Fibroblast growth factor receptor 2 tyrosine kinase is required for prostatic morphogenesis and the acquisition of strict androgen dependency for adult tissue homeostasis

The fibroblast growth factor (FGF) family consists of 22 members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells--the precursors of prostatic epithelial cells--for prostatic branching morphogenesis and prostatic growth. Most Fgfr2 conditional null (Fgfr2(cn)) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2(cn) prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2(cn) prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2(cn) prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate growth, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.     

fucosyltransferase1 and H-type complex carbohydrates modulate epithelial cell proliferation during prostatic branching morphogenesis

The prostate undergoes branching morphogenesis dependent on paracrine interactions between the prostatic epithelium and the urogenital mesenchyme. To identify cell-surface molecules that function in this process, monoclonal antibodies raised against epithelial cell-surface antigens were screened for antigen expression in the developing prostate and for their ability to alter development of prostates grown in serum-free organ culture. One antibody defined a unique expression pattern in the developing prostate and inhibited growth and ductal branching of cultured prostates by inhibiting epithelial cell proliferation. Expression cloning showed that this antibody binds fucosyltransferase1, an alpha-(1,2)-fucosyltransferase that synthesizes H-type structures on the complex carbohydrate modifications of some proteins and lipids. The lectin UEA I that binds H-type 2 carbohydrates also inhibited development of cultured prostates. These data demonstrate a previously unrecognized role for fucosyltransferase1 and H-type carbohydrates in controlling the spatial distribution of epithelial cell proliferation during prostatic branching morphogenesis. We also show that fucosyltransferase1 is expressed by epithelial cells derived from benign prostatic hyperplasia or prostate cancer; thus, fucosyltransferase1 may also contribute to pathological prostatic growth. These data further suggest that rare individuals who lack fucosyltransferase1 (Bombay phenotype) should be investigated for altered reproductive function and/or altered susceptibility to benign prostatic hyperplasia and prostate cancer.     

Collagen and the proliferation and differentiation of rat ventral prostate epithelial cells

The purpose of this study was to determine if a cause-and-effect relationship exists between androgen-induced changes in collagen and epithelial cell proliferation and/or differentiation in rat ventral prostate. Analyses of the temporal relationship between dihydrotestosterone (DHT)-induced changes in the synthesis and levels of collagen in the regressed ventral prostates of adult castrates demonstrated that, during the first 7 days of restoration of prostatic growth, androgen increased the synthesis as well as the degradation of collagen. Cis-hydroxyproline (CHP) treatment (2-200 mg/kg) during the first 7 days of androgen-stimulated prostatic growth, combined with maintenance of animals on a proline-free diet, produced a dose-dependent reduction in prostate weight and DNA content to a maximum of 50%. The epithelium was characterized by numerous disorganized layers of irregularly shaped and tightly packed cells, many of which had no contact with the basal lamina. There was a loss of epithelial lamina lucida and the development of a ragged lamina densa. Cis-hydroxyproline effects were reversible in that, following cessation of CHP treatment, the perturbed morphology, DNA content, and organ weight returned to the range of DHT-treated controls. Collagenous components seem to be important in supporting the normal androgen-dependent proliferation and differentiation of prostatic epithelial cells.     

Role of epithelial cell fibroblast growth factor receptor substrate 2alpha in prostate development, regeneration and tumorigenesis

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.     

Noggin is required for normal lobe patterning and ductal budding in the mouse prostate

Mesenchymal expression of the BMP antagonist NOGGIN during prostate development plays a critical role in pre-natal ventral prostate development and opposes BMP4-mediated inhibition of cell proliferation during postnatal ductal development. Morphologic examination of newborn Noggin^−/− male fetuses revealed genitourinary anomalies including cryptorchidism, incomplete separation of the hindgut from the urogenital sinus (UGS), absence of the ventral mesenchymal pad, and a complete loss of ventral prostate (VP) budding. Examination of lobe-specific marker expression in the E14 Noggin^−/− UGS rescued by transplantation under the renal capsule of a male nude mouse confirmed a complete loss of VP determination. More modest effects were observed in the other lobes, including decreased number of ductal buds in the dorsal and lateral prostates of newborn Noggin^−/− males. BMP4 and BMP7 have been shown to inhibit ductal budding and outgrowth by negatively regulating epithelial cell proliferation. We show here that NOGGIN can neutralize budding inhibition by BMP4 and rescues branching morphogenesis of BMP4-exposed UGS in organ culture and show that the effects of BMP4 and NOGGIN activities converge on P63⁺ epithelial cells located at nascent duct tips. Together, these studies show that the BMP-NOGGIN axis regulates patterning of the ventral prostate, regulates ductal budding, and controls proliferation of P63⁺ epithelial cells in the nascent ducts of developing mouse prostate.     

The role of Wnt5a in prostate gland development

The Wnt genes encode a large family of secreted glycoproteins that play important roles in controlling tissue patterning, cell fate and proliferation during development. Currently, little is known regarding the role(s) of Wnt genes during prostate gland development. The present study examines the role of the noncanonical Wnt5a during prostate gland development in rat and murine models. In the rat prostate, Wnt5a mRNA is expressed by distal mesenchyme during the budding stage and localizes to periductal mesenchymal cells with an increasing proximal-to-distal gradient during branching morphogenesis. Wnt5a protein is secreted and localizes to periductal stroma, extracellular matrix and epithelial cells in the distal ducts. While Wnt5a expression is high during active morphogenesis in all prostate lobes, ventral prostate (VP) expression declines rapidly following morphogenesis while dorsal (DP) and lateral lobe (LP) expression remains high into adulthood. Steroids modulate prostatic Wnt5a expression during early development with testosterone suppressing Wnt5a and neonatal estrogen increasing expression. In vivo and ex vivo analyses of developing mouse and rat prostates were used to assess the functional roles of Wnt5a. Wnt5a^−/− murine prostates rescued by organ culture exhibit disturbances in bud position and directed outgrowth leading to large bulbous sacs in place of elongating ducts. In contrast, epithelial cell proliferation, ductal elongation and branchpoint formation are suppressed in newborn rat prostates cultured with exogenous Wnt5a protein. While renal grafts of Wnt5a^−/− murine prostates revealed that Wnt5a is not essential for cyto- and functional differentiation, a role in luminal cell polarity and lumenization of the ducts was indicated. Wnt5a suppresses prostatic Shh expression while Shh stimulates Wnt5a expression in a lobe-specific manner during early development indicating that Wnt5a participates in cross-talk with other members of the gene regulatory network that control prostate development. Although Wnt5a does not influence prostatic expression of other Wnt morphogens, it suppresses Wif-1 expression and can thus indirectly modulate Wnt signaling. In summary, the present finds demonstrate that Wnt5a is essential for normal prostate development where it regulates bud outgrowth, ductal elongation, branching, cell polarity and lumenization. These findings contribute to the growing body of knowledge on regulatory mechanisms involved in prostate gland development which are key to understanding abnormal growth processes associated with aging.     

Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.     

Histochemical study of apoptotic epithelial cells depending on testosterone in primary cultured rat prostatic tissues

To clarify whether apoptosis can be induced in cultured rat prostatic epithelial cells, they were investigated at various time points, depending on different concentrations of testosterone. Ventral lobes of rat prostates were cultured as small pieces of tissues up to 14 days. They were examined by anti-Fas antibody immunostaining and also compared to findings revealed by in situ end-labelling (ISEL) technique. To clarify apoptotic nuclei at high resolution, the quick-freezing and deep-etching (QF-DE) method was also used, as reported before. The localization and appearance of Fas-positive cells were detected more widely and earlier than those of ISEL-positive cells, but both label-positive localizations were closely related to each other. In addition, they were detected more often in epithelial cells cultured with low testosterone concentrations. By the QF-DE method, chromatin fibers were found to be broken in spotty parts of apoptotic nuclei. We could control the concentration of testosterone in culture medium and detect the appearance of Fas antigen in cultured prostatic epithelial cells, followed by apoptotic changes. So, Fas and Fas-ligand system is one candidate for apoptosis in the prostate glands, depending on removal of hormonal testosterone.     

Altered prostatic epithelial proliferation and apoptosis, prostatic development, and serum testosterone in mice lacking cyclin-dependent kinase inhibitors

Normal prostatic development and some prostatic diseases involve altered expression of the cell-cycle regulators p27 and p21 (also known as CDKN1B and CDKN1A, respectively). To determine the role of these proteins in the prostate, we examined prostatic phenotype and development in mice lacking p27 and/or p21. In p27-knockout (p27KO) mice, epithelial proliferation was increased 2- and 3.8-fold in the ventral and dorsolateral prostate, respectively, versus wild-type (WT) mice, although prostatic weights were not different. Epithelial apoptosis was increased in p27KO mice and may account for the lack of a concurrent increase in weight. Testosterone deficiency observed in this group was not the cause of this increase, because vehicle- and testosterone-treated p27KO mice had similar percentages of apoptotic cells. Also observed was a trend toward a decreased functional epithelial cytodifferentiation, indicating a potential role of p27 in this process. Conversely, dorsolateral prostate and seminal vesicle (SV) of p21-knockout (p21KO) mice, and all prostatic lobes and SV of p21/p27 double-knockout mice, weighed significantly less compared to the WT mice, and their epithelial proliferation was normal. Decreased testosterone concentrations may contribute to the decreased prostatic weights. However, other factors may be involved, because testosterone replacement only partially restored prostatic weights. We conclude that loss of p27 increases prostatic epithelial proliferation and alters differentiation but does not result in prostatic hyperplasia because of increased epithelial cell loss. The p21KO mice showed phenotypes distinctly different from those of p27KO mice, suggesting nonredundant roles of p21 and p27 in prostatic development. Loss of p27 or of both p21 and p27 results in serum testosterone deficiency, complicating analysis of the prostatic effects of these cell-cycle regulators.     

Epithelial phenotypes in the developing human prostate.

An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13-42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate development.     

Morphological and histological study of castration-induced degeneration and androgen-induced regeneration in the mouse prostate

Degenerative and regenerative changes in the ductal architecture of the ventral and dorsolateral prostates (VP and DLP) of the adult mouse were investigated in microdissected specimens over a time-course of 14 days following castration and subsequently during 14 days of administration of testosterone propionate. After castration, about 35% of the ductal tips and branch-points were lost in distal regions (usually near the capsule) in both prostatic lobes. By contrast, in more proximal regions of the prostate (closer to the urethra), the ducts survived in an atrophic condition. The ductal morphology that had been lost in the distal regions completely regenerated after testosterone propionate was administered to the castrated males. In the VP, androgen replacement simply returned the gland to its former size with moderate ductal distension; in the DLP, excessive epithelial infoldings and ductal distension were elicited in the distal regions of the ducts after 14 days of treatment with testosterone propionate. These results suggest that androgenic responsiveness and dependency are different in distal versus proximal ducts. Distal ducts are exquisitely androgen-dependent and androgen-sensitive; in proximal regions, androgen-dependency is not as strict.     

Immunocytochemistry of epithelial markers in citral-induced prostate hyperplasia in rats.

Immunocytochemical characterization of several epithelial markers using the PAP technique was analyzed during different stages of induced prostatic hyperplasia in rats. Intact adolescent rats (42 days old) were treated with citral (3,7 dimethyl-2,6 octadienal) for 10, 30 and 100 days and their ventral prostate compared to untreated, matched-age animals. Among the epithelial markers studied the prostatic specific acid phosphatase was present in hyperplastic prostates of rats. The immunoreaction showed a fair correlation with the severity of lesion and duration of treatment. The prostatic specific antigen showed equally immunoreactive in both control and treated rats. The hyperplastic and normal rat prostates did not show immunoreactivity towards the other epithelial cell markers such as epithelial membrane antigen, carcinoembrionic antingen and alpha-fetoprotein antisera. It is concluded that prostatic specific acid phosphatase, and to a lesser extent prostatic specific antigen, might represent valuable markers for comparative studies of prostatic hyperplasia in rodents.     

Castration induced changes in dog prostate gland associated with diminished activin and activin receptor expression

This study was conducted to evaluate the effect of androgen ablation on dog prostate gland structure and the proliferation capacity of the prostatic cells and their association with the expression of Activin A and Activin RIIA receptor. The effect of androgen on the prostate gland was compared in intact and castrated dogs after one and two weeks. Specific primary antibodies were used to immunolocalize activin-A, activin receptor type II A and the proliferation marker (PCNA). The results showed that the glandular acini of the prostate gland of intact dogs are lined by tall columnar secretory cells and less abundant flattened basal cells and surrounded by a thin fibromuscular tissue. The cytoplasm of the glandular cells exhibited an intense immunoreaction for activin A and activin RIIA receptor while basal cells expressed PCNA. Castration induced a remarkable atrophy of the prostatic acini associated with a progressive loss of secretory epithelial cells, which showed a dramatic decrease to complete disappearance of Activin A and Activin RIIA receptor immunoreactions. The remaining cells of the atrophied acini continue to express PCNA and the inter-acinar fibromuscular tissue showed a remarkable increase in its mass and are induced to express PCNA. These results indicated that androgen is required for the survival of epithelial cells and to maintain growth-quiescent fibromuscular cells, while basal cell proliferation is androgen independent. The changes in the Activin A and Activin RIIA receptor localization and their association with the dynamic pattern of prostate gland regression after castration suggested that Activin A and Activin RIIA receptor expression are androgen dependent.     

Sonic hedgehog regulates prostatic growth and epithelial differentiation

The Sonic hedgehog (SHH)-signalling pathway mediates epithelial-mesenchymal interactions in several tissues during development and disease, and we have investigated its role in rat ventral prostate (VP) development. We have demonstrated that Shh and Ptc expression correlates with growth and development of the prostate and that their expression is not regulated by androgens in the VP. Prostatic budding was induced in response to testosterone in Shh null mouse urogenital sinus (UGS) explants grown in vitro and in rat UGS explants cultured with cyclopamine, suggesting that SHH-signalling is not critical for prostatic induction. SHH-signalling was disrupted at later stages of VP development (in vitro), resulting in a reduction in organ size, an increase in ductal tip number, and reduced proliferation of ductal tip epithelia. The addition of recombinant SHH to VPs grown in vitro caused a decrease in ductal tip number and expansion of the mesenchyme. In the presence of testosterone, inhibition of SHH-signalling accelerated the canalisation of prostatic epithelial ducts and resulted in ducts that showed morphological similarities to cribiform prostatic intraepithelial neoplasia (PIN). The epithelia of these ducts also demonstrated precocious and aberrant differentiation, when examined by immunohistochemistry for p63 and cytokeratin 14. In conclusion, we show that SHH-signalling is not essential for prostatic induction, but is important for prostatic growth, branching, and proliferation, and that androgen-stimulated growth in the absence of signalling from the SHH pathway results in aberrant epithelial differentiation.     

Effects of aging on growth factors gene and protein expression in the dorsal and ventral lobes of rat prostate

We hypothesize that various growth factors and their receptors gene and protein are modulated in dorsal and ventral lobes of aging prostate. To test this hypothesis, TGFbeta1, TGFbeta2 TGFbeta3, TGFbetaR-I, TGFbetaR-II, TGFalpha, EGF, EGFR, KGF and KGFR gene and protein expression were analyzed in dorsal and ventral lobes of aging rat prostates (1, 3, 6, 9, 12, 18, 24, and 28/30 months). KGF gene expression was very weak or absent in 1, 3, and 6 month old rat dorsal and ventral lobes of prostate whereas it re-expressed in 9, 12, 18, 24 and 30 month old rat prostate. All growth factors and their receptors expect KGF and EGFR were mainly localized in epithelium of ventral and dorsal lobes of aging rat prostates. EGF, TGFalpha, TGFbeta1, and TGFbetaR-I protein expression was lacking in stroma of dorsal and ventral lobes of 1, 3, 6, 9, 12/18 months old rat prostates. However, EGF, TGFbeta1 and TGFbetaR-I proteins re-expressed in stroma of 24 and 28 months old rat prostates. KGF protein expression was lacking in epithelium of dorsal and ventral lobes of all aging rat prostates. This is the first report to demonstrate differential gene and protein expression of growth factors in dorsal and ventral lobes is associated with aging rat prostate, suggesting their role in pathogenesis of prostatic diseases with aging.     

METTL3 promotes prostatic hyperplasia by regulating PTEN expression in an m6A-YTHDF2-dependent manner

Uncontrolled epithelial cell proliferation in the prostate transition zone and the hyper-accumulation of mesenchymal-like cells derived from the epithelial-mesenchymal transition (EMT) of prostatic epithelium are two key processes in benign prostatic hyperplasia (BPH). m⁶A RNA modification affects multiple cellular processes, including cell proliferation, apoptosis, and differentiation. In this study, the aberrant up-regulation of methylase METTL3 in BPH samples suggests its potential role in BPH development. Elevated m⁶A modification in the prostate of the BPH rat was partially reduced by METTL3 knockdown. METTL3 knockdown also partially reduced the prostatic epithelial thickness and prostate weight, significantly improved the histological features of the prostate, inhibited epithelial proliferation and EMT, and promoted apoptosis. In vitro, METTL3 knockdown decreased TGF-β-stimulated BPH-1 cell proliferation, m⁶A modification, and EMT, whereas promoted cell apoptosis. METTL3 increased the m⁶A modification of PTEN and inhibited its expression through the reading protein YTHDF2. PTEN knockdown aggravated the molecular, cellular, and pathological alterations in the prostate of BPH rats and amplified TGF-β-induced changes in BPH-1 cells. More importantly, PTEN knockdown partially abolished the improving effects of METTL3 knockdown both in vivo and in vitro. In conclusion, the level of m⁶A modification is elevated in BPH; the METTL3/YTHDF2/PTEN axis disturbs the balance between epithelial proliferation and apoptosis, promotes EMT, and accelerates BPH development in an m⁶A modification-related manner.Subject terms: Cell biology, Molecular biology     

Immunohistochemical comparative analysis of transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates.

Immunoreaction to TGF-alpha was limited to the basal epithelial cells of focal areas in the normal prostates. In benign prostatic hyperplasia (BPH) the immunostained areas were more widespread and immunolabelling was observed in both basal and columnar (secretory) cells of the epithelium. Some cells in the connective tissue stroma were also stained. In prostatic adenocarcinoma, epithelial immunostaining was even more extensive and intense than in BPH, and some stromal cells were also stained. Epidermal growth factor (EGF) immunostaining was only present in some basal cells in normal prostates. In BPH, this immunoreaction was strong in the basal cells and even stronger in the secretory cells. In prostatic cancer, the intensity of epithelial cell immunoreactivity was intermediate between that of normal prostates and that of BPH specimens. EGF-receptor immunostaining was focal and located in the basal cells in normal prostates. In BPH, labelling was also localized in basal cells but extended to wider areas. Some stromal cells appeared weakly labelled. In the prostatic carcinoma, both basal and columnar cells appeared stained and the number of immunolabelled stromal cells was higher than in BPH. The results presented suggest that, in normal conditions, EGF and TGF-alpha act as autocrine growth factors for the basal cells of the prostatic epithelium. In BPH this action is maintained and, in addition, the columnar cells start to secrete both factors which are bound by the basal cell receptors, giving rise to a paracrine regulation which probably overstimulates basal cell proliferation. In prostatic carcinoma, besides these regulatory mechanisms, the acquisition of EGF-receptors by the secretory cells develops an autocrine regulation which might induce their proliferation.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Immunoexpression of tumour necrosis factor-alpha and its receptors 1 and 2 correlates with proliferation/apoptosis equilibrium in normal, hyperplasic and carcinomatous human prostate

Immunohistochemical and semiquantitative study of TNF-alpha, its receptors types 1 (TNFR1) and 2 (TNFR2), cell proliferation (Ki-67 nuclear antigen), and apoptosis (Tunel method) was carried out in human prostates, in normal healthy conditions, as well as in benign prostatic hyperplasia (BPH) and prostatic carcinoma (PC). Cell proliferation was higher in BPH than in normal prostates, and even higher in PC, mainly in neoformations showing a microglandular pattern. The apoptotic index was similar in BPH and normal prostates, and increased significantly in PC with independence of the pattern. In BPH, immunoreaction to TNF-alpha decreased as compared with that of normal prostates, while immunoreactions to both TNF-alpha receptors increased. This suggests a feedback downregulation of the factor, and that the low TNF-alpha activity in BPH are compensated by the increased amount of receptors. In PC, immunoreaction to TNF-alpha and its two receptors increased markedly, suggesting that the TNF-induced effects are also increased. Contrarily to cell proliferation immunoexpression, PC reaction to TNFR2 was stronger in the papillar pattern than in the micrograndular pattern, and this suggests an inverse correlation between TNFR2 expression and cell proliferation.