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.

Relating drug response to epigenetic and genetic markers using a region-based kernel score test.

In GAW20, we investigated the association of specific genetic regions of interest (ROIs) with log-transformed triglyceride (TG) levels following lipid-lowering medication using epigenetic and genetic markers. The goal was to incorporate kernels for cytosine-phosphate-guanine (CpG) markers and compare the kernels to a purely parametric model. Post-treatment TG levels were investigated for post-methylation data at CpG sites and region-specific SNPs and adjusted for pre-treatment TG levels and age, in independent individuals only (real data: n = 150; simulated data, replicate 84: n = 111). In both data sets, our single-CpG-marker results using kernels and linear regression were in good agreement. In the real data, we investigated the introns of the CPT1A gene previously reported as associated with TG levels as separate ROIs, and were able to find hints of an association of cg17058475 and cg00574958 with post-treatment TG levels. In the simulated data, we investigated a total of 10 regions, in which the 5 causal and 5 non-causal markers lie, respectively, with increased methylation variances, yielding plausible results for the 3 window sizes. Overall, this indicates that kernels for CpG markers are feasible. An interaction regression model for the causal SNP with the nearest CpG marker identified an effect for the SNPs with the three greatest heritabilities simulated. The simulation model assumed full SNP effect only for unmethylated regions decreasing to zero in the case of full methylation. Thus, in the context of a clear candidate setting, interaction between epigenetic and genetic data may enhance information, albeit nominally, even with small sample sizes. Relieving the burden of multiple testing, developing kernels further to analyze data from multiple omics jointly is well warranted.

Pathway-Based Kernel Boosting for the Analysis of Genome-Wide Association Studies.

The analysis of genome-wide association studies (GWAS) benefits from the investigation of biologically meaningful gene sets, such as gene-interaction networks (pathways). We propose an extension to a successful kernel-based pathway analysis approach by integrating kernel functions into a powerful algorithmic framework for variable selection, to enable investigation of multiple pathways simultaneously. We employ genetic similarity kernels from the logistic kernel machine test (LKMT) as base-learners in a boosting algorithm. A model to explain case-control status is created iteratively by selecting pathways that improve its prediction ability. We evaluated our method in simulation studies adopting 50 pathways for different sample sizes and genetic effect strengths. Additionally, we included an exemplary application of kernel boosting to a rheumatoid arthritis and a lung cancer dataset. Simulations indicate that kernel boosting outperforms the LKMT in certain genetic scenarios. Applications to GWAS data on rheumatoid arthritis and lung cancer resulted in sparse models which were based on pathways interpretable in a clinical sense. Kernel boosting is highly flexible in terms of considered variables and overcomes the problem of multiple testing. Additionally, it enables the prediction of clinical outcomes. Thus, kernel boosting constitutes a new, powerful tool in the analysis of GWAS data and towards the understanding of biological processes involved in disease susceptibility.

Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

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.

Routine packing of simple cutaneous abscesses is painful and probably unnecessary.

OBJECTIVES: The objective was to determine whether the routine packing of simple cutaneous abscesses after incision and drainage (I&D) confers any benefit over I&D alone. METHODS: In a prospective, randomized, single-blinded trial, subjects with simple cutaneous abscesses (less than 5 cm largest diameter) underwent incision, drainage, irrigation, and standard abscess preparation in the usual manner. Subjects were then randomized to either packing or no-packing. Visual analog scales (VAS; 100 mm) of pain were recorded in the emergency department (ED). All patients received trimethoprim-sulfamethoxazole (TMP-SMX), ibuprofen, and narcotic prescriptions, recorded twice daily VAS pain scores, and returned in 48 hours at which time dressings and packing, if present, were removed and a physician blinded to the randomization and not part of the initial visit repeated measurements and determined the need for further intervention. RESULTS: Forty-eight subjects were included in the final analysis. There were no significant differences in age, sex, abscess location, or initial pain scores between the two groups. There was no significant difference in need for a second intervention at the 48-hour follow-up between the packed (4 of 23 subjects) and nonpacked (5 of 25 subjects) groups (p = 0.72; relative risk = 1.3, 95% confidence interval [CI] = 0.4 to 4.2). Patients in the group that received packing reported higher pain scores immediately postprocedure (mean difference = 23.8 mm; p = 0.014, 95% CI = 5 to 42 mm) and at 48 hours postprocedure (mean difference = 16.4 mm; p = 0.03, 95% CI = 1.6 to 31.2 mm), as well as greater use of ibuprofen (mean difference = 0.32; p = 0.12, 95% CI = -1.4 to 2.0) and oxycodone/acetaminophen (mean difference = 2.19; p = 0.03, 95% CI = 0.2 to 4.1). CONCLUSION: In this pilot study, not packing simple cutaneous abscesses did not result in any increased morbidity, and patients reported less pain and used fewer pain medications than packed patients.

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.

Basic fibroblast growth factor modulates the expression of glycophorin A and c-kit and inhibits erythroid differentiation in K562 cells.

Basic fibroblast growth factor (bFGF) is produced by bone marrow stromal cells as well as by normal and leukemic hematopoietic cells. In this study, we examine the direct effects of bFGF on erythroid differentiation in K562 cells in order to determine whether bFGF can promote the expression of a primitive phenotype. Low levels of bFGF inhibited erythroid differentiation as evidenced by decreased expression of glycophorin A and increased expression of c-kit. bFGF also increased both the numbers and the sizes of colonies of K562 cells in soft agar assays. The addition of TGF-beta to these cells induced erythroid differentiation which resulted in an increase in glycophorin A and a decrease in c-kit. The simultaneous addition of bFGF and TGF-beta to K562 cells prevented both the TGF-beta-mediated increase in glycophorin A expression and the decrease in c-kit expression associated with erythroid differentiation. bFGF antagonised the TGF-beta-mediated promotion of erythroid differentiation in K562 cells in a dose dependent manner and these two cytokines counteracted each other on an approximately molar basis. These results indicate that bFGF alone increases expression of c-kit and promotes a primitive phenotype in K562 cells. In addition, bFGF counteracts the effects of differentiation-inducing cytokines, such as TGF-beta, on hematopoietic cells. It is therefore possible that enhanced production of bFGF by leukemic cells could contribute to their neoplastic phenotype by opposing the effects of negative regulators or cytokines that induce differentiation.

Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells.

Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells.