We have hypothesized that epithelial growth, branching, and canalization in the rodent ventral prostate (VP) would require matrix remodeling, and hence matrix metalloproteinase (MMP) activity. Therefore, the aim of this study was to evaluate the impact of blocking MMP-2, using whole organ culture. siRNA was employed to inhibit MMP-2 expression, and this was compared to GM6001's (a broad-spectrum MMP inhibitor) inhibition of general MMPs. These blocks impaired VP morphogenesis. MMP-2 silencing reduced organ size, epithelial area, and the number of tips, as well as caused a dilation of the distal parts of the epithelium. Histology, 3-D reconstruction, biochemistry, and second harmonic generation (SHG) revealed that MMP-2 silencing affected VP architecture by interfering in epithelial cell proliferation, lumen formation, and cellular organization of both epithelium and stroma, besides intense accumulation of collagen fibers. These data suggest that MMP-2 plays important roles in prostate growth, being directly involved with epithelial morphogenesis. Developmental Dynamics 239:737-746,

Androgens regulate prostate physiology, and exert their effects through the androgen receptor. We hypothesized that androgen deprivation needs additional transcription factors to orchestrate the changes taking place in the gland after castration and for the adaptation of the epithelial cells to the androgen-deprived environment, ultimately contributing to the origin of castration-resistant prostate cancer. This study was undertaken to identify transcription factors that regulate gene expression after androgen deprivation by castration (Cas). For the sake of comparison, we extended the analysis to the effects of administration of a high dose of 17β-estradiol (E2) and a combination of both (Cas+E2). We approached this by (i) identifying gene expression profiles and enrichment terms, and by searching for transcription factors in the derived regulatory pathways; and (ii) by determining the density of putative transcription factor binding sites in the proximal promoter of the 10 most up- or down-regulated genes in each experimental group in comparison to the controls Gapdh and Tbp7. Filtering and validation confirmed the expression and localized EVI1 (Mecom), NFY, ELK1, GATA2, MYBL1, MYBL2, and NFkB family members (NFkB1, NFkB2, REL, RELA and RELB) in the epithelial and/or stromal cells. These transcription factors represent major regulators of epithelial cell survival and immaturity as well as an adaptation of the gland as an immune barrier in the absence of functional stimulation by androgens. Elk1 was expressed in smooth muscle cells and was up-regulated after day 4. Evi1 and Nfy genes are expressed in both epithelium and stroma, but were apparently not affected by androgen deprivation.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Epithelial growth, branching, and canalization are important morphogenetic events of the rodent ventral prostate (VP) that take place during the first postnatal week. In this study, we evaluated the effect of knocking out MMP-2 (MMP-2 2/2 ), by examining developmental and structural aspects of the VP in MMP-2 2/2 mice. Neonate (day 6) MMP-2 2/2 mice showed fewer epithelial tips, a lower epithelial cell proliferation rate, and also reticulin fiber accumulation. The VP of adult MMP-2 2/2 mice showed lower relative weight, smaller epithelial and smooth-muscle cell volume, and a larger amount of thicker reticulin fibers. No differences in cell proliferation or apoptotic index were noted between adult MMP-2 2/2 and wild-type mice. MMP-9 was found in the adult MMP-2 2/2 , but not in the wild-type. In conclusion, MMP-2 function is essential for the epithelial morphogenesis of the mouse VP, and expression of MMP-9 is not sufficient for acquisition of the normal adult histology. Developmental Dynamics 239:2386-2392,

Heparanase-1 (HPSE-1) is an endoglycosidase that cleaves heparan sulfate. The physiological functions of HPSE-1 include embryo development, hair growth, wound healing, tumor growth, angiogenesis, metastasis, and inflammation. HPSE-1 expression was found to increase temporarily in the rat ventral prostate (VP) after castration. The promoter region of the Hpse-1 gene has estrogen-responsive elements, suggesting that the gene is regulated by estrogens. In this study, we investigated the expression of HPSE-1 in the VP of 90-day-old rats after neonatal exposure to a high dose of 17β-estradiol. HPSE-1 was not found by immunohistochemistry in the epithelium of estrogenized animals. To determine whether inhibition of Hpse-1 expression in the epithelium was due to pre- or post-transcriptional regulation, epithelial cells were isolated by centrifugation in Percoll gradient and the presence of Hpse-1 mRNA was investigated by RT-PCR. Hpse-1 mRNA was not detected in the estrogenized animals. Considering that Hpse-1 transcription could be inhibited by DNA methylation, we used the methylation-sensitive restriction enzyme HpaII and PCR to show that a single CCGG site at position +185 was more frequently methylated in the epithelium of estrogenized than in control animals. Immunohistochemistry for 5-methylcytidine revealed that the epithelial cell nuclei in estrogenized animals were heavily methylated. These results suggest that Hpse-1 expression was blocked in the epithelial cells of the VP, by estrogen imprinting by a pre-transcriptional mechanism involving DNA methylation.