TGF-ß and stem cell factor regulate cell proliferation in the proximal stem cell niche.

BACKGROUND: Stem cells are located in specific regulatory environments termed niches, which modulate the survival and proliferation of the cells through a variety of both mitogenic and inhibitory cytokines. In the murine prostate, stem cells are located in the proximal region of prostatic ducts. We examined the regulation of murine prostate cells in the stem cell niche by transforming growth factor beta (TGF-ß) and stem cell factor (SCF). METHODS: Prostate cells from the proximal and distal regions of prostatic ducts were cultured in the presence and absence of TGF-ß and SCF, both on collagen-coated wells and in collagen gels. Cell growth on collagen was assessed by determining cell number. Cell growth in collagen gels was quantified by determining the number, size and complexity of prostatic ducts. The basal and luminal phenotype of the cells was determined by immunohistochemistry. RESULTS: Endogenous TGF-ß inhibited proliferation and promoted differentiation of proximal cells towards a luminal phenotype. It also inhibited duct-forming capacity and promoted differentiation of prostatic ducts towards a luminal phenotype. Addition of SCF enhanced proximal cell proliferation on collagen-coated wells and duct formation in collagen gels. Proliferation was further increased by ablation of endogenous TGF-ß. CONCLUSION: Proliferation and the basal/luminal cell composition of cells isolated from the proximal region of prostatic ducts, the stem cell niche, is regulated in part by opposing effects of SCF and endogenous TGF-ß.

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.

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.

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.

Axin2 expression identifies progenitor cells in the murine prostate.

BACKGROUND: We previously reported that prostatic stem/progenitor cells are concentrated in the proximal region of prostatic ducts and express stem cell antigen 1 (Sca-1). As Wnt signaling is important for the maintenance of stem cells, we determined whether Sca-1 expressing cells also express Axin2, as Axin2 expression is highly suggestive of active Wnt signaling. METHODS: Axin2 promoter reporter mice were used for whole mount and fluorescence activated cell sorting (FACS) analysis to determine its expression in the prostate. Axin2 expressing cells were also examined for the co-expression of Sca-1. We also used a chemical activator of Wnt signaling, BIO, to determine the effects of Wnt signaling on the growth of primary prostate cells in vitro. RESULTS: We show that Axin2 expression is present in all lobes and is regulated by androgens with the highest Axin2 expression in the lateral and dorsal prostate. Furthermore, a fraction of Axin2 expressing cells co-express Sca-1, suggesting that some progenitor cells have active Wnt signaling. Lastly, we demonstrate that activation of the Wnt pathway may result in increased growth, consistent with a role for Wnt signaling in maintenance and/or expansion of the progenitor cell population. CONCLUSION: Axin2 expressing cells that co-express Sca-1 are present in all prostate lobes suggesting that progenitor cells reside within the Wnt active population. An understanding of the basic biology of signaling pathways mediating growth in the prostate may lead to rational therapies to treat benign prostatic hyperplasia and prostate cancer.

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.

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.

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.

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.

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.

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.

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.