In addition to hormonal regulation, the prostate is clearly under paracrine and autocrine regulation, as has been demonstrated by epithelial/mesenchymal recombination studies in vivo14,16,240,241 and coculture/conditioned media studies in vitro.242-244 Some growth factors such as nerve-growth factor (NGF)/trfc and NGF-P receptors245,246 as well as hepatocyte growth factor/met recep-tors247-249 operate as classic paracrine mediators, with ligand produced by the prostatic stroma and the receptors present on the basal and/or luminal epithelial cells. In contrast, TGF-P250,251 and IGF-II134 have the necessary components to utilize both paracrine and autocrine (epithelial to epithelial or stromal to stromal) pathways. The role played by these growth factor pathways in pro-static physiology and disease as well as their integration into the other prostatic regulatory pathways are currently being actively researched. This review focuses on three of the better characterized systems in the prostate that represent two strictly local pathways: (1) EGFs (generally stimulatory to proliferation); (2) TGF-Ps (generally inhibitory for proliferation); and (3) IGFs, which act by both endocrine and local pathways.
Epidermal Growth Factor Family Pathway The EGFR family of membrane-localized tyrosine kinases has to date four members: (1) erbB-1/EGFR (170 kDa); (2) erbB-2/neu/HER-2 (185 kDa); (3) erbB-3 (160 kDa); and (4) erbB-4 (170 kDa).183,252 Depending on the particular ligand bound and the complement of receptors present, homo- or heterodimers of receptor subunits are formed following ligand binding.183,252 To date, no ligand has been identified for P185erbB appears to not have an active tyrosine kinase domain.183 This forces these two receptors to form obligate heterodimers with other members of the EGFR family for ligand-directed intracellular signaling. In the human prostate, all four EGFR/erbB family members have been identified in the epithelial compartment, with all but P170erbB-4 being expressed primarily in the basal epithelium;
P170erbB has been reported to be expressed in both the basal and secretory epithelium.253-257 There are also persistent reports of some EGFR expression in the prostatic stroma.258,259 Multiple ligands for the EGFR family have been detected in the normal human prostate, including EGF,260 and heparin-binding (HB)-EGF,261 which have been localized to the stroma. Immunoreactive TGF-a has been identified inconsistently in a small proportion of normal human prostatic epithelial cells.257,258 The presence or absence of TGF-a in the stroma of the normal human prostate is still uncertain.257,258 Together, these results suggest that the EGFR/erbB family acts as a paracrine pathway from the stroma to the epithelium. Unlike the other ligand of the EGFR/erbB family, neu differentiation factor (NDF/hereg-ulin) has been localized in the stromal, basal, and secretory epithelium.254
In rats, EGFR P185erbB-2 and P160erbB-3 have been identified in normal prostates and cell lines.202,262,263
Both EGFR and TGF-a have been localized to the lumi-nal epithelium of the ventral prostate in the rat; thus the EGFR-TGF-a axis may operate, unlike in the human prostate, as an autocrine rather than a paracrine path-way.264,265 There has been no TGF-a detected in the stroma of any of the three lobes.264 Epidermal growth factor is also expressed by the rat prostatic epithelium, with the highest levels of expression in the dorsal prostate, although some expression has also been detected in the ventral lobes.266,267 There has been no EGF detected in the rat prostatic stroma.266
Interest in EGF in relation to the prostate is in part due to its role in proliferation and its use as an additive to tissue-culture medium. Epidermal growth factor stimulates the proliferation of prostatic epithelial cells in vitro268,269 and following orthotopic injection.270 It has also been reported to stimulate the growth of fetal prostates and adult human fibroblasts.271 The EGFR family is also believed to play a role in PIN and PCa, as there are frequently changes in levels of expression and a shift of localization of expression with these conditions. Prob ably the best documented change is the dramatic increase in P185erbB-2 expression seen in PCa.253 The P185erbB-2 is able to form active heterodimers with all three other EGFR/erbB receptors, and it can be activated by a variety of ligands, including NDF, EGF, and TGF-a.183,272 An upregulation of this receptor would therefore dramatically increase the activation pathways available to the cell. Experiments with nontumorigenic epithelial cells from the rat ventral prostate have demonstrated that increased expression of P185erbB-2 can induce tumorigenesis and metastasis.262 Each of P185erbB-2 EGFR, P160erbB-3, and TGF-a has been reported, albeit inconsistently, to show increased expression in the luminal epithelium in PIN or PCa as well as in human prostate epithelial cell lines derived from PCa.255,256,273-276 It is possible that acquisition of an autocrine loop for the EGFR/erbB pathway may be a key event in the progression of PCa. The decreased expression of EGF in PCa compared to the normal prostate has also led to speculation that a shift in available ligands (e.g., decreased EGF, increased TGF-a) may also play a role in the progression of PCa.256,274
Transforming Growth Factor-P Inhibition of prostatic growth and the subsequent induction of either differentiation or apoptosis also appear to be regulated by paracrine factors.133,277-283 Of these pathways, the TGF-P family (TGF-P1, -2, and -3) has been the most intensively studied. The TGF-Ps are classically thought of as inhibitors of epithelial growth and inducers of apopto-sis in epithelial cells from several organs.185,284 They are secreted in a latent form that must subsequently be acti-vated.185,246,284-286 Activation can occur through several mechanisms, including integrins avp 1,285 IGFR-II,287 and extracellular proteases.185 Based on the localization of receptors and ligand, TGF-P, at least in the human prostate, should be able to act by both paracrine and autocrine pathways. The receptor is a transmembrane heterodimer with serine/threonine kinase activity.185,284 In the human prostate, both TGFR-I and TGFR-II are present in the glandular epithelial cells and colocalize with cells coexpressing smooth muscle a-actin in the stroma.251,288 Immunoreactivity of the ligand, TGF-P 1, has also been localized to both the epithelial and stromal compartments.250 Immunoreactivity of TGF-P2 and -3 is preferentially localized in the epithelium, with TGF-P3 localized to the basal epithelial cells.289 Both receptors as well as TGF-P 1 and -3 have been detected in the ventral prostate of the rat.286,290,291
The TGF-Ps and their receptors were one of the first proteins shown to be negatively regulated by androgens, as their expression was dramatically upregulated immediately following castration and downregulated with subsequent androgen replacement.180,198,291,292 Continuous perfusion with TGF-P 1 in vivo reduces wet weights of the ventral prostate without reducing serum testosterone, implying that the effects of TGF-Ps are downstream of those of testosterone.293 In vitro, exposure to TGF-P 1 induces apoptosis in NRP-154 cells and primary cultures of prostatic epithelial cells as well as inhibiting the growth of primary cultures of epithelial cells from the rat ventral prostate.294,295 However, it should be noted that in both studies results were found to be highly dependent on culture conditions. The TGF-Ps also inhibit the proliferation of prostatic stromal cells296,297 and induce differentiation of primary stromal cultures from a fibroblastic phenotype (expression of fibronectin and vimentin) to the smooth muscle phenotype (expression of a-smooth muscle actin).185,296 Epidermal growth factor can attenuate the TGF-Ps' induction of smooth muscle phenotype,269 their ability to induce apoptosis,295 and their inhibition of pro-liferation.298 The TGF-Ps are able to inhibit androgen and EGF-stimulated growth; treatment of castrated rats with EGF increases the expression of TGF-P 1 mRNA in prosta-tic stromal cells, suggesting a feedback loop between growth stimulators and inhibitors.192,290
Paradoxically, the overexpression of TGF-Ps in tumorigenic prostate cell lines has been shown to induce greater tumor growth, be associated with induction of carcinomas and hyperplasia in the myc+ras transgenic mice prostate model, and to possibly induce hyperplasia in murine recombinant prostates.241,284,299 The pheno-typic changes by which TGF-P promotes tumorigenesis are likely to be multiple, including alterations in the expression of extracellular matrix components, integrins, protease inhibitors, and metalloproteases.185,300-302
Insulin-Like Growth Factor The IGF pathway in the prostate differs from that of other growth factors in that it has both endocrine (IGF-I) and paracrine (IGF-II) components.184,303 IGFR-I, which is activated by IFG-I > IGF-II >>> insulin, is present on human prostatic epithelial cells and adenoma cell lines.135,304 As judged by in situ hybridization and RT-PCR, IGFR-I is present at low levels in the prostatic stroma.135,305 The IGFR-II component has been less well characterized and has been reported to be present only on the adenoma cell line DU 145278 and on benign human stromal cells.305 The primary source of serum and presumably prostatic IGF-I is the liver.184,303 The IGF-I protein has not been identified in the conditioned media of primary cultures of human prostate fibroblasts or epithelial cells134,305,306 or in prostatic carcinoma cell lines.279,307
In the rat, however, IGF-I has been identified in the prosta-tic stroma308,309 and IGF-II has been detected in the pro-static epithelium,135 stroma,306 and in the conditioned media of prostatic stromal and epithelial cells.306 It thus may act as an autocrine (epithelial to epithelial) or paracrine (stromal to epithelial) regulator in the rat.
The activity of the IGF pathway is also modulated by binding proteins, originating both in the sera and locally.134,184 As judged by combinations of protein (immunohistochemistry, Western blot) and mRNA (Northern blot, in situ hybridization), both prostatic fibroblasts and epithelial cells express IGFBP-1 through IGFBP-7.134,306,310-314 The ability to liberate serum-derived IGF-I from its carrier proteins (primarily IGFBP-3) is another mechanism by which this pathway is regulated. At least three prostatic enzymes—PSA,133 cathepsin D,315 and urokinase279—are capable of releasing IGF-I from IGFBP-3. Whether the basally secreted PSA or the increased expression of urokinase and cathepsin D in PCa101,128,316,317 actually liberate enough IGF-I to stimulate the growth of the tumor remains to be determined (see Figure 14-4).
Several in vivo and in vitro experiments have demonstrated that the IGF pathway can have an effect on pro-static growth. The prostate is vestigial in IGF-I null mice,318 and systemic treatment of IGF-I in rats increases wet weight of the ventral and dorsal lateral prostate by increasing the proportion of epithelial cells.319 In vitro, both IGF-I and IGF-II increase growth of prostatic epithelial cells and PCa cell lines.134,320,321 Also, expression of antisense message to IGFR-I reduces tumor size and metastatic capabilities of the PA-III rat adenocarcinoma model.322 Using the Noble rat model, Wang and Wong308 reported that during the induction of adenoma, expression of IGF-I shifted from the prostatic stroma to the epithelium, suggesting, as has been demonstrated for EGFR/TGF-a, a shift from a paracrine to autocrine pathway for this growth factor during tumorigenesis. In contrast, IGFBPs are generally reported to inhibit growth in most but not all cases.133,278,279,323 Probably the best studied of the prostatic IGFBPs, IGFBP-3 has been reported to induce apoptosis of cell lines derived from PCa by both ligand-dependent and -independent pathways.282,324
Clinically, there has been a great deal of interest in determining if increased serum and altered IGFBP profiles327 can be used to predict, diagnose, or follow PCa. In vivo, IGF-I expression in the liver is regulated by growth hormone that acts at this level to increase IGF-I and somatostatin expression, both of which act at the anterior pituitary to inhibit the release of growth hormone. Similar to the use of luteinizing hormone-releasing hormone (LHRH) agonists to inhibit testosterone expression by the testis, somatostatin agonists are being explored as potential therapeutics for PCa.328,329
Neurogenic Regulation Traditionally, investigation of the role of neurogenic regulation in the prostate has been limited to the obstructive symptoms of BPH330,331and the addition of the prostatic secretions to the ejaculate.98,332 Its role in regulation of prostatic function has not been well studied, although evidence is accruing that it may play a broader role than previously suspected. The reader is referred to Walsh and colleagues,333 Higgins and Gosling,334 and McVary and colleagues335 for descriptions of the gross distribution of nerves and ganglia within the human prostate. Synap-tomenal complexes appear to be limited to the prostatic stroma.336,337 Alpha-adrenergic innervation predominates in both the rat and human prostate, although mus-
carinic335,338 and nonadrenergic noncholinergic path-ways334,336,337,339-341 are also present.
Alpha1-adrenergic receptors are localized primarily to the stromal compartment in both rats and humans.342-344 Alpha2-adrenergic receptors have also been identified in the prostate, particularly in the vicinity of blood vessels344 and in the prostatic capsule.345,346 Based on genetic and agonist/antagonist studies, three a 1 subtypes have been identified: a 1A (previously a 1c), oc 1B, and a 1D (previously a 1A and a 1A/d).347,348 Both RT-PCR and in situ hybridization studies have identified all three subtypes in the human prostate.349-351 Based on mRNA, protein expression, ligand-binding, and isometric contraction studies, the a 1A subtype is by far the most predominant receptor in the human prostate.349,350,352-355 The role played by these receptors in prostatic contraction is illustrated by the clinical use of a 1-adrenergic blockers in relieving the obstructive symptoms of BPH.330,331 The agonist/antagonist binding profile for prostatic a 1A receptors differs from that found in several other tissues. It has been hypothesized that the prostatic a 1A-receptor is a unique a 1A subtype, another as yet uncharacterized receptor, or is subjected to unique regulatory pathways.352,354,355 The a 1C-adrenergic receptors are localized predominantly, but not exclusively, within the stroma.349-351 Studies have found a 1D (oc 1A/d) receptors in both the stromal and epithelial compart-ments.349,350 In contrast, the 0C1B receptors are predominately localized to the glandular epithelium.349 In addition to expelling the contents of the prostatic acini during ejaculation as has been found in the rat,98,332 the adrenergic pathway may also play a role in promoting prostatic smooth muscle differentiation356 and in survival of the prostate stroma and epithelium.335,357
Beta-adrenergic receptors are present in the prostatic capsule345,346 but, as judged by ligand-binding and contraction studies, are all but absent from the parenchyma of the human prostate.335,346 In contrast, P2 receptors are readily detectable in the rat ventral prostate.358 Beta2-adrenergic agonists stimulate cAMP accumulation in epithelial-cell primary cultures359 and have been reported to upregulate the expression of PBP message and protein in denervated prostates and in recombinant prostatic graphs in the renal capsule.357,360 Unlike the a 1-adrenergic agonists,98,332,361 P 2-adrenergic agonists do not appear to regulate exocrine secretion.98
The muscarinic cholinergic receptor system is also present in both rats and humans species although this has been much less extensively studied.335,338 In human pro-static adenomas, the M1 subtype has been localized to the prostatic epithelium and is the predominant muscarinic receptor detected by immunoprecipitation and ligand-binding studies.344,362 The presence of muscarinic receptors in the stroma is still a matter of controversy,344,362 but the M2-receptor subtype has been detected primarily on cultures of prostatic smooth muscle.363 In the ventral prostate, the M3 subtype predominates,364 although the Ml subtype has also been detected on the prostatic epithelium.365 In humans, muscarinic agonists stimulate the contraction of the prostatic capsule but not of the parenchyma.346 Muscarinic receptors (M3 subtype) have also been reported to stimulate proliferation of primary cultures of epithelial cells obtained from radical prostatectomies.366 In rats, muscarinic agonists induce a sustained release of prostatic secretory products, which appears to occur by mechanisms other than smooth muscle contraction.98,332
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