are associated with multiple mechanisms including apoptosis, aromatase expression and paracrine regulation via prostaglandin E2. Selective estrogen receptor modulators or other agents that can influence intraprostatic estrogen levels might conceivably be potential therapeutic targets for the treatment of BPH. Ho, C. K. M. & Habib, F. K. Nat. Rev. Urol. 8, (2011); doi: /nrurol Introduction the human prostate begins its morphogenesis at about weeks of gestation and prostatic growth continues while fetal plasma androgen levels remain high. 1 the fetal testis secretes testosterone into the circulation at sufficient levels to stimulate the differentiation and growth of the urogenital sinus tissues, leading to formation of the prostate gland. 2 after birth, plasma testosterone decreases to a low baseline level 3 and the prostate does not resume growth until puberty, when large amounts of androgens secreted from the testes stimulate prostatic cells to undergo morphofunctional maturation, giving rise to the various histological zones and functional tubuloalveolar glands. the human prostate reaches its full size of approximately 20 g and mature morphology at years of age. 4 Benign prostatic hyperplasia (BPH) is a pathological condition characterized by nonmalignant enlargement of the prostate gland, common in elderly men. Patients with BPH typically present with lower urinary tract symptoms (luts), which can be classified as either urinary tract obstructive symptoms such as hesitancy, inter mittent stream and straining, or urinary bladder irritation symptoms such as frequency, urgency and urge incontinence. urinary retention, whether acute or chronic, is also common. medical management of BPH and luts typically involves the reduction of prostate size by hormonal treatment, adrenergic blockade of urinary bladder tone or both. Histologically, BPH is characterized by hyperplasia of both epithelial and stromal tissues within the prostate Competing interests The authors declare no competing interests. gland. 5 in a review of studies including more than a thousand human prostate specimens, 50% of men between 51 and 60 years of age showed pathological features of BPH. 6 the percentage of men with histologically identifiable BPH at autopsy increases every year between 41 and 90 years of age. 4 autopsy studies performed in different parts of the world demonstrate similar age specific prevalence of BPH in many countries in europe, asia and the us. 7,8 Histological hyperplasia of the prostate is commonly observed in older men, so much so that some urologists and researchers consider it a natural occurrence during the process of prostate development and aging. only two factors are generally considered essential for the development of BPH, namely the presence of a testis, or more precisely androgens, and aging. to date, the exact etiology of BPH remains unresolved despite research data from a plethora of studies. the three main hypotheses embryonic reawakening, stem cell theory, and various hormonal theories are briefly summarized below. 7 During embryonic development, the prostate develops from the urogenital sinus under the influence of androgens secreted from the fetal testis, and prostate epithelial cells are directed towards functional differentiation by stromal signaling. 9 mcneal 10 proposed the embryonic reawakening hypothesis of BPH in 1978, which states that stroma derived factors associated with aging might induce glandular budding and branching, giving rise to new alveoli and eventually hyperplastic nodules. 10 epithelium stroma interactions might also be mediated by abnormal levels of growth factors (including Department of Biochemistry, Raigmore Hospital, ld Perth Road, Inverness IV2 3UJ, UK (C. K. M. Ho). Prostate Research Group, Room FU501, Chancellor s Building, 49 Little France Crescent, University of Edinburgh, Edinburgh EH16 4SB, UK (F. K. Habib). Correspondence to: F. K. Habib f.k.habib@ed.ac.uk nature reviews urology volume 8 JanuarY

2 Key points Androgens play a permissive role in the pathogenesis of benign prostatic hyperplasia (BPH) Inhibition of 5α-reductase activity is currently the mainstay of hormonal treatment of BPH Increasing evidence from epidemiological, animal and in vitro studies supports a role for estrogens in the pathogenesis of BPH Estrogen receptors ERα and ERβ mediate proliferative and antiproliferative effects of estrogens on prostate cells, respectively Some androgens are weak ligands for ERs but might have potent agonistic effects on prostate cells because of high tissue concentrations The prevalence of ERα and ERβ in hyperplastic prostate raises the potential of selective estrogen receptor modulators as potential therapeutic agents for BPH epidermal growth factor, basic fibroblast growth factor and transforming growth factor β [tgf β]) from either the epithelial or stromal compartment, resulting in the reawakening of embryonic cellular growth potential, and leading to hyperplasia. 11 the latter notion is supported by a transgenic mouse model, in which over expression of FGF3 resulted in epithelial hyperplasia of the murine prostate gland. 12 Furthermore, under hypoxic in vitro conditions, human prostate stromal cells in culture secrete increased levels of growth factors (such as FGF7 and tgf β) than normoxic cells, suggesting local hypoxia might be one of the triggers of embryonic reawakening of the prostatic stroma. 13 in 1989, isaacs and Coffey 7 proposed the stem cell theory of BPH pathogenesis, suggesting that an increase in the number of prostatic stem cells or an increase in the clonal expansion of existing stem cells into transit amplifying cells might be responsible. Precise distribution of stem cells in the human prostate is currently unclear, although both epithelial and stromal stem cell units are believed to be present in the adult prostate. 14 Biomarkers characteristic of stem cells have been identified in cultured stromal cells derived from human BPH tissues. 15 stem cells in the adult prostate might expand both the stromal and epithelial compartments in response to as yet unidentified stimuli, resulting in hyperplasia of the prostate. multiple hormonal theories of BPH etiology have been proposed and they are mainly focused on the actions of androgens, estrogens or both. the essential role of androgens in the development of normal prostate is widely accepted. a central role for androgens in the development of BPH was concluded from observations that castra tion significantly improved the symptoms of BPH. in addition, 5α dihydrotestosterone (DHt) concentra tions are significantly higher in hyperplastic than normal prostate tissues, although no difference in andro stenedione or testosterone concentrations between the two types of tissues have been detected. 16 recently, the abnormal prostate development observed in patients deficient in 5α reductase enzyme has highlighted the importance of 5α reduced androgens, such as DHt, in the normal growth and development of this gland. 17 Despite a widely accepted role for androgens in prostate development and growth, it is not entirely clear why BPH develops at a stage in life when plasma levels of androgens are gradually decreasing. in addition, multiple lines of new evidence from epidemiological, animal and in vitro studies support a role for estrogens in the pathogenesis of BPH. increasing estrogen:androgen ratios (in plasma and possibly also in prostate tissues) with advancing age has also been proposed as a factor for BPH. in this review, we will briefly summarize the biochemistry of androgens and estrogens in relation to the normal and hyperplastic human prostate. we will then discuss the existing epidemiological data on the association between increased levels of these sex steroids and BPH. the distribution of androgen receptor (ar), estrogen receptor (er), 5α reductase and aromatase in both normal and hyperplastic prostate tissues will be examined, and recent data on the mechanisms by which estrogens exert their actions in the prostate will be discussed. Finally, therapeutic inhibition of 5α reductase and aromatase enzymes directly involved in the metabolism and biosynthesis of androgens and estrogens in the prostate for the management of BPH will be discussed. Androgen and estrogen signaling Sex steroids in men sex steroid hormones, including androgens and estrogens, are synthesized from a common sterol pre cursor, cholesterol, by the concerted actions of multiple enzymes (Figure 1). in the normal male, testes are the major source of circulating androgens and the principal testicular androgen is testosterone. Besides testosterone, the testes also secrete small amounts of other sex steroids including androstenedione, DHt, estradiol and estrone. testicular function is regulated by two pituitary gonadotropins follicle stimulating hormone and luteinizing hormone (lh). upon stimulation by lh, leydig cells in the testis produce testosterone, which is secreted into the bloodstream. Plasma levels of the two gonadotropins are regulated by gonadotropin releasing hormone from the hypothalamus. the testis exerts an influence on the hypothalamic pituitary axis by negative feedback mechanisms involving androgens, estrogens and inhibin B. 18 Besides the testes, another important source of circulating androgens in man is the adrenal gland. major adrenal androgens include dehydroepiandrosterone (DHea), DHea sulfate and androstenedione. although these adrenal androgens are weak androgens compared with testosterone, they can be converted to more active steroid metabolites and thus indirectly alter prostate growth and function. the most potent androgen in men, DHt, is believed to be largely converted from testosterone in peripheral tissues such as prostate and skin by the action of 5α reductase isozymes (Figure 1). of the circulating estrogens in men, 75 90% are believed to derive from peripheral conversion of testosterone and androstenedione to estradiol and estrone, respectively (Figure 1), in adipose, brain, bone and other tissues. Conversion in the testes is responsible for 10 25% of circulating estrogens. 19 the two natural estrogens in man, estradiol and estrone, can be interconverted by enzymes belonging to the family of 17β hydroxysteroid dehydrogenases. 30 JANUARY 2011 volume 8

3 Cholesterol H Androgens DHEA Estrogens H 3βHSD 5α-Androstanedione Androstenedione Estrone 5αR Aromatase H 17βHSD 17βHSD 17βHSD DHT H Testosterone H Estradiol H 5αR Aromatase Figure 1 Biosynthesis and metabolism of the major androgens and estrogens in human prostate. Cholesterol is the precursor of all steroid hormones. All enzymes required for the conversion of DHEA, which is the most abundant steroid in the bloodstream (mainly secreted by the adrenal gland), to active steroid hormones including estradiol, testosterone and DHT, are expressed in the human prostate. Abbreviations: 5αR, 5α-reductase; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; HSD, hydroxysteroid dehydrogenase. H in plasma, most of the circulating steroid hormones are bound to carrier proteins. the major binding proteins are albumin, sex hormone binding globulin (shbg) and cortisol binding globulin. For example, approximately 2% of circulating testosterone is free or unbound in men, whereas the rest is either tightly bound to shbg or loosely bound to albumin. Free testosterone is thought to constitute the active hormone and provides a better measure of testosterone status in the body than total testo sterone level. measurement of free testosterone by the gold standard method of equilibrium dialysis is laborious and impractical for routine laboratory practice. to this end, many equations have been proposed to estimate plasma free testosterone levels in men using the measured concentrations of total testosterone, albumin and shbg. 20,21 steroid hormones, including androgens and estrogens, exert their effects on target gene expression by binding to their cognate intracellular receptors, which function as hormone inducible transcription factors. Androgen and estrogen receptors steroid receptors, including the ar and er, share a similar design of structural and functional domains (Figure 2). the n terminal activation function 1 (af 1) domain of ar is constitutively active in truncated receptor fragments containing no ligand binding domain (lbd). By contrast, the C terminal activation function 2 (af 2) domain operates in a ligand dependent manner and is more conserved in sequence than af 1 among steroid hormone receptors. 22 length of the ar polypeptide varies because of two trinucleotide repeat polymorphic stretches in exon 1 of the AR gene (Figure 2); a polyglutamine stretch and a polyglycine stretch are encoded by polymorphic CaG and GGC trinucleotide repeats, respectively. the number of CaG repeats, and thus length of glutamine repeats in the ar polypeptide, is inversely related to both basal and ligand induced transactivation activity of ar. 23 ar is expressed in many adult and fetal tissues, with the highest concentrations in reproductive tissues such as testis, prostate and seminal vesicle. 24,25 two isoforms of ar protein have been reported; ar a and ar B are encoded by a single gene. the shorter, n terminally truncated ar a isoform is produced by translation initiation at an alternate site the first internal methionine residue (met 188) (Figure 2). 25 in a study examining ar expression in adult and fetal reproductive tissues including the prostate, full length ar B was the predominant or only detectable isoform, accounting for 80% or more nature reviews urology volume 8 JanuarY

4 AR-B AR-A ERα ERβ 1 Gln (n) AF-1 DBD Hinge LBD/AF A/B C D E/F 1 1 Gly (n) Gly (n) (18%) (97%) (30%) (59%) Figure 2 Schematic representation of human ARs and ERs. General organization of domains A to F is shared by all steroid receptors including ARs and ERs. The N-terminal A/B domain is involved in transactivation, whereas the C-domain recognizes and binds to DNA sequences of target genes. The C-terminus (E/F domains) mediates multiple functions including ligand binding, dimerization and transactivation. AR-A and AR-B are encoded by a single AR gene with two different translational start sites separated by 187 amino acids. Polyglutamine (Gln) and polyglycine (Gly) tracts of variable lengths at the N-terminus of AR result in variable total lengths of AR-A and AR-B (approximately 732 and 919 amino acids, respectively). ERα and ERβ are products of two different genes. Percentages in parentheses indicate amino acid homology between structural domains of the two ER subtypes. Their nearly identical DBDs indicate that the two ER subtypes can bind to similar target genes, whereas low amino acid homologies in other domains suggest dissimilar ligand preference and transactivation functions between ERα and ERβ. Abbreviations: AF-1/-2, activation function 1/2; AR, androgen receptor; DBD, DNA-binding domain; ER, estrogen receptor; LBD, ligand-binding domain. Compiled using data from studies by williams and Franklin, 129 Katzenellenbogen et al. 151 and Gregory et al of the total ar immunoreactivity. 25 Currently, it is not known whether the two isoforms perform different physio logical functions. Both testosterone and DHt can readily bind to ar, while androstenedione and DHea have much lower affinities for ar. the relative binding affinities of the common circulating androgens are shown in table 1. Despite their ability to bind to the same receptor, DHt and testosterone are believed to have distinct roles in normal development. During embryogenesis, testosterone is essential for the development of wolffian duct structures such as the epididymis and vas deferens, whereas DHt is required for normal prostate formation and masculinization of male external genitalia. mechanisms by which the two androgens exert different physiological functions via the same ar are not fully understood. multiple mechanisms have been proposed, including differential ar binding affinity and dissociation, recruitment of different steroid receptor coregulators 29,30 and differential response owing to diverse androgen response element sequences. 31 erα and erβ are encoded by two distinct genes located on different chromosomes. the two er subtypes have the same arrangement of functional domains with the most conserved region being the Dna binding domain (97% identity), consistent with the receptors binding to similar Dna response elements. 32 However, af 1 domains share 18% homology only, suggesting that they might transcriptionally activate distinct estrogen responsive genes. 33 the lbds are relatively well conserved (59% identity), which explains their similar affinities for various estrogens and estrogenic compounds. the er subtypes have dissimilar relative binding affinities for a number of naturally occurring estrogenic compounds (table 1). some steroids traditionally considered weak androgens, such as 5 androstenediol and 3β androstanediol have moderate affinities for both er subtypes (table 1). an important metabolite of DHea in the prostate, 7α hydroxy dehydroepiandrosterone (7α oh DHea), has also been shown to bind and activate erβ in vitro; erβ and the enzyme CYP7B, which converts DHea to 7α oh DHea, are both expressed in the prostate epithelium, suggesting a role for CYP7B in regulating the estrogenic environment in prostate. 34 Androgen signaling in BPH The role of androgens androgens are widely accepted to be essential for normal prostate development and constitute an indispensible component of the pathophysiology of BPH. this notion is supported by clinical observations in patients with BPH treated by androgen withdrawal; prostate glands of patients with BPH markedly decreased in size upon either orchiectomy 35 or administration of the antiandrogen flutamide. 36 Given the androgen dependence of BPH, it is not clear why this condition is common at a stage of life when plasma androgen levels are declining with advancing age. multiple studies have examined possible relationships between the risk of BPH and circulating levels of androgens including testosterone and DHt but no consistent relationship has been established For example, in the third national Health & nutrition examination survey (nhanes iii) plasma total testosterone and calculated free testosterone concentrations were not different between control subjects and men with luts. However, those patients with luts were reported to have higher plasma levels of androstanediol glucuronide (a metabolite of DHt) than controls. 39 By contrast, higher total and bioavailable testosterone levels were associated with reduced BPH risk in the Prostate Cancer Prevention trial. 40 Hypogonadism is a relatively common condition in the aging man and provides a useful model for the study of androgen deficiency and prostate physiology. men with untreated androgen deficiency were reported to have reduced total and central prostate volumes measured by transrectal ultrasonography compared with age matched controls. Decreased total and central prostate volumes were also observed in men with treated androgen deficiency despite prolonged restoration of physiological testo sterone concentrations. 42 in comparison, healthy young men who were long term anabolic steroid abusers were shown to have approximately 20% higher central prostate volume but similar total prostate volume compared with age matched controls. the ratio of central to peripheral prostate volume was increased by 77% in anabolic steroid abusers, suggesting that the 32 JANUARY 2011 volume 8

5 central prostate is particularly sensitive to stimulation by supra physiological concentrations of androgens. 43 not all studies to date support the hypothesis that plasma androgen levels are directly proportional to prostate volume or severity of luts. nevertheless, chronic androgen deficiency owing to hypogonadism can be associated with reduced prostate size, whereas supraphysiological androgen levels can lead to enlargement of the prostate. although a role for androgens in causing BPH is debatable, it is generally accepted that they play at least a permissive role. also, plasma levels of androgens do not necessarily reflect their concentrations within the prostate gland. 44 DHt concentrations in BPH tissues are generally higher than testosterone concentrations, whereas the reverse is true in plasma. 45,46 in fact, the prostate not only responds to androgens derived from plasma but is also an organ rich in androgenmetabolizing enzymes including 5α reductase, which converts testosterone to the more potent DHt. an early report of higher intraprostatic DHt concentrations in BPH tissues than in normal tissues led to the hypothesis that local accumulation of DHt within the prostate can cause BPH. 16 However, walsh et al. 47 demonstrated that DHt levels were not higher in BPH than normal prostate tissues and argued that the previously reported differences in prostatic DHt levels were artifacts caused by differences in sample collection methods. 47 Androgen metabolism in the prostate a myriad of androgen metabolizing enzymes have been detected in the human prostate, including 5α reductase, 17β hydroxysteroid dehydrogenase, 3α hydroxysteroid dehydrogenase, 3β hydroxysteroid dehydrogenase and cytochrome P450 19a1 (CYP19a1; commonly referred to as aromatase) (Figure 1); among these, 5α reductase is probably the enzyme most relevant to normal prostate development and the treatment of BPH. 48 within the prostate, androgen metabolizing enzymes expressed in different cell types might function in concert with one another to produce a particular androgen metabolite; this is, to a certain extent, analogous to the zonal arrangement of steroidogenic enzymes in the adrenal gland and ovary. labrie et al. 49 proposed that DHea is converted to testosterone in basal cells of the prostatic epithelium, whereas testosterone is converted to DHt by 5α reductase within basal cells or luminal epithelial cells. 49 Both testo sterone and DHt can bind to ar, which is predominantly expressed in the luminal epithelium (Figure 3). whether this type of local steroid metabolism can act as a crosstalk signaling mechanism between various compartments of the prostate and thus contribute to the development of hyperplasia awaits further investigation. Ar involvement in BPH many studies have demonstrated ar immuno reactivity in most nuclei of the luminal epithelium in normal and hyperplastic human prostate tissues, compared to only a small number or none of the basal epithelial cells showing immunostaining. some stromal cell nuclei were also immunoreactive (table 2) to date, no consistent Table 1 Relative binding affinities of ligands for AR and ERs 29, Compound relative binding affinity Ar (rat) Erα (human) Erβ difference in ar expression levels between normal prostate and BPH tissues has been identified. Because BPH is an androgen dependent condition and the number of CaG repeat lengths in exon 1 of the AR gene is inversely related to transcriptional activity of ar, Giovannucci et al. 55 examined the hypothesis that men with shorter AR gene CaG repeat lengths have an increased risk of BPH. odds ratio (or) for BPH surgery or enlarged prostate gland in men with 19 or fewer CaG repeats was 1.92 (95% Ci ) compared with those having 25 or more CaG repeats. 55 in contrast to the above study on health professionals in the us, other studies conducted in Japan, 56 the netherlands 57 and scotland (F. K. Habib, unpublished work) have not identified any difference in the number of CaG repeats in exon 1 between patients with BPH and age matched controls. whether the number of CaG repeats in the AR gene is related to risk of BPH remains unresolved. 5α-reductase two distinct isozymes of 5α reductase type 1 (5αr1) and type 2 (5αr2) have been identified in humans Both isozymes can convert testosterone to DHt and androstenedione to 5α androstanedione (Figure 1). several other steroids including progestogens and glucocorticoids are also substrates of the two isozymes. 61,62 testosterone is the most important physiological substrate for both 5α reductase isozymes. 62 the human 5αr1 gene (SRD5A1) is located on chromo some 5 and consists of 5 exons. the gene encodes a protein of 259 amino acids with a predicted Human Estradiol * 100* 100* Estrone < NR 37 Estriol < NR 21 Diethylstilbestrol Tamoxifen Progesterone <0.01 <0.01 < Androstenediol Androstenediol NR 0.6 3β-Androstanediol 12 3 NR 7 3α-Androstanediol NR 0.3 DHEA NR α-Androstanedione NR <0.01 NR <0.01 Androstenedione 1.24 <0.01 NR <0.01 DHT NR 0.17 Methyltrienolone NR NR NR Testosterone 100* <0.01 <0.01 <0.01 *Relative binding affinities of testosterone for ARs and estradiol for ER subtypes are arbitrarily set at 100. Ligand-binding domains of human ARs and rat ARs are identical, 156 whereas ligand-binding domains of human ERβ and rat ERβ share 93% homology. 157 Abbreviations: AR, androgen receptor; DHEA, dehydroepiandrosterone; DHT, 5α-dihydrotestosterone; ER, estrogen receptor; NR, not reported. rat nature reviews urology volume 8 JanuarY

6 Luminal epithelium Basal epithelium Basement membrane Stroma Testosterone Lumen AR DHT 5αR1 Testosterone DHT 5αR2 Aromatase Testosterone Estradiol ERβ Estradiol Estradiol ERβ Apoptosis Apoptosis Cell proliferation Figure 3 Actions of ER subtypes in the major prostate cell types. ERα and ERβ are differentially expressed in the two major compartments of the prostate; ERα is mainly but not exclusively localized to the stroma, whereas ERβ has been identified in the epithelium and some stromal cells in most studies. Experimental evidence indicates that ERα mediates cell proliferation, while activation of ERβ results in apoptosis. Aromatase is predominantly localized to the stromal compartment and regulation of its expression or activity represents a potential therapeutic target for treatment of benign prostatic hyperplasia. Abbreviations: 5αR, 5α-reductase; AR, androgen receptor; DHT, dihydrotestosterone; ER, estrogen receptor. ERα Table 2 Distribution of signaling components in BPH tissues Signaling protein Prostate compartment luminal epithelium Basal epithelium Stroma AR ++ ± ± 5αR αR2 ± ++ ± ERα ERβ ± Aromatase ± ± + ++, abundant expression; +, weak expression; ±, expressed in only some cells. Abbreviations: 5αR, 5α-reductase; AR, androgen receptor; BPH, benign prostatic hyperplasia; ER, estrogen receptor. mol ecular weight of 29 kd. over 40% of the amino acids are hydrophobic and only 60% are identical to those of the rat 5αr1 isozyme. 58 the human 5αr2 gene (SRD5A2; chromosome 2) also consists of 5 exons and encodes a hydrophobic protein of 254 amino acids, of which 50% are identical to those of 5αr1 and 77% identical to those of the rat type 2 isozyme. 59 5α-Reductase in prostate development all tissues within the prostatic capsule are thought to derive from the urogenital sinus, the only exceptions being the intraprostatic vasa deferentia and ejaculatory ducts, which are of wolffian duct origin. normal prenatal development of the prostate and wolffian ducts requires androgen stimulation. Differentiation of the wolffian ducts into seminal vesicles and epididymides is completed in the human male embryo by approximately 13 weeks of development, before the capacity to produce DHt is acquired by these tissues. 63 while wolffian duct differentiation is testosterone dependent, DHt is essential for the development of the prostate and virilization during fetal growth, and mediates sexual maturation during puberty, including development of the external genitalia and growth of facial hair. 63 5αr2 is the predominant isozyme detectable in fetal genital skin and male accessory sex glands including the prostate. 64 the importance of DHt and 5αr2 in normal prostate development is best illustrated by a form of male pseudohermaphroditism as a result of 5αr2 deficiency. affected individuals have normal male internal wolffian duct derived structures (epididymides, vasa deferentia and seminal vesicles), confirming that wolffian duct development is DHt independent, but ambiguous or female external genitalia and rudimentary prostates. 17,59,65 transrectal ultrasonography and mri demonstrate that adult pseudohermaphrodites with 5αr2 deficiency have significantly smaller prostates than age matched control males, 17 which appear as plate like soft tissue structures on imaging studies. transrectal ultrosonography guided prostate biopsies revealed fibrous connective tissue and smooth muscle but no epithelial tissue. 66 the enzyme 5αr2 is therefore essential for the normal development of human prostate through its role in converting testosterone into DHt, which in turn stimulates the morphological and functional maturation of the prostate. no 5αr1 deficiency has yet been identified in humans. Intraprostatic distribution of isozymes Both isozymes of 5α reductase are expressed in BPH tissues (table 2). strong and uniform mrna expression of both 5αr1 and 5αr2 has been demonstrated in the epithelial component of BPH specimens; staining was particularly intense in the basal cells and was weaker and more variable in the stroma. 67,68 in another study, 5αr1 mrna was localized to many epithelial cells and few stromal cells in BPH tissues, whereas intensity of 5αr2 mrna labeling was similar in epithelium and stroma. 69 using quantitative real time PCr, significantly higher mrna levels of 5αr1 than 5αr2 were detected in BPH tissues. 70 with regard to zonal distribution, both 5αr1 and 5αr2 mrnas have been detected in all of the peripheral, transitional and central zones of normal prostate. 69 immunohistochemical studies have shown strong 5αr1 reactivity in the epithelium of normal and hyperplastic glands. 70,71 5αr2 has been localized mainly to the epi thelial compartment but immunoreactivity in the stroma has also been reported. three reports on normal and hyper plastic prostate tissues demonstrated 5αr2 immuno staining predominantly in basal epithelial cells and either absent or less intense in the luminal cells; some stromal cells were immunoreactive but less so than basal cells By contrast, another study reported strong 5αr2 enzyme expression in the stroma of both adult hyperplastic tissues and fetal prostates of weeks gestation, with little immuno reactivity in basal or luminal epithelial cells JANUARY 2011 volume 8

7 taken together, the above studies indicate that both stromal and epithelial cells of the human prostate express 5αr1 and 5αr2. therefore, the one cell type one isozyme distribution pattern of 5αr1 and 5αr2 previously observed in the rat prostate 75 cannot be applied to men. 5αr2 is thought to be the predominant isozyme in the adult human prostate and is responsible for converting the bulk of testosterone to 5α reduced metabolites in BPH tissues. 67,76,77 expression of the two isozymes might also exhibit variability within different regions of the prostate, among different individuals or according to the developmental status of the gland. Clinical inhibition of 5α-reductase activity the notion that 5α reductase activity is altered in the hyperplastic prostate is contentious. 78,79 semiquantitative reverse transcription PCr studies revealed that mrna expression of both 5αr1 and 5αr2 in BPH specimens was slightly but significantly increased compared with normal tissues. 69 issacs et al. 78 reported higher 5α reductase activity in hyperplastic tissues, with lower activity of 3α hydroxysteroid dehydrogenase and 3β hydroxysteroid dehydrogenase (which catabolize DHt) when compared with normal prostate tissues. 78 In vitro, 5α reductase activity in fibroblasts cultured from BPH tissues was approximately eight fold higher than in fibroblasts from normal tissues. 80 However, another study reports no difference in 5α reductase activity between normal and hyperplastic tissues. 79 whether BPH tissues have different 5α reductase activity, and thus DHt concentrations, to normal prostate is unclear. However, it has been proven that treatment of patients with BPH with a 5α reductase inhibitor decreases both intraprostatic DHt concentrations and prostate size. 77 two inhibitors of 5α reductase activity are currently available for the clinical treatment of BPH; by inhibiting the conversion of testosterone to DHt. Finasteride and dutasteride both markedly reduce plasma and intraprostatic DHt concentrations, accompanied by slightly increased testosterone levels in plasma. 81,82 Finasteride, a competitive inhibitor of 5αr2 with relatively low affinity for 5αr1, 83 has been used in clinical practice since the early 1990s and is now one of the most commonly prescribed drugs for the management of BPH. multiple clinical studies have examined the efficacy and safety of finasteride. a systematic review of randomized trials demonstrated that finasteride produced greater improvements in total symptom score, maximum urinary flow rate, and prostate volume compared with placebo controls. However, adverse effects including sexual dysfunction, impotence, ejaculation disorder and decreased libido were more common in men who had received finasteride for 12 months than control individuals. 84 the efficacy and safety of dutasteride, a non selective 5α reductase inhibitor, have been investigated in three randomized clinical studies, which compared men receiving 0.5 mg dutasteride daily with a placebo group. at 24 months, serum DHt levels were reduced from baseline by more than 90%, and the total prostate and transition zone volumes were reduced by more than 20%. symptom score and maximal flow rate improved significantly, resulting in reduced risk of acute urinary retention and BPH related surgical intervention compared with placebo. 85 Gynecomastia, impotence, and decreased libido were more commonly reported by men treated with dutasteride than the placebo group. in a clinical study comparing the effects of finasteride and dutasteride, 399 patients with BPH were randomized to receive dutasteride (at a dose of 0.01 mg, 0.05 mg, 0.5 mg, 2.5 mg or 5.0 mg daily), finasteride (5 mg daily), or placebo for 24 weeks. Dutasteride 5 mg daily and dutasteride 0.5 mg daily decreased serum DHt concentra tions by 98.4% and 94.7% respectively, compared with a 70.8% reduction of DHt by finasteride 5 mg daily (P <0.001). 86 an increasing number of plant extracts (phytotherapeutic agents) such as saw palmetto, south african star grass, stinging nettle, african plum and rye pollen, are available and purported to relieve luts, treat BPH or both. a comprehensive review of the use of herbal extracts in the treatment of BPH is beyond the scope of this article and the reader is referred to other reviews on the topic. 87,88 an example of such phytotherapeutic agents is a lipidosterolic extract of the fruit of saw palmetto, Permixon (Pierre Fabre médicament, Castres, France), containing predominantly free fatty acids; it was shown in clinical studies to improve urological symptoms and urinary flow measures in patients with BPH. 89,90 the exact mechanism of action of Permixon is not known, although it has been shown to inhibit both isozymes of 5α reductase in a baculovirus directed insect cell system expressing either of the two isozymes, 91 and in human BPH derived cells in primary culture. 92 other endocrinerelated effects of this plant extract include inhibition of DHt binding to ar 93 and reduction of the concentrations of nuclear estrogen receptors in prostate tissues removed from patients with BPH. 94 Estrogen signaling in BPH The role of estrogens in the 1970s, animal models of BPH that demonstrated a synergistic effect between estrogens and androgens in inducing glandular prostatic hyperplasia in castrated dogs 95,96 first led to the hypothesis that estrogens might also induce prostatic hyperplasia in man. when considering a role for estrogens in the pathogenesis of BPH, it is important to examine both direct and indirect effects of estrogens on the prostate. on the one hand, high levels of circulating estrogens can indirectly cause regressive changes in the prostate, believed to be mediated by the inhibition of pituitary gonadotropin secretion, leading to decreased testicular output of androgens and lowered plasma androgen levels. 43 on the other hand, estrogens might exert their effects directly on prostate cells, mediated by their cognate receptors expressed in the prostate. these direct effects of estrogens will be further discussed below. Epidemiological evidence Cross sectional and longitudinal studies have shown that with advancing age, plasma androgen levels in nature reviews urology volume 8 JanuarY

8 men decrease gradually while estrogen levels remain constant or decrease slightly, resulting in increasing estrogen:androgen ratios in plasma. 38, in addition, plasma estradiol levels are positively correlated to body mass index and subcutaneous fat mass, most likely as a result of increased total aromatase activity and thus conversion of androgens to estrogens in adipose tissues. many studies have reported a relationship between plasma estrogen levels and the risk of BPH ,101 in the Physicians Health study, multivariate analyses demonstrated a strong trend for increasing risk of BPH with increasing estradiol levels (highest quintile versus lowest quintile or 3.56; P = 0.009); the excess risk associated with high estradiol level was confined to men with relatively low androgen levels. 37 in another study, increased prostate volume (>30 ml) was associ ated with estradiol level (upper tertile versus lower tertile or 3.5; 95% Ci ) in men with bioavailable testo sterone levels above sample median, whereas no association between estradiol level and prostate volume was detected in those with bioavailable testosterone levels less than the sample median. 38 the latter finding is suggestive of a synergistic effect between testo sterone and estradiol in the pathogenesis of BPH. in the nhanes iii study, increased risk of luts in men was positively correlated with estradiol levels (upper tertile versus lower tertile or 1.78; 95% Ci ) and estradiol:testosterone ratios (upper tertile versus lower tertile or 2.52; 95% Ci ). 39 By contrast, in the Prostate Cancer Prevention trial, mean total estradiol levels were slightly (3.3%) lower in patients with BPH than age matched controls, whereas no difference was detected in free estradiol or estrone levels between the two groups. 40 Besides estradiol, weak estrogens have also been implicated in causing BPH. For example, 5 androstenediol has relatively low affinity for erα and erβ compared to estradiol (table 1) but mean plasma levels of 5 androstenediol (2.2 nmol/l) were found to be more than 20 fold higher than estradiol (0.064 nmol/l) and estrone (0.098 nmol/l) in males with BPH, 102 suggesting that 5 androstenediol might have an estrogenic role in males. 7α hydroxy DHea, a metabolite of DHea in the prostate, was also shown to have weak estrogenic activity 34 and its serum concentrations are much higher than estradiol in healthy males. 103 in summary, most but not all studies have demon strated an association between estrogen levels in blood and risk of BPH or luts. Estrogen effects on prostate cell proliferation many studies have examined the effects of estradiol on prostate cells in primary culture estradiol has been shown to stimulate the proliferation of stromal cells derived from both BPH tissue 105, and normal prostate. 106,109 these proliferative effects were antagonized by the antiestrogens fulvestrant 107,109 and tamoxifen, 105 suggesting that estradiol increases stromal cell proliferation via an er mediated mechanism. By contrast, estradiol did not affect the proliferation of BPH derived prostate fibroblasts in another study. 104 the in vitro proliferation of epithelial cells derived from BPH tissues 107 and normal prostate 106 was also demonstrated to be unaltered by estradiol treatment. taken together, results from most published studies indicate that estradiol increases the proliferation of prostate stromal cells but not epithelial cells via an er mediated mechanism. the differential effects of estradiol on the two main prostate cell types in vitro are in concordance with a previous in vivo study, which showed that estradiol treatment increased the volume of the prostate stromal compartment but decreased that of the epithelial compartment in male rats that had been castrated and supplemented with testosterone. 110 Possible interaction between androgen signaling and estrogen signaling on prostate stromal cell growth in vitro was investigated by another study. 106 treatment of stromal cells with a fixed concentration of estradiol ( mol/l) plus increasing concentrations of DHt ( mol/l) increased cell numbers above controls (no DHt) in a dose dependent manner, as did treatment with a fixed DHt concentration ( mol/l) plus increasing concentrations of estradiol ( mol/l). these observations lend support to the hypothesis that androgen and estrogen synergistically augment prostate cell growth in vitro. Expression and function of Er subtypes estrogens exert their effects on target gene expression via binding to specific intracellular ers, which function as hormone inducible transcription factors. the different effects of estradiol on stromal and epi thelial cells have been hypothesized to depend on differ ential er expression in the two main prostate cell types. localization of the two er subtypes in human prostate has been reported in numerous studies and is summarized in table 2. 34, to date, the majority of studies have detected erα expression mainly, but not exclusively, in the stroma of normal prostate and BPH tissues ,115,116 at least five studies have reported the expression of erβ in prostate epithelium; some stromal cells were also found to be erβ immuno reactive in most studies. 34, Differential localization of the two er subtypes in prostate epithelial and stromal cell compart ments remains to be clarified by further studies, but reports to date have identified erα and erβ predominantly, but not exclusively, in the prostate stroma and epithelium, respectively (Figure 3). the expression of multiple variants of erα 117,118 and erβ 119,120 has been reported in human prostate tissues and prostate derived cell lines. Current nomenclature of these er splice and deletion variants is not uniform in the literature. erβδ5 is an alternatively spliced isoform of erβ with a truncated C terminal ligand binding domain and was found to inhibit estradiol transactivation of both erα and erβ. 121 erβ2, also known as erβcx, is another alternative splice variant of wild type erβ, also designated erβ1; it shares identical n terminal and Dna binding domains with erβ1 but differs from the latter by the substitution of amino acids at the C terminus with a unique 27 amino acid sequence. 120 erβ2 is believed to regulate erβ1 mediated action by forming heterodimers with erβ1 and reducing liganddependent activity. 119,120 the co localization of erβ2 36 JANUARY 2011 volume 8

9 (erβcx) and wild type erβ1 in BPH tissues suggests that erβ2 might regulate erβ signaling in prostate cells. 116 Functionally, animal studies have revealed that estrogens exert proliferative and apoptopic effects on prostate cells via erα mediated and erβ mediated mechanisms, respectively. However, few studies have investigated the differential role of the two er subtypes in the human prostate. activation of erβ with the erβ selective agonist 8β ve2 in the benign human epithelial cell lines BPH 1 and rwpe 1, resulted in apoptosis. 122 when human BPH specimens were grafted into male mice, increased apoptosis was detected in both epithelial and stromal cells in the human xenografted tissue in erβ agonist treated mice but not vehicle treated mice. in the same study, increased apoptosis of the Du145 prostate cancer cell line was observed in 8β ve2 treated cells and abrogated by sirna knockdown of tumor necrosis factor α (tnf α), suggesting that erβ mediated apoptosis is mediated by tnf α. 122 in another study, estrogenstimulated prolifera tion of the normal human stromal cell line wpmy 1 was decreased by transfection of small hairpin rna to knock down erα mrna levels, suggesting that the proliferative effect of estradiol on prostate stromal cells is mediated by erα. 108 selective estrogen receptor modulators (serms) are drugs that can modulate er activity, with effects ranging from nearly full estrogenic to almost pure anti estrogenic. 123 serms including tamoxifen, 4 hydroxytamoxifen, toremifene and raloxifene suppressed proliferation of normal human prostate epithelial 124 and stromal cells in vitro. 124,125 if human prostate cell growth in vivo can be proven to be stimulated and suppressed by erα and erβ, respectively (Figure 3), then conceivably a serm that inhibits erα but activates erβ activity would be a potential therapeutic agent for the management of BPH. Besides ers and ars, another type of sex steroid receptor, the progesterone receptor (Pr), is also found in both epithelium and stroma of human prostate tissues Pr shares some amino acid homology with both ars and ers. 129 although its expression in the prostate is responsive to estrogen stimulation, 130 little is known about the role of progesterone and its receptor in prostate pathophysiology. whether progesterone signaling interacts with androgens and estrogens in the human prostate awaits further investigation. Aromatase the human aromatase gene (CYP19A1) is located on chromosome 15 and consists of 10 exons, spanning more than 120 kb of genomic Dna. 131 the coding region of CYP19A1 spans nine exons beginning with exon 2. tissue specific expression of aromatase is determined in part by alternative use of tissue specific promoters, which give rise to mrna with unique 5' noncoding ends. 132,133 a number of untranslated exons with unique promoter regions have been identified, 131,134 and several splice variants have been identified in mrna prepared from both BPH and cancer tissue samples, 132,135 but there is no consensus on the predominant splice variant expressed in normal and hyperplastic prostate tissues. aromatase catalyzes the conversion of androgen precursors to aromatic estrogens, and is associated with nadph cytochrome P450 reductase. 136,137 the essentially ubiquitous nadph cytochrome P450 reductase enzyme transfers reducing equivalents from nadph to any microsomal cytochrome P450 with which it comes into contact. 136 this reaction results in aromatization of the a ring to give the phenolic structure characteristic of estrogens. 133 Both androstenedione and testosterone are substrates for aromatase. However, 5α reduced steroids such as DHt and 5α androstanedione cannot serve as precursors because 5α reduction of the a ring prevents the completion of aromatization. in men, estradiol is thought to be formed primarily by aromatization of testo sterone in adipose tissue. Aromatase in the prostate aromatase is expressed in a wide variety of human tissues including adipose tissue, various regions of the brain, placenta, ovary and testis. 132 aromatase mrna expression has also been identified in human prostate tissues. 107,132,135,138 in contrast to the uncertain distribution of er subtypes, immunostaining of aromatase in human prostate is less controversial. Both normal prostate and BPH tissues showed pronounced aromatase immunoreactivity in the stroma in two studies, 135,139 and the epithelium was also stained when a higher antibody concentration was used. takase et al., 140 on the other hand, identified aromatase immunostaining in both stromal cells and luminal cells of the epithelium in BPH specimens. 140 immunoreactivity in stromal cells was also found to have marked heterogeneity among different regions of individual prostate specimens. 135 taken together, most studies have localized aromatase to the stromal compartment of human prostate, suggesting that regulation of aromatase expression and thus local conversion of androgens to estrogens might play a role in estrogen signaling within the stroma. the issue of aromatase enzyme activity in the human prostate was controversial until recently. although three studies have failed to detect any aromatase activity in BPH or prostate cancer tissues, androgen aromatiza tion in normal, BPH and cancerous prostate tissues was shown by other investigators. 107,135, aromatase activity was also detected in primary cultures of human prostate stromal cells. 107,144 Discrepancies in results from previous studies are probably owing to the relatively low aromatase activity in the prostate compared to 5α reductase isozymes and other androgen metabolizing enzymes. recently, aromatase mrna expression in prostate stromal cells derived from BPH tissues was found to be inducible by prostaglandin e2 and prostate epithelial cell conditioned culture medium. 147 these effects were abrogated by a CoX 2 specific inhibitor, suggesting that epithelial cells regulate aromatase expression in stromal cells via a paracrine mechanism that involves the secretion of prostaglandin e Prostaglandin e2 mediated regula tion of aromatase in stromal cells has been further confirmed by another study demonstrating that er related nature reviews urology volume 8 JanuarY