Regional expression of transforming growth factor-alpha in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement

The prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-alpha (TGFalpha), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFalpha in the rat ventral prostate during postnatal development to adulthood. TGFalpha-immunopositive cells were located throughout the ductal epithelium from postnatal days 5-20. By day 45 and thereafter, regional variation in TGFalpha expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFalpha in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFalpha staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFalpha in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFalpha expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFalpha in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFalpha is not regulated by androgen in this region of the ventral prostate. In summary, TGFalpha expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFalpha may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.     

Modifying psychotropic drug prescription patterns: a follow-up survey

Blood flow to the rat ventral prostate (VP), dorsolateral prostate (DP), and Dunning R3327 prostatic tumors was measured at different times up to 7 days after castration, using the microsphere method. In the VP organ weight was decreased from day 3 onwards. Blood flow was, however, already significantly decreased from day 1. The reduced blood flow in VP in 1-3 and 7-day castrated animals could be reversed by testosterone treatment. Organ weight was slightly decreased but blood flow was unaffected by castration in DP. Castration left Dunning tumor volume and blood flow unaffected. Using immunohistochemistry, androgen receptors were observed in epithelial and stromal cells in VP, DP and Dunning tumors, but not in blood vessels. Castration is known to induce apoptosis in the VP, but not in the DP or in Dunning tumors. This suggests that a reduction in blood flow might be an important component for the castration-induced involution and apoptosis in prostatic tissue. The reason why castration reduces blood flow only in the VP, and not in the DP or Dunning tumor is unknown.     

Expression and degradation of rat androgen receptor following castration, testosterone replacement and antiandrogens administration: analysis by Western blot and immunohistochemistry

To elucidate the autoregulation of androgen receptor (AR) by androgen and antiandrogen, Western blot analysis and immunohistochemical study were performed. Castration reduced the immunodetected AR content, and nuclear staining was lost without cytoplasmic staining. Testosterone (T) supplement restored AR content. Quick response of AR content restoring following single administration of T was observed 48 hours after castration. The recovery of AR content detected by Western blot under each condition was accompanied by recovery of the reduced unclear staining intensities in the epithelia. Neither steroidal nor non-steroidal antiandrogens, chlormadinone acetate and flutamide, altered the AR content in normal rat ventral prostate 5, 12, 24 or 48 hours after single administration. Furthermore, neither of the drugs at various doses altered AR levels 12 hours after single administration. In summary, the rat AR is upregulated by androgen. Single administration of antiandrogens have no effect on immunodetected AR content.     

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.     

Changing patterns of gene expression identify multiple steps during regression of rat prostate in vivo

The rat ventral prostate is an androgen-dependent organ that undergoes dramatic cell death upon removal of testosterone by surgical castration. Several well characterized criteria, such as nuclear condensation, organelle blebbing, and DNA fragmentation, have been used to demonstrate that most of this cell loss is due to programmed cell death, or apoptosis, of the secretory epithelial cells. In addition to changes in morphology, it is well known that cells undergoing apoptosis show alterations in gene expression, and it is widely assumed that many of these genes are directly involved in the mechanism of programmed cell death. Using poly A+ RNA derived from normal rat prostate as well as from the regressing prostates of castrated rats, we have used a PCR-based subtractive hybridization approach to generate complementary DNA (cDNA) libraries greatly enriched in cDNAs strongly regulated during rat prostate regression. Several hundred of the genes represented in these libraries appear to be strongly regulated during prostate regression and most of these are prostate specific. Sequence analysis indicates that up to 30% of these clones are similar or identical to genes of known function, approximately 20% are similar to expressed sequence tags (ESTs), and as many as 50% of these clones have not been characterized previously. Analysis of selected clones using in situ hybridization indicates that they are expressed specifically in prostate epithelial cells, and that certain of these clones are regulated temporally in a pattern consistent with apoptosis. The patterns of gene expression include: 1) genes whose expression decreases uniformly after removal of androgen, indicative of androgen sensitive genes; 2) genes whose expression increases in apoptotic prostate cells and in other tissues, suggesting a class of genes generally involved in apoptosis; 3) and genes whose expression increases in individual regressing prostate epithelial cells, suggesting a class of prostate specific genes associated with apoptosis.     

Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells

The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.     

The long view: how genetic discoveries will aid healthcare reform

A rat model was used to study the role of renal insulin-like growth factor-I (IGF-I) and circulating IGF-I and IGF binding proteins (IGFBPs) in early posttransplantation renal hypertrophy and overall body growth during high-dose immunosuppression. Seven days of prednisolone and cyclosporin A (CsA) immunosuppression was administered to rats following unilateral nephrectomy compared with sham-operated controls. Immunosuppression of nephrectomized and control rats was followed by a reduction in body weight (BW) compared with placebo treatment. In addition, immunosuppression inhibited kidney IGF-I accumulation and compensatory renal growth in uninephrectomized rats, but caused no change in kidney weight or IGF-I levels in control rats. Immunosuppression induced a sustained significant increase in circulating IGFBP-3 and 30-kd IGFBPs in uninephrectomized and controls rats, whereas serum IGF-I levels were unchanged. In a supplementary study separating the effects of the two immunosuppressants, the kidney IGF-I changes and renal growth were primarily affected by CsA, while the changes in IGFBPs appeared to be caused by prednisolone treatment. In conclusion, immunosuppression with prednisolone and CsA was followed by less kidney IGF-I accumulation and compensatory renal growth compared with placebo treatment. In addition, a sustained increase in circulating levels of IGFBP-3 and 30-kd IGFBPs was observed, which may be involved in the growth impairment observed following immunosuppressive treatment.     

Blockade of androstenedione-induced stimulation of androgen-sensitive parameters in the rat prostate by combination of Flutamide and 4-MA

In order to mimic the human situation in which adrenal steroid precursors are converted to the active androgen dihydrotestosterone (DHT) in prostatic tissue, we have used castrated rats supplemented with the precursor steroid androstenedione (delta 4-dione) released from Silastic implants. While it is well known that the action of DHT can be partially neutralized by antiandrogens which compete for binding to the androgen receptor, we have used 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone into DHT, in order to decrease intraprostatic DHT levels and thus facilitate the action of the antiandrogen. Animals were treated for 7 days with Flutamide (FLU, 2 mg) or 4-MA (4 mg) injected subcutaneously, twice daily, alone or in combination. 4-MA administered alone caused a 54% inhibition of delta 4-dione-stimulated ventral prostate weight while FLU exerted a 74% inhibitory effect and 4-MA+FLU further improved inhibition to 81%. We then measured, by in situ hybridization, the levels of prostatic mRNAs encoding the C1 and C3 components of the prostatic binding protein (PBP) which are highly specific and sensitive markers of androgen action. PBP-C3 mRNA levels fell by 95% following castration while treatment with delta 4-dione completely reversed the effect of castration. Administration of FLU or 4-MA independently caused 33% and 10% decreases, respectively, of PBP-C3 mRNA levels stimulated by delta 4-dione while the combination of both compounds further inhibited PBP-C3 mRNA levels to reach a 55% inhibition. Similar effects were observed on PBP-C1 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning electron microscopy of the accessory sex glands of the adult male rat

A systematic, comparative study of the accessory sex glands of the adult male rat after androgen withdrawal was carried out. The changes were investigated by using scanning electron microscopy at different intervals after surgical castration. The main common signs of epithelial cell involution were flattening of the cell surface, reduction of the size and number of microvilli, some blurring of the cell borders, cessation of secretory activity and diminution of the luminal volume of the glands. Overall, confident signs of atrophy were evident after one week, and complete epithelial involution was reached by the third week. The epithelial cell atrophy was accompanied by a relative stromal hyperplasia. The new observation seems to be that the process of stroma consolidation is progressing for a considerable time subsequent to the completion of the epithelial involution. This phenomenon is particularly evident in the dorsal prostate, the seminal vesicle and the coagulating gland.     

Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells

Calreticulin was identified in a screen for androgen-response genes in the rat ventral prostate. Northern blot and Western blot analyses in the rat model showed that both calreticulin messenger RNA and protein are down-regulated by castration and up-regulated by androgen replacement in the prostate. Northern blot analysis showed that calreticulin expression level in the prostate is much higher than that in seminal vesicles, heart, brain, muscle, kidney, and liver. The regulation of calreticulin expression by androgen is only observed in the prostate and seminal vesicles, two male secondary sex organs. The induction of calreticulin by androgen in prostate organ culture partially resists protein synthesis inhibition, suggesting that calreticulin is a direct androgen-response gene. In situ hybridization and immunohistochemistry studies showed that calreticulin is an intracellular protein in prostatic epithelial cells. Because calreticulin is a major intracellular Ca++-binding protein with 1 high-affinity and 25 low-affinity Ca binding sites, our observations suggest that calreticulin is a promising candidate that mediates androgen regulation of intracellular Ca++ levels and/or signals in prostatic epithelial cells. The expression of calreticulin is also regulated by androgen in the mouse and human prostate, suggesting that androgen regulation and function of calreticulin in the prostate are conserved evolutionarily.     

Differential expression and biological effects of insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis. The bioactivity of IGF-I is modulated by a group of high affinity, specific binding proteins (IGF-binding proteins; IGFBPs) that are present in the interstitial fluid. Previously, we have reported that porcine VSMCs synthesize and secrete IGF-I and several forms of IGFBPs, including IGFBP-2, IGFBP-4, and IGFBP-5. In this study, we examined the role of autocrine/paracrine secreted IGF-I in controlling the expression of IGFBP-4 and IGFBP-5 as well as the effects of these IGFBPs in modulating the cellular replication response to IGF-I. The concentrations of IGFBP-4 in the conditioned medium increased significantly from <50 ng/ml to 742 +/- 105 ng/ml. This increase was associated with a decrease in the activity of an IGF-I-regulated IGFBP-4 protease. In contrast, the synthesis of IGFBP-5 was inversely correlated with culture density, and its concentration decreased from 792 +/- 91 to 44 +/- 14 ng/ml. IGFBP-5 mRNA in sparse cultures was 3-fold higher compared with those in confluent cultures. This culture density-dependent change in IGFBP-5 mRNA correlated closely with endogenous IGF-I levels. Since treatment of VSMC with exogenous IGF-I increased IGFBP-5 mRNA levels, we neutralized the effect of endogenously secreted IGF-I with an anti-IGF-I antibody to determine if it would alter IGFBP-5 mRNA abundance. This resulted in a 4.4-fold decrease in IGFBP-5 mRNA levels. When added together with IGF-I, exogenous IGFBP-4 inhibited IGF-I-induced DNA synthesis in a concentration-dependent manner. IGFBP-5, on the other hand, potentiated the effect of IGF-I. Therefore, IGFBP-4 and IGFBP-5 appear to be differentially regulated by autocrine/paracrine IGF-I through distinct mechanisms. These two proteins, in turn, play opposing roles in modulating IGF-I action in stimulating VSMC proliferation.     

Androgen-dependent protein interactions within an intron 1 regulatory region of the 20-kDa protein gene

The 20-kDa protein gene is androgen regulated in rat ventral prostate. Intron 1 contains a 130-base pair complex response element (D2) that binds androgen (AR) and glucocorticoid receptor (GR) but transactivates only with AR in transient cotransfection assays in CV1 cells using the reporter vector D2-tkCAT. To better understand the function of this androgen-responsive unit, nuclear protein interactions with D2 were analyzed by DNase I footprinting in ventral prostate nuclei of intact or castrated rats and in vitro with ventral prostate nuclear protein extracts from intact, castrated, and testosterone-treated castrated rats. Multiple androgen-dependent protected regions and hypersensitive sites were identified in the D2 region with both methods. Mobility shift assays with 32P-labeled oligonucleotides spanning D2 revealed specific interactions with ventral prostate nuclear proteins. Four of the D2-protein complexes decreased in intensity within 24 h of castration. UV cross-linking of the androgen-dependent DNA binding proteins identified protein complexes of approximately 140 and 55 kDa. The results demonstrate androgen-dependent nuclear protein-DNA interactions within the complex androgen response element D2.     

Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells

IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-I and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays using total RNA demonstrated that MC express all of the IGFBPs. [125I]IGF-I Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-beta are implicated in glomerular hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng/ml), TGF-beta (2 ng/ml) and forskolin (10(-5) M) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-beta, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-I-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-beta is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs. Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-beta and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.     

Effect of castration on serum concentrations of gonadal hormones, insulin-like growth factor-I and its binding proteins in male pigs

Ten male pigs (Large White x Landrace), 7 months old, were randomly allocated to two experimental groups. Five of them were castrated and the other five served as controls. Sera were collected on the day of castration and 1, 5, 6 and 7 weeks after castration for hormone assay. There was a significant rise in the splenic and pancreatic weights in the castrates (P < 0.01). The weights of prostate, seminal vesicles and bulbourethral glands were significantly decreased (P < 0.01) in the castrates, which is attributed to a fall in testosterone levels (P < 0.001). The fall in oestradiol concentrations (P < 0.001) in castrates confirms that the testis is the major source of oestrogens in males. Although there was no significant change in the body weight, serum IGF-I levels were elevated in the castrates as compared to the controls after 5, 6 and 7 weeks (P < 0.001). IGFBP bands of 43 and 39 kda predominate in both control and experimental groups indicating that castration had no effect on the IGFBP pattern. It is suggested that the increase in IGF-I levels may be due to uncoupling of GH/IGF-I axis induced by the decrease in steroid concentrations due to castration.     

Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12

Androgen has an important role in development of the prostate, and the actions of androgen are mediated, in part, by locally produced growth factors. These growth factors are postulated to mediate stromal-epithelial interaction in the prostate to maintain normal tissue physiology. Transforming growth factor-alpha (TGF-alpha) is one of the growth factors that can stimulate prostatic growth. The expression of TGF-alpha is thought to be regulated by androgen. The expression of epidermal growth factor receptor (EGFR), which is the receptor of TGF-alpha and EGF, also may be regulated by androgen. The hormonal and developmental regulation of TGF-alpha and EGFR messenger RNA (mRNA) levels in isolated epithelial and stromal cells from rat ventral prostate was investigated. The expression of mRNA for TGF-alpha and EGFR was analyzed by a quantitative RT-PCR (QRT-PCR) procedure developed. Observations from this assay demonstrated that both epithelial and stromal cells expressed the mRNA for TGF-alpha and EGFR. TGF-alpha mRNA expression was constant during postnatal, pubertal, and adult development of the prostate. EGFR mRNA expression was elevated at the midpubertal period and decreased with age. After castration of 60-day-old adult rats, both TGF-alpha and EGFR mRNA were significantly enhanced. TGF-alpha mRNA expression was stimulated by EGF in stromal cells (4.5-fold increase) but was not changed by any treatment in epithelial cells. EGFR mRNA levels were stimulated by EGF and keratinocyte growth factor treatment and inhibited by testosterone treatment in epithelial cells. Stromal cell EGFR mRNA levels were not affected by any treatment. Both testosterone and EGF stimulated incorporation of 3H-thymidine into prostatic stromal and epithelial cells. Anti-TGF-alpha antibody significantly inhibited testosterone-stimulated 3H-thymidine incorporation into stromal cells and epithelial cells. Immunocytochemical localization of TGF-alpha and EGFR demonstrated expression on the luminal surface of epithelial cells within prostatic ducts, and minimal expression was observed in stromal cells. Results indicate that testosterone does not directly regulate TGF-alpha mRNA levels but does inhibit EGFR mRNA levels. Interestingly, anti TGF-alpha antibody suppressed the effect of testosterone on 3H-thymidine incorporation into prostatic stromal and epithelial cells. This finding suggests that testosterone may act indirectly on prostatic cells to influence TGF-alpha actions. TGF-alpha mRNA levels were influenced by EGF in stromal cells only, and EGFR mRNA levels were influenced by testosterone, EGF, and keratinocyte growth factor in epithelial cells. These observations suggest that regulation of TGF-alpha and EGFR is distinct between the cell types. In conclusion, a network of hormonally controlled growth factor-mediated stromal-epithelial interactions is needed to maintain prostate development and function.     

Suppression of caveolin expression induces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancer cells

Although prostate cancer cells are often initially sensitive to androgen ablation, they eventually lose this response and continue to survive, grow and spread in the absence of androgenic steroids. The mechanism(s) that underlie resistance to androgen ablation therapy remain mostly unknown. We have demonstrated that elevated caveolin protein levels are associated with human prostate cancer progression in pathological specimens. Here we show that suppression of caveolin expression by a stably transfected antisense caveolin-1 cDNA vector converted androgen-insensitive metastatic mouse prostate cancer cells to an androgen-sensitive phenotype. Orthotopically grown tumors and low-density cell cultures derived from antisense caveolin clones had increased apoptosis in the absence of androgenic steroids, whereas similarly grown tumors and cells from vector (control) clones and parental cells were not sensitive to androgens. Studies using a representative antisense caveolin clone showed that selection for androgen resistance in vivo correlated with increased caveolin levels, and that adenovirus-mediated caveolin expression blocked androgen sensitivity. Our results identify a new candidate gene for hormone-resistant prostate cancer in man and indicate that androgen insensitivity can be an inherent property of metastatic prostate cancer.