Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Role of p63 and basal cells in the prostate

The prostate contains two major epithelial cell types - luminal and basal cells - both of which develop from urogenital sinus epithelium. The cell linage relationship between these two epithelial types is not clear. Here we demonstrate that luminal cells can develop independently of basal cells, but that basal cells are essential for maintaining ductal integrity and the proper differentiation of luminal cells. Urogenital sinus (UGS) isolated from p63(+/+) and p63(-/-) embryos developed into prostate when grafted into adult male nude mice. Prostatic tissue that developed in p63(-/-) UGS grafts contained neuroendocrine and luminal cells, but basal cells were absent. Therefore, p63 is essential for differentiation of basal cells, but p63 and thus basal cells are not required for differentiation of prostatic neuroendocrine and luminal epithelial cells. p63(-/-) prostatic grafts also contained atypical mucinous cells, which appeared to differentiate from luminal cells via activation of Src. In the response to castration, regression of p63(-/-) prostate was inordinately severe with almost complete loss of ducts, resulting in the formation of residual cystic structures devoid of epithelium. Therefore, basal cells play critical roles in maintaining ductal integrity and survival of luminal cells. However, regressed p63(-/-) prostate did regenerate in response to androgen administration, indicating that basal cells were not essential for prostatic regeneration.     

Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation

Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.     

Estrogen receptor β activation impairs prostatic regeneration by inducing apoptosis in murine and human stem/progenitor enriched cell populations

Androgen depletion is the primary treatment for prostate disease; however, it fails to target residual castrate-resistant cells that are regenerative and cells of origin of prostate cancer. Estrogens, like androgens, regulate survival in prostatic cells, and the goal of this study was to determine the advantages of selective activation of estrogen receptor β (ERβ) to induce cell death in stem cells that are castrate-resistant. Here we show two cycles of short-term ERβ agonist (8β-VE2) administration this treatment impairs regeneration, causing cystic atrophy that correlates with sustained depletion of p63+ basal cells. Furthermore, agonist treatment attenuates clonogenicity and self-renewal of murine prostatic stem/progenitor cells and depletes both murine (Lin(-)Sca1(+)CD49f(hi)) and human (CD49f(hi)Trop2(hi)) prostatic basal cells. Finally, we demonstrate the combined added benefits of selective stimulation of ERβ, including the induction of cell death in quiescent post-castration tissues. Subsequent to castration ERβ-induces further apoptosis in basal, luminal and intermediate cells. Our results reveal a novel benefit of ERβ activation for prostate disease and suggest that combining selective activation of ERβ with androgen-deprivation may be a feasible strategy to target stem cells implicated in the origin of prostatic disease.     

Regulation of trespin expression by modulators of cell growth,differentiation, and apoptosis in prostatic epithelial cells

We recently identified a novel rat ov-serpin, Trespin, which inhibits the trypsin-like serine proteinase plasmin and is expressed in several tissues, including prostate. In this report Trespin expression was studied in prostatic cell lines, NRP-152, NRP-154, and DP-153, derived from the Lobund-Wistar rat. Northern blots revealed Trespin mRNA is expressed in NRP-152 and DP-153 basal epithelial cell lines but not in the luminal line, NRP-154. Similarly, Trespin levels drop >30-fold following transdifferentiation of NRP-152 cells toward a luminal variant, further suggesting Trespin expression is specific for basal prostatic epithelial cells. Trespin expression in NRP-152 cells is up-regulated by dexamethasone (Dex) and insulin-like growth factor-I (IGF-I), each of which stimulate growth and prevent differentiation and apoptosis. However, Dex (alone) facilitates loss of Trespin by TGF-beta, yet enhances the ability of LR(3)-IGF-I to reverse such loss, similar to the pattern of apoptosis induced by TGF-beta. Likewise, several apoptosis inducers markedly decrease Trespin mRNA levels. HEK293 cells stably overexpressing Trespin display increased cell proliferation and partial resistance to growth inhibition and phosphorylation of c-Jun induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Together these data strongly suggest that Trespin has critical functions tied to the regulation of growth, differentiation, and apoptosis of prostatic epithelial cells.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

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.     

Identification of a stem cell candidate in the normal human prostate gland

Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial-stromal interaction was maintained, a remarkable increase of Ck6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p<0.0001). Within the prostate organ culture model the increase of cytokeratin 6a-expressing cells significantly correlated with increased proliferation index (r = 0.7616, p = 0.0467). The Ck6a+ cells were capable of differentiation as indicated by their expression of luminal cell markers such as ZO-1 and prostate specific antigen (PSA). Our data indicate that Ck6a+ cells represent a prostatic epithelial stem cell candidate possessing high potential for proliferation and differentiation. Since the development of benign prostatic hyperplasia and prostate carcinogenesis are disorders of proliferation and differentiation, the Ck6a+ cells may represent a major element in the development of these diseases.     

Development and limitations of lentivirus vectors as tools for tracking differentiation in prostate epithelial cells

To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133⁺ cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133⁺ cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation.     

Protein profile of basal prostate epithelial progenitor cells—stage‐specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin^?Sca‐1⁺ CD49f⁺ Trop2^high‐phenotype) and human (Lin^? CD49f⁺ TROP2^high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f⁺/TROP2^high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9⁺/CD24⁺/CD29⁺/CD44⁺/CD47⁺/CD49f⁺/CD104⁺/CD147⁺/CD326⁺/Trop2^high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f⁺ TROP2⁺ basal prostate progenitor cells. Transplantation experiments suggest that CD49f⁺ TROP2^high SSEA‐4^high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f⁺ TROP2^high or CD49f⁺ TROP2^high SSEA‐4^low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.     

Prostate Sphere-forming Stem Cells Are Derived from the P63-expressing Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, including basal, luminal, and neuroendocrine cells. Multiple methods have been used to identify P-SCs in adult prostates. These include in vivo renal capsule implantation of a single epithelial cell with urogenital mesenchymal cells, in vitro prostasphere and organoid cultures, and lineage tracing with castration-resistant Nkx3.1 expression (CARN), in conjunction with expression of cell type-specific markers. Both organoid culture and CARN tracing show the existence of P-SCs in the luminal compartment. Although prostasphere cells predominantly express basal cell-specific cytokeratin and P63, the lineage of prostasphere-forming cells in the P-SC hierarchy remains to be determined. Using lineage tracing with P63^CreERT2, we show here that the sphere-forming P-SCs are P63-expressing cells and reside in the basal compartment. Therefore we designate them as basal P-SCs (P-bSCs). P-bSCs are capable of differentiating into AR⁺ and CK18⁺ organoid cells, but organoid cells cannot form spheres. We also report that prostaspheres contain quiescent stem cells. Therefore, the results show that P-bSCs represent stem cells that are early in the hierarchy of overall prostate tissue stem cells. Understanding the contribution of the two types of P-SCs to prostate development and prostate cancer stem cells and how to manipulate them may open new avenues for control of prostate cancer progression and relapse.     

Cell hierarchy and lineage commitment in the bovine mammary gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin⁻ epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24^medCD49f^pos (putative stem cells, puStm), CD24^negCD49f^pos (Basal), CD24^highCD49f^neg (putative progenitors, puPgt) and CD24^medCD49f^neg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched.     

Epcam, CD44, and CD49f distinguish sphere-forming human prostate basal cells from a subpopulation with predominant tubule initiation capability

Background

Human prostate basal cells expressing alpha-6 integrin (CD49f^Hi) and/or CD44 form prostaspheres in vitro. This functional trait is often correlated with stem/progenitor (S/P) activity, including the ability to self-renew and induce differentiated tubules in vivo. Antigenic profiles that distinguish tubule-initiating prostate stem cells (SCs) from progenitor cells (PCs) and mature luminal cells (LCs) with less regenerative potential are unknown.

Methodology/Principle Findings

Prostasphere assays and RT-PCR analysis was performed following FACS separation of total benign prostate cells based upon combinations of Epcam, CD44, and/or CD49f expression. Epithelial cell fractions were isolated, including Epcam⁺CD44⁺ and Epcam+CD44+CD49f^Hi basal cells that formed abundant spheres. When non-sphere-forming Epcam⁺CD44⁻ cells were fractionated based upon CD49f expression, a distinct subpopulation (Epcam⁺CD44⁻CD49f^Hi) was identified that possessed a basal profile similar to Epcam⁺CD44⁺CD49f^Hi sphere-forming cells (p63⁺AR^LoPSA⁻). Evaluation of tubule induction capability of fractionated cells was performed, in vivo, via a fully humanized prostate tissue regeneration assay. Non-sphere-forming Epcam⁺CD44⁻ cells induced significantly more prostate tubular structures than Epcam⁺CD44⁺ sphere-forming cells. Further fractionation based upon CD49f co-expression identified Epcam⁺CD44⁻CD49f^Hi (non-sphere-forming) basal cells with significantly increased tubule induction activity compared to Epcam⁺CD44⁻CD49f^Lo (true) luminal cells.

Conclusions/Significance

Our data delineates antigenic profiles that functionally distinguish human prostate epithelial subpopulations, including putative SCs that display superior tubule initiation capability and induce differentiated ductal/acini structures, sphere-forming PCs with relatively decreased tubule initiation activity, and terminally differentiated LCs that lack both sphere–forming and tubule-initiation activity. The results clearly demonstrate that sphere-forming ability is not predictive of tubule-initiation activity. The subpopulations identified are of interest because they may play distinct roles as cells of origin in the development of prostatic diseases, including cancer.     

Prostate epithelial stem cell culture

The prostate gland is the site of the second most common cancer in men in the UK, with 9,280 deaths recorded in 2000. Another common disease of the prostate is benign prostatic hyperplasia and both conditions are believed to arise as a result of changes in the balance between cell proliferation and differentiation. There are three types of prostatic epithelial cell, proliferative basal, secretory luminal, and neuroendocrine. All three are believed to be derived from a common stem cell through differentiation along different pathways but the mechanisms behind these processes is poorly understood. In particular, there has until recently been very little information about prostate stem cell growth and differentiation. This review will discuss ways of distinguishing these prostate cell types using markers, such as keratins. Methods available for the culture of prostate epithelial cells and for the characterisation of stem cells both in monolayer and three-dimensional models are examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of epithelial ductal branching in the prostate by sonic hedgehog is indirectly mediated by stromal cells

Sonic hedgehog (Shh), a vertebrate homologue of the Drosophila segment-polarity gene hedgehog, has been reported to play an important role during normal development of various tissues. Abnormal activities of Shh signaling pathway have been implicated in tumorigenesis such as basal cell carcinomas and medulloblastomas. Here we show that Shh signaling negatively regulates prostatic epithelial ductal morphogenesis. In organotypic cultures of developing rat prostates, Shh inhibited cell proliferation and promoted differentiation of luminal epithelial cells. The expression pattern of Shh and its receptors suggests a paracrine mechanism of action. The Shh receptors Ptc1 (Patched1) and Ptc2 were found to be expressed in prostatic stromal cells adjacent to the epithelium, where Shh itself was produced. This paracrine model was confirmed by co-culturing the developing prostate in the presence of stromal cells transfected with a vector expressing a constitutively active form of Smoothened, the real effector of the Shh signaling pathway. Furthermore, expression of activin A and TGF-beta1 that were shown previously to inhibit prostatic epithelial branching was up-regulated following Shh treatment in the organotypic cultures. Taken together, these results suggest that Shh negatively regulates prostatic ductal branching indirectly by acting on the surrounding stromal cells, at least partly via up-regulating expression of activin A and TGF-beta1.