Molecular signatures of the primitive prostate stem cell niche reveal novel mesenchymal-epithelial signaling pathways.

BACKGROUND: Signals between stem cells and stroma are important in establishing the stem cell niche. However, very little is known about the regulation of any mammalian stem cell niche as pure isolates of stem cells and their adjacent mesenchyme are not readily available. The prostate offers a unique model to study signals between stem cells and their adjacent stroma as in the embryonic prostate stem cell niche, the urogenital sinus mesenchyme is easily separated from the epithelial stem cells. Here we investigate the distinctive molecular signals of these two stem cell compartments in a mammalian system. METHODOLOGY/PRINCIPAL FINDINGS: We isolated fetal murine urogenital sinus epithelium and urogenital sinus mesenchyme and determined their differentially expressed genes. To distinguish transcripts that are shared by other developing epithelial/mesenchymal compartments from those that pertain to the prostate stem cell niche, we also determined the global gene expression of epidermis and dermis of the same embryos. Our analysis indicates that several of the key transcriptional components that are predicted to be active in the embryonic prostate stem cell niche regulate processes such as self-renewal (e.g., E2f and Ap2), lipid metabolism (e.g., Srebp1) and cell migration (e.g., Areb6 and Rreb1). Several of the enriched promoter binding motifs are shared between the prostate epithelial/mesenchymal compartments and their epidermis/dermis counterparts, indicating their likely relevance in epithelial/mesenchymal signaling in primitive cellular compartments. Based on differential gene expression we also defined ligand-receptor interactions that may be part of the molecular interplay of the embryonic prostate stem cell niche. CONCLUSIONS/SIGNIFICANCE: We provide a comprehensive description of the transcriptional program of the major regulators that are likely to control the cellular interactions in the embryonic prostatic stem cell niche, many of which may be common to mammalian niches in general. This study provides a comprehensive source for further studies of mesenchymal/epithelial interactions in the prostate stem cell niche. The elucidation of pathways in the normal primitive niche may provide greater insight into mechanisms subverted during abnormal proliferative and oncogenic processes. Understanding these events may result in the development of specific targeted therapies for prostatic diseases such as benign prostatic hypertrophy and carcinomas.

PINing down the origin of prostate cancer.

The epithelium that lines the surface of prostate glands contains several cell types, including luminal secretory cells and basal cells of unclear function. Despite the fact that prostate tumors contain cells with a luminal phenotype and lack basal cells, a recent report indicates that the cell of origin for human prostate cancer is a basal cell and not a luminal cell. In contrast, another study indicates the reverse. It is possible that both basal and luminal stem/progenitor cells may independently give rise to prostate cancer; a comparison of the molecular signatures of the target cells of transformation with those of prostate tumors may aid in predicting the phenotypes of tumors with aggressive characteristics.

High aldehyde dehydrogenase activity: a novel functional marker of murine prostate stem/progenitor cells.

We have shown previously that prostatic stem/progenitor cells can be purified from isolated prostate ducts, based on their high expression of the Sca-1 surface antigen. We now report that high levels of aldehyde dehydrogenase (ALDH) activity are present in a subset of prostate epithelial cells that coexpress a number of antigens found on stem/progenitor cells of other origins (CD9, Bcl-2, CD200, CD24, prominin, Oct 3/4, ABCG2, and nestin). Almost all of these cells expressing high levels of ALDH activity also express Sca-1 and a third of them express high levels of this antigen. The cells with high levels of ALDH activity have greater in vitro proliferative potential than cells with low ALDH activity. Importantly, in an in vivo prostate reconstitution assay, the cells expressing high levels of ALDH activity were much more effective in generating prostatic tissue than a population of cells with low enzymatic activity. Thus, a high level of ALDH activity can be considered a functional marker of prostate stem/progenitor cells and allows for simple, efficient isolation of cells with primitive features. The elucidation of the role of ALDH in prostate stem/progenitor cells may lead to the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia.

Molecular signatures of prostate stem cells reveal novel signaling pathways and provide insights into prostate cancer.

BACKGROUND: The global gene expression profiles of adult and fetal murine prostate stem cells were determined to define common and unique regulators whose misexpression might play a role in the development of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: A distinctive core of transcriptional regulators common to both fetal and adult primitive prostate cells was identified as well as molecules that are exclusive to each population. Elements common to fetal and adult prostate stem cells include expression profiles of Wnt, Shh and other pathways identified in stem cells of other organs, signatures of the aryl-hydrocarbon receptor, and up-regulation of components of the aldehyde dehydrogenase/retinoic acid receptor axis. There is also a significant lipid metabolism signature, marked by overexpression of lipid metabolizing enzymes and the presence of the binding motif for Srebp1. The fetal stem cell population, characterized by more rapid proliferation and self-renewal, expresses regulators of the cell cycle, such as E2f, Nfy, Tead2 and Ap2, at elevated levels, while adult stem cells show a signature in which TGF-beta has a prominent role. Finally, comparison of the signatures of primitive prostate cells with previously described profiles of human prostate tumors identified stem cell molecules and pathways with deregulated expression in prostate tumors including chromatin modifiers and the oncogene, Erg. CONCLUSIONS/SIGNIFICANCE: Our data indicate that adult prostate stem or progenitor cells may acquire characteristics of self-renewing primitive fetal prostate cells during oncogenesis and suggest that aberrant activation of components of prostate stem cell pathways may contribute to the development of prostate tumors.

Endothelial cells support the growth of prostate tissue in vivo.

INTRODUCTION: The contribution of vascular endothelial cells to prostate growth has not been investigated. We examined whether endothelial cells support growth of prostate tissue when co-inoculated with prostate epithelial cells under the renal capsule. METHODS: Vascular endothelial cells were isolated from mice and co-inoculated under the renal capsule with a prostate luminal or basal epithelial cell line. After 60 days, kidneys were examined for growth of prostate tissue. Prostatic tissues were examined by immunohistochemistry for expression of cytokeratins 5 and 8, and vascular density was determined. To determine if increased expression of VEGF-A would increase prostatic growth, transfected endothelial cells overexpressing VEGF-A were co-inoculated with the prostate luminal or basal epithelial lines. RESULTS: Co-inoculation of endothelial cells and prostate luminal or basal epithelial cells resulted in significant growth of prostatic tissue, whereas inoculation of any of the cell lines alone resulted in little growth. The growths from co-inoculation of endothelial cells and luminal epithelial cells contained duct-like structures that stained with antibodies to cytokeratin 8, whereas those from co-inoculation of endothelial cells and basal epithelial cells contained cords of cells that stained with antibodies to cytokeratin 5. Overexpression of VEGF-A had no effect on growth of the prostatic tissues. CONCLUSION: Endothelial cells contribute to the growth of prostatic epithelial cells.

Vascular density is highest in the proximal region of the mouse prostate.

BACKGROUND: The proximal region of the prostatic ducts harbor the prostatic epithelial stem cells. As stem cell niches in other organs are highly vascularized, we determined if the proximal region is more highly vascularized than the remaining regions of the prostate. The effect of androgen on vascular density in the different prostatic regions was also examined. METHODS: Sections from prostates were immunostained with antibodies to CD31, and the vascular density in proximal, intermediate, and distal regions was calculated by image analysis software. Vascular density was compared in prostates from castrated mice that received daily inoculations of testosterone or vehicle alone for 3 days. To examine the role of angiogenic factors in the response to androgen, some animals were also treated with soluble VEGF receptor-2-Fc or Tie-2--Fc fusion proteins, which inhibit the activities of VEGF and angiopoietins, respectively. The endothelial proliferative response to androgen was determined by double staining sections with antibodies to CD31 and Ki-67. RESULTS: In prostates from intact mice, vascular density was highest in the proximal region and lowest in the distal region. Administration of testosterone to castrated mice increased vascular density to the greatest extent in the distal and intermediate regions. The increase in vascular density required VEGF and the angiopoietins. Endothelial cell proliferation was less sensitive to androgen in the proximal region than the remainder of the prostate. CONCLUSIONS: Vascular density is highest in the proximal region of the prostate, but the proximal vessels are less responsive to testosterone.

Vascular endothelial growth factor and angiopoietin are required for prostate regeneration.

BACKGROUND: The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens. METHODS: Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2. RESULTS: Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells. CONCLUSION: VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens.

Prostate cells express two isoforms of fibroblast growth factor receptor 1 with different affinities for fibroblast growth factor-2.

BACKGROUND: Fibroblast growth factor receptor 1 (FGFR1) mRNA can be alternatively spliced to generate isoforms containing (FGFR1alpha) or lacking (FGFR1beta) the first immunoglobulin-like domain. We examined which isoforms are expressed by cultured prostate cells, their affinities for FGF-2, and the effect of heparin on FGF-2 binding. METHODS: FGFR1 isoform expression was examined by RT-PCR. FGFR1alpha and FGFR1beta were expressed in CHO cells mutant in heparan sulfate synthesis, and their affinities for FGF-2, FGF-1, FGF-4, and FGF-6 were determined in the presence and absence of heparin. RESULTS: FGFR1alpha was expressed in luminal epithelial cells, whereas FGFR1beta was expressed in basal epithelial and smooth muscle cells. FGFR1beta bound FGF-2 with three-fourfold higher affinity than FGFR1alpha both in the presence and absence of heparin. Heparin increased affinity of both receptor isoforms for FGF-2 approximately four-fivefold. CONCLUSIONS: Prostate smooth muscle and basal epithelial cells are likely to be more sensitive than luminal epithelial cells to the low concentrations of FGFs present in vivo.

Proximal prostatic stem cells are programmed to regenerate a proximal-distal ductal axis.

Prostate carcinoma and benign prostatic hypertrophy may both originate in stem cells, highlighting the importance of the characterization of these cells. The prostate gland contains a network of ducts each of which consists of a proximal (adjacent to the urethra), an intermediate, and a distal region. Here, we report that two populations of cells capable of regenerating prostatic tissue in an in vivo prostate reconstitution assay are present in different regions of prostatic ducts. The first population (with considerable growth potential) resides in the proximal region of ducts and in the urethra, and the survival of these cells does not require the presence of androgens. The second population (with more limited growth potential) is found in the remaining ductal regions and requires androgen for survival. In addition, we find that primitive proximal prostate cells that are able to regenerate functional prostatic tissue in vivo are also programmed to re-establish a proximal-distal ductal axis. Similar to their localization in the intact prostate, cells with the highest regenerative capacity are found in the proximal region of prostatic ducts formed in an in vivo prostate reconstitution assay. The primitive proximal cells can be passaged through four generations of subrenal capsule grafts. Together, these novel findings illustrate features of primitive prostate cells that may have implications for the development of therapies for treating proliferative prostatic diseases.

Retinal blood vessels develop in response to local VEGF-A signals in the absence of blood flow.

The role of hemodynamic forces and other signals from circulating blood in guiding the development of the retinal vasculature was examined by following the growth of these vessels in organ cultures. Retinal vascular development in organ cultures was monitored by immunofluorescent staining of retinal whole-mounts using antibodies against ICAM-2, a specific marker for endothelial cells and by vascular adenosine disphosphatase activity. Under culture conditions, the retinal vasculature from mice at postnatal day 3 (P3) grew from the optic nerve area to the edge of the retina in a manner similar to that observed in vivo. Both inner and outer vascular plexuses formed in retinal explants. Within the first few days of organ culture, the initial uniform meshwork of blood vessels was reorganized into arterioles, venules, and capillaries. As in animals, the initial retinal vascular plexus contained abundant vessels, and afterward some vessels regressed leading to the formation of a mature vascular bed. Changes in vascular density due to blood vessel growth and remodeling were confirmed by RT-PCR and Western blot analyses of ICAM-2 mRNA and protein levels, respectively. In addition, during in vitro retinal vascularization, arterioles acquired mural cell coverage, as shown by positive staining for alpha-smooth muscle actin. Thus, blood flow and blood-derived signals were not required for the development and maturation of retinal vessels. In contrast, stability of blood vessels in retinal explants was tightly regulated by endogenous levels of vascular endothelial growth factor-A (VEGF-A). VEGF-A was expressed in the explants throughout the culture period, and addition of neutralizing antibodies against VEGF-A to the organ culture caused a severe regression of blood vessels from the vascular front toward the optic nerve. In contrast, addition of anti-FGF-2 antibodies had no effect on the developing vasculature. Thus, retinal vascular development is dependent on local VEGF-A signals rather than systemic signals.

TGF-{beta} maintains dormancy of prostatic stem cells in the proximal region of ducts.

We have previously shown that prostatic stem cells are located in the proximal region of mouse prostatic ducts. Here, we show that this region responds differently to transforming growth factor (TGF)-beta than the distal ductal region and that under physiological conditions androgens and TGF-beta are crucial overall regulators of prostatic tissue homeostasis. This conclusion is supported by the observations showing that high levels of TGF-beta signaling are present in the quiescent proximal region of ducts in an androgen-replete animal and that cells in this region overexpress Bcl-2, which protects them from apoptosis. Moreover, androgen ablation reverses the proximal-distal TGF-beta signaling gradient, leading to an increase in TGF-beta signaling in the unprotected distal region (low Bcl-2 expression). This reversal of TGF-beta-mediated signaling accompanies apoptosis of cells in the distal region and gland involution after androgen withdrawal. A physiological TGF-beta signaling gradient (high proximally and low distally) and its functional correlates are restored after androgen replenishment. In addition to highlighting the regulatory role of androgens and TGF-beta, these findings may have important implications for the deregulation of the stem cell compartment in the etiology of proliferative prostatic diseases.

Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue.

We previously showed that prostatic stem cells are concentrated in the proximal regions of prostatic ducts. We now report that these stem cells can be purified from isolated proximal duct regions by virtue of their high expression of the cell surface protein stem cell antigen 1 (Sca-1). In an in vivo prostate reconstitution assay, the purified Sca-1-expressing cell population isolated from the proximal region of ducts was more effective in generating prostatic tissue than a comparable population of Sca-1-depleted cells (203.0 +/- 83.1 mg vs. 11.9 +/- 9.2 mg) or a population of Sca-1-expressing cells isolated from the remaining regions of ducts (transit-amplifying cells) (31.9 +/- 24.1 mg). Almost all of the proliferative capacity of the proximal duct Sca-1-expressing cell population resides within the fraction of cells that express high levels of Sca-1 (top one-third), with the proximal region of prostatic ducts containing 7.2-fold more Sca-1(high) cells than the remaining regions. More than 60% of the high-expressing cells coexpress alpha6 integrin and the anti-apoptotic factor Bcl-2, markers that are also characteristic of stem cells of other origins. Further stratification of the phenotype of the stem cells may enable the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia.

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2).

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.

Stromal/epithelial interactions of murine prostatic cell lines in vivo: a model for benign prostatic hyperplasia and the effect of doxazosin on tissue size.

BACKGROUND: One of the major constraints in elucidating the mechanisms involved in the etiology of benign prostatic hyperplasia (BPH) is the lack of suitable model systems that are readily manipulable in vitro and in vivo. To address this issue, we have used murine prostatic cell lines to establish a novel in vivo model for studying prostatic cell interactions. METHODS: Luminal, basal, and smooth muscle (SM) cell lines were inoculated alone or in combinations under the renal capsule of intact or castrated male mice, and the growth and composition of prostatic tissue in the absence or presence of doxazosin was determined. RESULTS: Both the luminal and basal cell lines reconstituted prostatic tissue if co-inoculated under the renal capsule with normal SM cells, whereas none of the lines formed significant tissue when inoculated alone. Luminal cells produced and secreted prostatic secretory products. The growth of prostatic tissue formed from co-inoculation of basal and SM cells was androgen responsive. In addition, a significant reduction in prostatic tissue was noted in animals treated with doxazosin. CONCLUSION: We have established an in vivo model that uses prostatic epithelial and SM cell lines for investigating cellular interactions between epithelial and SM cells that regulate prostatic growth and function. This model will be useful for delineating the mechanisms by which prostatic cells interact and in determining the efficacy of new approaches aimed at interfering with prostatic stromal/epithelial interactions that result in abnormal cellular proliferation.

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis.

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.

Differentiation and stromal-induced growth promotion of murine prostatic tumors.

BACKGROUND: We have derived a panel of p53-null prostatic "basal" and "luminal" epithelial cell lines and their ras transformed counterparts to study stromal/epithelial interactions and the properties of tumors arising from "basal" and "luminal" cells. METHODS: Previously derived normal murine prostatic "basal" epithelial (PE-B-1) and "luminal" epithelial (PE-L-1) cell lines were transformed with N-Ras. These lines and a spontaneously transformed "luminal" cell line were inoculated subcutaneously or orthotopically into athymic mice, alone or in combination with normal prostatic smooth muscle cells (SMC). RESULTS: All transformed lines formed subcutaneous tumors. SMC significantly enhanced the growth rate of the tumors arising from the "basal" and one of the "luminal" cell lines. The transformed "basal" line gave rise to tumors expressing both "basal" and "luminal" cytokeratins. CONCLUSIONS: Prostatic SMC promote the growth of transformed epithelial cells, suggesting that prostatic stroma may promote tumor development. Furthermore, transformed "basal" cells give rise to tumors containing "luminal" cells, suggesting that although most human tumors have a "luminal" phenotype, they may originate from transformed "basal" cells.

Lack of ERK activation and cell migration in FGF-2-deficient endothelial cells.

The formation of blood capillaries from preexisting vessels (angiogenesis) and vascular remodeling secondary to atherosclerosis or vessel injury are characterized by endothelial cell migration and proliferation. Numerous growth factors control these cell functions. Basic fibroblast growth factor (FGF-2), a potent angiogenesis inducer, stimulates endothelial cell proliferation, migration, and proteinase production in vitro and in vivo. However, mice genetically deficient in FGF-2 have no apparent vascular defects. We have observed that endothelial cell migration in response to mechanical damage in vitro is accompanied by activation of the extracellular signal-regulated kinase (ERK) pathway, which can be blocked by neutralizing anti-FGF-2 antibodies. Endothelial cells from mice that are genetically deficient in FGF-2 neither migrate nor activate ERK in response to mechanical wounding. Addition of exogenous FGF-2 restores a normal cell response, which shows that impaired migration results from the genetic deficiency of this growth factor. Injury-induced ERK activation in endothelial cells occurs only at the edge of the wound. In addition, FGF-2-induced ERK activation mediates endothelial cell migration in response to wounding without a significant effect on proliferation. These data show that FGF-2 is a key regulator of endothelial cell migration during wound repair.

Androgens modulate the balance between VEGF and angiopoietin expression in prostate epithelial and smooth muscle cells.

BACKGROUND: The vasculature of the prostate responds to androgens. Androgens most likely affect the vasculature indirectly by modulating the expression of angiogenic factors in the cells of the prostate. Most studies to date have examined the production of angiogenic factors by the prostate luminal epithelium. Here we examine the effects of androgen on production of three angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2, by the three major cell types in the prostate. METHODS: The ability of androgen to modulate VEGF, angiopoietin-1, and angiopoietin-2 production in cultured mouse prostate luminal epithelial, basal epithelial, and smooth muscle cells (SMCs) was assessed by Western blot and RT-PCR. RESULTS: The production of VEGF was modulated by androgens in both luminal epithelial and prostate SMCs but not in basal epithelial cells. However, in prostate luminal epithelial cell cultures, VEGF was predominately secreted apically, suggesting that in vivo most of the epithelium-derived VEGF is unavailable to the underlying blood vessels. In addition, prostate luminal epithelial cells produced angiopoietin-2, an angiogenesis inhibitor. In contrast, prostate SMCs produced angiopoietin-1, a positive modulator of angiogenesis. Synthesis of the angiopoietins did not respond to androgen treatment. CONCLUSIONS: Prostate smooth muscle may play an important role in regulating vascular responses to androgen.