David J. Zelner

EXPRESSION AND LOCALIZATION OF TRANSFORMING GROWTH FACTOR-BETA RECEPTORS TYPE I AND TYPE II IN THE RAT VENTRAL PROSTATE DURING REGRESSION

Of the three ubiquitously expressed transforming growth factor-beta (TGF beta) receptors, only type I and type II receptors contain serine/threonine kinase activity and have a direct role in TGF beta signal transduction. In the prostate, it has been reported that the level of type III receptor expression increases transiently after castration. However, the relationship between the TGF beta signaling receptors, type I and type II, and androgen is currently unclear. Thus, in the present study, we made an initial attempt to elucidate the effect of androgen on type I and type II receptor expression in the rat ventral prostate by measuring the levels of messenger RNA (mRNA) and protein at specific time points after castration up to 10 days. Within 3 days after castration, an increase in type II receptor mRNA was observed in the prostate, and the level continued to rise until 7 days postcastration (approximately 8-fold increase). Between days 7-10 postcastration, no significant change in the level of type II receptor mRNA was observed. Testosterone administration immediately after castration abolished the induction of type II receptor mRNA during the same 10-day period. Western blot analysis performed for type II receptor showed a similar result, in that the level of type II receptor protein increased approximately 5-fold by day 10 postcastration. In a similar manner to the expression of type II receptor mRNA, the level of type I receptor mRNA increased steadily until day 7 postcastration (approximately 6-fold increase). Between days 7-10 postcastration, the level of type I receptor mRNA did not change significantly. As with type II receptor mRNA, the induction of type I receptor mRNA was suppressed when testosterone was administered immediately after castration. To localize the expression of TGF beta receptor type II, immunohistochemical studies were performed. The results of these studies demonstrated a preferential localization of type II receptor in the prostatic epithelial cells and an increased staining intensity for the receptor after castration. Taken together, these data indicate that TGF beta signaling receptors, type I and type II, are under negative androgenic regulation at the transcriptional level and that TGF beta may be an important regulator of a stromal-epithelial interaction in the rat ventral prostate.

Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21waf1/cip1†

Inhibition of protein kinase C (PKC) and modulation of transforming growth factor‐β (TGF‐β) are both associated with tamoxifen treatment, and both appear to be important in the regulation of prostate cancer cell growth. Investigations were performed which sought to measure the efficacy, and to elucidate the mechanism of growth inhibition by tamoxifen, in hormone‐refractory prostate cancer.

Characterization and localization of nitric oxide synthase isoforms in the BB/WOR diabetic rat.

PURPOSE Erectile dysfunction is a common pathological development in individuals with diabetes mellitus. Nitric oxide synthase (NOS) is essential for regulation of normal penile erection and NOS protein activity has been shown to be altered with diabetes. Several different isoforms and subtypes of NOS exist. However, little is known about how the distribution and abundance of these isoforms are altered with diabetes. We characterized the distribution and abundance of NOS isoforms and explored how they are altered with diabetes and result in erectile failure. MATERIALS AND METHODS In situ hybridization and quantitative reverse transcriptase-polymerase chain reaction were done to measure the abundance and distribution of NOS-Ia, NOS-Ib, NOS-Ic, NOS-II and NOS-III in control and diabetic (BB/WOR) rats. Protein was localized by immunohistochemical analysis and alterations in protein abundance with diabetes were examined by Western blot analysis. RESULTS NOS-I, NOS-II and NOS-III were observed in the endothelium lining the cavernous spaces and in the epithelium of the urethra. NOS-I protein was also present in the nerves of control and diabetic penes. We observed an increase in NOS-II expression around the dorsal nerves of diabetic penes, a decrease in NOS-III expression in diabetic pelvic ganglia and a decrease in NOS-Ib expression in the diabetic penis. NOS-I protein abundance was significantly decreased in diabetic pelvic ganglia. CONCLUSIONS To our knowledge this is the first report of regional differences in the distribution of NOS-III in the urethra and altered NOS-Ib gene expression with diabetes.

Prostatic ductal system in rats : Tissue-specific expression and regional variation in stromal distribution of transforming growth factor-β1

Regional variations in stromal‐epithelial interactions, mediated through soluble growth factors, may be responsible for differences in epithelial growth and death observed between regions of the rat prostatic ductal system. Since transforming growth factor‐β1 (TGF‐β1) can induce prostatic epithelial cell death in vitro and in vivo, we examined the localization and production of TGF‐β1 with respect to the functional regions of the rat prostatic ductal system.

Analysis of NOS isoform changes in a post radical prostatectomy model of erectile dysfunction

Optimal treatment of erectile dysfunction (ED) following radical prostatectomy remains a subject of much controversy and is a significant concern for prostate cancer patients requiring surgical intervention. Neural stimulation involving nitric oxide synthase (NOS) is a crucial aspect of the normal erection process. In this study NOS isoform interaction was evaluated to improve our understanding of molecular changes pertaining to erection post radical prostatectomy. Bilateral cavernous nerve (CN) resected and control adult male Sprague-Dawley rats were killed 7, 14 and 21 days after injury. RT-PCR, in situ hybridization, Western blot and immunohistochemical analysis were used to evaluate changes in NOS isoform expression and distribution. NOS-I protein was dramatically decreased after CN injury while NOS-III and NOS-II remained unchanged. A profound decrease in smooth muscle and endothelium was observed in the corpora. To our knowledge this is the first report of differential altered NOS isoform protein abundance under conditions which mimic radical prostatectomy. These results show the importance of maintaining at least partial innervation of the penis after surgical intervention and that endothelial and smooth muscle changes resulting from loss of innervation may account for the ED observed in prostatectomy patients.

Sonic hedgehog Cascade Is Required for Penile Postnatal Morphogenesis, Differentiation, and Adult Homeostasis

Abstract The penis is unique in that it undergoes morphogenesis and differentiation primarily in the postnatal period. For complex structures such as the penis to be made from undifferentiated precursor cells, proliferation, differentiation, and patterning are required. This process involves coordinated activity of multiple signals. Sonic hedgehog (Shh) forms part of a regulatory cascade that is essential for growth and morphogenesis of many tissues. It is hypothesized that the penis utilizes regulatory mechanisms similar to those of the limb and accessory sex organs to pattern penile postnatal morphogenesis and differentiation and that the Shh cascade is critical to this process. To test this hypothesis, Shh, BMP-4, Ptc, and Hoxa-10 localization and function were examined in Sprague-Dawley rat penes by means of quantitative reverse transcription polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting. These genes were expressed in the penis during postnatal morphogenesis in a spatially and temporally restricted manner in adjacent layers of the corpora cavernosal sinusoids. The function of Shh and BMP-4 is to establish and maintain corpora cavernosal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and targets of its signaling in maintaining penile homeostasis in the adult is significant because disruption of Shh signaling affects erectile function. This is the first report that demonstrates the significant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. These studies provide valuable insight that may be applied to improve treatment options for erectile dysfunction.

Altered Sonic Hedgehog Signaling Is Associated with Morphological Abnormalities in the Penis of the BB/WOR Diabetic Rat

Abstract Erectile dysfunction (ED) is a common and debilitating pathological development that affects up to 75% of diabetic males. Neural stimulation is a crucial aspect of the normal erection process. Nerve injury causes ED and disrupts signaling of the Sonic hedgehog (Shh) cascade in the smooth muscle of the corpora cavernosa. Shh and targets of its signaling establish normal corpora cavernosal morphology during postnatal differentiation of the penis and regulate homeostasis in the adult. Interruption of the Shh cascade in the smooth muscle of the corpora cavernosa results in extensive changes in corpora cavernosal morphology that lead to ED. Our hypothesis is that the neuropathy observed in diabetics causes morphological changes in the corpora cavernosa of the penis that result in ED. Disruption of the Shh cascade may be involved in this process. We tested this hypothesis by examining morphological changes in the penis, altered gene and protein expression, apoptosis, and bromodeoxyuridine incorporation in the BB/WOR rat model of diabetes. Extensive smooth muscle and endothelial degradation was observed in the corpora cavernosa of diabetic penes. This degradation accompanied profound ED, significantly decreased Shh protein in the smooth muscle of the corpora cavernosa, and increased penile Shh RNA expression in the intact penis (nerves, corpora, and urethra). Localization and expression of Shh targets were also disrupted in the corpora cavernosa. Increasing our understanding of the molecular mechanisms that regulate Shh signaling may provide valuable insight into improving treatment options for diabetic impotence.

ABSENCE OF EXPRESSION OF TRANSFORMING GROWTH FACTOR-beta TYPE II RECEPTOR IS ASSOCIATED WITH AN AGGRESSIVE GROWTH PATTERN IN A MURINE RENAL CARCINOMA CELL LINE, RENCA

Transforming growth factor-beta1 (TGF-beta1) inhibits the proliferation of many cancer cells. However, tumor cells frequently become resistant to this inhibitory effect due to the absence of TGF-beta receptor (TbetaR) expression. This study reports the nature of TGF-beta sensitivity in an aggressive murine renal carcinoma cell line, Renca, investigated in a series of experiments. The growth of Renca cells, in tissue culture, was not sensitive to the inhibitory effect of TGF-beta1 with doses ranging from 0.1 to 10 ng./ml., nor was this cell line sensitive to the effect of TGF-beta1 in inducing the expression of plasminogen activator inhibitor-I. Renca cells expressed TGF-beta1 mRNA and protein, as determined by RT-PCR and ELISA, respectively. The level of TGF-beta1 production by Renca cells was moderate, thus eliminating the possibility that endogenous TGF-beta1 production might be masking the effect of TGF-beta sensitivity. Furthermore, Renca cells expressed TbetaR-I mRNA, but did not express TbetaR-II mRNA, suggesting that the absence of this receptor may be the cause of TGF-beta insensitivity. Additionally, a vector containing the TbetaR-II cDNA was transiently transfected into Renca cells. The inhibitory effect of TGF-beta1 was introduced in Renca cells after transfection with this receptor. At the same time, the growth rate of these cells diminished significantly when compared with that of the wild type Renca cells, as judged by the rate of [3H]-thymidine incorporation in the absence of any exogenous TGF-beta1. These observations demonstrated that Renca cells lack the functional TbetaR-II and suggest that their aggressive growth pattern is due, at least in part, to their insensitivity to TGF-beta.

Review of impotence literature protein and gene expression of nitric oxide synthase isoforms I and III in the rat penile shaft

Nitric oxide synthase (NOS) plays a key role in penile smooth muscle relaxation through the regulation of nitric oxide (NO). NO is a major neurotransmitter in the autonomic nervous system, and alteration of its activity has been implicated in erectile dysfunction. The objectives of this study were twofold: 1) to demonstrate and localize the NOS protein isoforms I and III in the normal rat penis, and 2) to identify and quantitate NOS I and III gene expression in the normal rat penis. The gene and protein product of NOS isoforms I and III are expressed in rat penile tissue. Protein expression of NOS I was confined primarily to neuronal tissue, while NOS III protein expression was identified primarily in both cavernosal smooth muscle and endothelium. The presence of both NOS I and III was confirmed in the penile shaft by Western blot. Quantitation of NOS I and III gene expression by reverse transcription-polymerase chain reaction revealed NOS III to be more highly expressed than that of NOS I in the rat penile shaft. NOS I and III protein and gene products are both expressed in normal rat penile tissue. Protein expression is localized primarily to neuronal tissue for NOS I, whereas NOS III is localized primarily to cavernosal smooth muscle and endothelium. NOS III gene expression is greater than that of NOS I in the normal rat penile shaft. These findings support the possibility that penile erection is regulated by different NOS isoforms released from neural, endothelial, and smooth muscle sources.