TY - JOUR
AU1 - Yin, Lijuan
AU2 - Li, Jingjing
AU3 - Liao, Chun-Peng
AU4 - Jason Wu, Boyang
AB - Abstract Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5+ or p63+ basal cells, accompanied by lower Sca-1 expression in p63+ basal cells, but intact differentiated CK8+ luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133+/CD44+/CD24− stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Mice lacking monoamine oxidases, including both MAOA and MAOB isoforms, exhibit prostate atrophy, reduced CK5+ basal cells and p63+/Sca-1+ prostate basal/progenitor cell composition. Genetic silencing or pharmacological inhibition of monoamine oxidases in human prostate epithelial cells also impairs the regenerative capacity of prostate progenitor cells to form spheres. Open in new tabDownload slide Open in new tabDownload slide Monoamine oxidases, Prostate atrophy, Prostate stem/progenitor cells Significance Statement Prostate atrophy is a common phenomenon in men, with an increasing frequency with age, and has clinical implications in both benign and malignant prostate diseases. This article reports that abrogation of monoamine oxidases (MAOs) causes the development of prostate atrophy with reduced prostate basal and stem/progenitor cell activity in adult mice. This finding provides insight into the maintenance of prostate structure and function. This study also supports the application of clinically used MAO inhibitors to prevent prostatic hyperplasia and prostate cancer by suppressing prostate stem cell populations. Introduction Monoamine oxidases (MAOs) catalyze the oxidative deamination of a spectrum of biogenic and dietary amines, including monoamine neurotransmitters such as serotonin and dopamine [1]. The degradation of monoamines by MAOs produces hydrogen peroxide as a byproduct, which is a major source of oxidative stress [1, 2]. The two MAO isoforms, MAOA and MAOB, differ in substrate preferences and inhibitor specificities [2]. MAO dysfunctions have long been implicated in a variety of neuropsychiatric disorders such as depression and aggression, as demonstrated consistently in both humans and genetically modified mice [3-6]. MAO inhibitors are currently used in the clinic to treat depressive and neurodegenerative disorders [7]. The prostate is richly supplied with nerves, including mixed autonomic postganglionic neurons, which are crucial in regulating the physiology, morphology, and growth maturation of the prostate [8-10]. The autonomic nervous system in the prostate is mediated by neurotransmitters, neuromodulators, and neuropeptides that facilitate the crosstalk between nerve cells and prostate cells [8, 11]. Emerging evidence from recent studies has brought into focus the function of MAOs in normal prostate physiology and prostate cancer development. For instance, MAOA is expressed in the basal epithelial cells of normal prostate glands and prevents basal epithelial cells from differentiating into secretory cells [12]. MAOA was also recently shown by several groups, including us, to correlate with prostate cancer progression and serve as a therapeutic target in prostate cancer [13-16]. Nevertheless, the biological consequences of MAO ablation in the prostate remain largely unclear. Prostate stem cells play important roles in normal prostate development and adult tissue maintenance [17, 18]. The prostate contains three cell types: secretory luminal cells, basal cells, and rare neuroendocrine cells [19]. Developmental studies combining mouse models and prostate regeneration assays reveal that the mouse prostate originates from an ancestral basal stem cell population, which is in agreement with the evidence for the human prostate where the basal cell layer harbors regenerative stem cells [20-23]. Lineage-tracing studies also suggest the presence of lineage-restricted stem/progenitor cells in the luminal cell layer [24, 25], which is supported by human evidence revealing a common clonal origin for basal and luminal cells using in situ lineage tracking of human prostatic epithelial stem cell fate [26]. The regulation of prostate stem cells is complex, and involves a network mediated by both intrinsic genetic regulators and environmental cues [27, 28]. There have been no studies to date reporting a direct relationship of MAOs with prostate stemness properties and stem-like cell behavior. In this study, we report that mice lacking both MAOA and MAOB exhibit smaller prostate mass and prostate atrophy, accompanied by a reduction in the basal/progenitor cell capacity and epithelial cell proliferation. These findings provide new insights into the maintenance of the structural and functional integrity of the prostate gland. Materials and Methods Mice Wild-type (WT) and MAOA/B-knockout (KO) mouse tissues were kindly provided by Dr. Jean C. Shih (Department of Pharmacology and Pharmaceutical Sciences, University of Southern California). Mice were housed in the animal research facility at the University of Southern California and fed a normal chow diet. Generation of MAOA/B-KO mice was reported in a previous study [29]. Mice were genotyped for confirmation prior to use. Urogenital tissues including prostates were excised from 6-month-old WT and MAOA/B-KO male mice and weighed for comparison. Tissue Microarrays Prostate tissue microarrays containing 24 cases of normal prostate tissue (BNS19011) and 70 cases of prostate hyperplasia (PR804) were obtained from US Biomax and stained with antibodies specific against MAOA (H-70, Santa Cruz) or MAOB (kindly provided by Dr. Oliver Cases from Curie Institute, Paris, France) using our published protocol [15]. Histology and Immunohistochemical Analyses Prostate tissues of different lobes were fixed in 10% neutral-buffered formalin and embedded in paraffin for hematoxylin and eosin (H&E) or immunohistochemistry (IHC) staining following our published protocol [15]. Tissue sections were stained with rabbit anti-MAOA, rabbit anti-MAOB, rabbit anti-CK5 (Poly19055, Covance), mouse anti-CK8 (TROMA-I, Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-p63 (D-9, Santa Cruz), rabbit anti-p63 (H-137, Santa Cruz), mouse anti-Sca-1 (D7, BD Biosciences), rabbit anti-Ki-67 (Cell Signaling), mouse anti-α smooth muscle actin (αSMA) (1A4, Sigma-Aldrich), rabbit anti-androgen receptor (AR) (N-20, Santa Cruz), mouse anti-neurofilament-H (NF-H) (RMdO 20, Cell Signaling), rabbit anti-serotonin (ab10385, Abcam), or rabbit cleaved caspase-3 (Asp175, Cell Signaling) antibodies. HRP-conjugated goat antibodies against rabbit and mouse IgG (Dako) were used to develop specific stains with 3′3-diaminobenzidine. Image acquisition was performed by Nikon camera and software. For fluorescence visualization, sections were stained with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) or Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Life Technologies). Sections were further counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in mounting medium (Vector Laboratories) and examined by Zeiss Axio Observer Z1 fluorescence microscope. For multiplexed quantum dot labeling (mQDL) analysis of CK8, CK5 and p63 in mouse prostates, sections were sequentially stained with each protein using a previously published protocol [30]. Image acquisition was performed with a CRi spectral imaging system (Caliper Life Sciences) with built-in Nuance v3.1 software following the manufacturer's recommended protocol. Cell-based average of signal intensity counts for individual protein expression in epithelial or stromal components of each sample was analyzed by inForm software (PerkinElmer), an automated image analysis software enabling per-cell analysis of IHC samples, after areas of interest were defined using manual tissue segmentation by a pathologist [16, 30]. For analyzing CK8+, CK5+, p63+, p63+/Sca-1+, or Ki-67+ cells in mouse prostates, ventral and dorsolateral prostates were used as these lobes exhibit an atrophic phenotype in MAOA/B-KO mice compared with WT mice. Five ventral prostate lobes and 5 dorsolateral prostate lobes from 5 mice in each group were used for analysis. Cell analysis was performed by acquiring at least 5 adjacent nonoverlapping images per section, 5 sections per prostate lobe, with each section in a thickness of 5 µm and 100 µm apart, which represented an average of ∼100,000 cells per mouse. Cell Culture and Reagents The human normal prostate epithelial RWPE-1 cell line was obtained from the American Type Culture Collection. RWPE-1 cells were cultured in keratinocyte serum free medium (K-SFM) supplemented with bovine pituitary extract and EGF (Life Technologies). Human MAOA, MAOB, and nontargeting control shRNA lentiviral particles were purchased from Sigma-Aldrich. Clorgyline and deprenyl were purchased from Sigma-Aldrich. Generation of MAOA/B-Knockdown (KD) Cells Stable shRNA-mediated KD of MAOA and MAOB was achieved by sequential infection of cells with lentiviral particles expressing shRNAs against MAOA or MAOB, which carry a neomycin or a puromycin resistant gene respectively. Selection of MAOA-KD stable cells was achieved by 3-week G418 treatment (500 µg/ml) followed by 2-week puromycin selection (2 µg/ml) to obtain MAOA/B-KD stable cells. Lentiviral particles expressing a scrambled control shRNA were used in stable control cells. MAOA/B KD was validated by luminescence-based MAO-Glo assay (Promega) following the manufacturer's instructions. Established stable cells were maintained in culture medium supplemented with G418 and puromycin at the same doses for selection. Prostate Sphere Assay RWPE-1 cells subjected to MAOA/B KD or MAO inhibitor treatment were plated in 24-well plates with an ultra-low attachment surface (Corning) in serum-free prostate epithelial basal medium (PrEBM, Lonzo) supplemented with B-27 supplement (Life Technologies), EGF (20 ng/ml, BD Biosciences), bFGF (10 ng/ml, Life Technologies), insulin (5 µg/ml, Sigma-Aldrich), and 0.4% bovine serum albumin (Sigma-Aldrich). Floating spheres were grown for 9 days, counted, and dissociated with Accutase (Millipore) for secondary sphere expansion under the same conditions. Phase contrast images were obtained using the EVOS FL Cell Imaging System (Life Technologies). Flow Cytometric Analysis RWPE-1 cells or spheres were dissociated by Accutase and suspended in phosphate-buffered saline (PBS) supplemented with 0.5% BSA for staining with antibodies for 15 minutes at 4°C. The antibodies used were FITC-conjugated anti-CD44 (clone BJ18, BioLegend), PerCP/Cy5.5-conjugated anti-CD24 (clone ML5, BioLegend), and PE-conjugated anti-CD133 (clone AC133, Miltenyi Biotec). Fluorescence activated cell sorting (FACS) was performed using the BD FACSCanto II and the FACSAria III (BD Biosciences) for analysis and isolation of the CD133+/CD44+/CD24– cell population, respectively. Western Blotting Analysis Cells were extracted with radioimmunoprecipitation assay buffer in the presence of a protease and phosphatase inhibitor cocktail (Thermo Scientific), and blots were performed as described previously [31] using primary antibodies against MAOA, MAOB, CK8 (E432, Millipore), CK5 (RCK103, Santa Cruz), or β-Actin (AC-15, Santa Cruz). Immunoblots were further subjected to morphometric analysis by ImageJ software (NIH). Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (PCR) Total RNA was isolated from control and MAOA/B-KD RWPE-1 cells using an RNeasy Mini Kit (Qiagen) and reverse-transcribed to cDNA by M-MLV reverse transcriptase (Promega) following the manufacturer's instructions. qPCR was conducted using SYBR Green PCR Master Mix and run with Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). PCR conditions included an initial denaturation step of 3 minutes at 95°C, followed by 40 cycles of PCR consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. The PCR data were analyzed by 2–ΔΔCT method [32]. All primer sequences used are as follows: ALDH1A1 forward 5′-AGTGCCCCTTTGGTGGATTC-3′, ALDH1A1 reverse 5′-AAGAGCTTCTCTCCACTCTTG-3′, TROP2 forward 5′-TGACCTCCAAGTGTCTGCTG-3′, TROP2 reverse 5′-GTCGTAGAGGCCATCGTTGT-3′, CD166 forward 5′-CAACGTGTTTGAGGCACCTAC-3′, CD166 reverse 5′-TGAAATGCAGTCACCCAACTTT-3′, CK8 forward 5′-GGTTCCCGCATCAGCTCCTC-3′, CK8 reverse 5′-TCCACCTCCAGGACAAGGGG-3′, CK5 forward 5′-TGGAGCTGGTGGTGGCTTTG-3′, CK5 reverse 5′-GGTCTTGATCTGCTCGCGCT-3′, β-Actin forward 5′-TTGTTACAGGAAGTCCCTTGCC-3′, and β-Actin reverse 5′-ATGCTATCACCTCCCCTGTGTG-3′. Statistical Analysis Data are presented as the mean ± SEM as indicated in figure legends. All comparisons were analyzed by unpaired two-tailed Student's t test. A p value < .05 was considered statistically significant. Results MAOA/B Ablation Causes Smaller Prostate Mass and Prostate Atrophy To investigate the role of MAOs in the prostate, we used MAOA/B-KO mice where a spontaneous MAOA mutation occurred in MAOB-KO mice, resulting in the absence of protein and catalytic activity of both isoforms [29]. Male urogenital systems, including the prostate, seminal vesicles, bladder, and urethra, were collected from 6-month-old male adult mice. MAOA/B-KO urogenital organs showed a significant reduction in mass by ∼20% as compared with the WT controls (Fig. 1A, 470.5 ± 45.9 mg vs. 381.1 ± 33.3 mg for WT and MAOA/B-KO, respectively, n = 5 mice per group). Next, prostates, including all three types of lobes (ventral, dorsolateral, anterior) were separated from other urogenital tissues. MAOA/B-KO prostates also weighed less compared with the WT prostates (Fig. 1B, 84.7 ± 8.2 mg vs. 64.8 ± 5.7 mg for WT and MAOA/B-KO respectively, n = 5 mice per group). We did not observe significant changes in mouse body weight between the two groups (30.6 ± 4.1 g vs. 28.9 ± 2.4 g for WT and MAOA/B-KO respectively, n = 5 mice per group). As expected, MAOA and MAOB both showed negative expression in MAOA/B-KO prostates across all different cell types, including epithelial and stromal cells, compared with the WT prostates as examined by IHC (Fig. 1C). Histological analysis revealed distinctive histological changes in MAOA/B-KO prostates in comparison with the WT controls. Compared with the columnar shape of the cells in the WT ventral and dorsolateral prostates, the cells in the MAOA/B-KO ventral and dorsolateral prostates were thin with decreased amounts of cytoplasm, confirming epithelial atrophy. This phenotypic difference between the two groups was not evident in the anterior prostates (Fig. 1D). We further found a significant reduction in cell number in the MAOA/B-KO prostatic epithelia but not stroma compared with the WT controls by nuclear counts (210 ± 89 cells/gland, n = 623 vs. 177 ± 65 cells/gland, n = 639 for the WT and MAOA/B-KO respectively; p < .01), which may account for the smaller prostate mass in MAOA/B-KO mice relative to WT mice. These results in aggregate indicate a direct effect of MAOs on the mass and morphology of prostate glands. Figure 1 Open in new tabDownload slide MAOA/B ablation results in smaller prostate mass and prostate atrophy. (A, B): Weights of urogenital systems (A) and prostates (B, including ventral, dorsolateral, and anterior lobes) in 6-month-old male WT and MAOA/B-KO mice (n = 5 mice/group). (C): IHC of MAOA and MAOB in WT and MAOA/B-KO mouse prostates. Scale bars represent 20 µm. (D): H&E staining of WT and MAOA/B-KO mouse prostates at low (upper panel, ×100) and high (lower panel, ×400) magnification. Scale bars represent 20 µm. Data represent the mean ± SEM. **, p < .01. Abbreviations: AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; WT, wild-type. Figure 1 Open in new tabDownload slide MAOA/B ablation results in smaller prostate mass and prostate atrophy. (A, B): Weights of urogenital systems (A) and prostates (B, including ventral, dorsolateral, and anterior lobes) in 6-month-old male WT and MAOA/B-KO mice (n = 5 mice/group). (C): IHC of MAOA and MAOB in WT and MAOA/B-KO mouse prostates. Scale bars represent 20 µm. (D): H&E staining of WT and MAOA/B-KO mouse prostates at low (upper panel, ×100) and high (lower panel, ×400) magnification. Scale bars represent 20 µm. Data represent the mean ± SEM. **, p < .01. Abbreviations: AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; WT, wild-type. MAOA/B Ablation Results in Reduced Basal and Progenitor Cells in the Prostate To investigate the potential biological consequences of MAO ablation in prostates, we examined the cellular composition of prostate epithelium in MAOA/B-KO mice. The prostate epithelium is composed of basal and luminal cells arranged in two layers. Basal cells appear triangular and flattened in shape. The basal cell layer is aligned between the basement membrane and the luminal cells while keeping the luminal cells in contact with the basement membrane [19]. We observed a 31% decrease in CK5+ basal cell composition in MAOA/B-KO ventral and dorsolateral prostates relative to the WT controls by IHC (Fig. 2A, 2B, 35.0% ± 6.4% vs. 24.1% ± 3.4% for WT and MAOA/B-KO respectively). The CK8+ luminal cell layer remained intact in response to MAO ablation (Fig. 2A, 2B). We also used an unbiased approach to quantitatively evaluate individual CK expression at the single cell level, allowing the assessment to take into account both the proportion of cells stained and staining intensity [16, 30]. We demonstrated a similar 32% decrease in CK5 intensity counts per cell in MAOA/B-KO ventral and dorsolateral prostates compared with the WT controls. No significant changes were seen in CK8 intensity counts by MAO ablation (Fig. 2C). Moreover, we confirmed our explicit CK staining in mouse prostates using p63 as an alternative basal cell marker. p63 staining clearly distinguished CK5+ basal cells from CK8+ luminal cells, where p63 staining was present in most CK5+ basal cells but not in CK8+ luminal cells (Supporting Information Fig. S1). Next, we analyzed p63 expression in the basal cells. p63, a transcription factor belonging to the p53 superfamily, has been shown to be required for the normal development of several epithelial tissues, including the prostate. It is also essential for the maintenance and proliferative potential of stem cell populations in various organ systems such as the prostate [20, 33]. p63+ basal cells in the MAOA/B-KO prostates displayed a flattened, spindle shaped nucleus compared with the control prostates (Fig. 2D). We also found a 37% reduction in the percentage of p63+ basal cells in the MAOA/B-KO ventral and dorsolateral prostates compared with the WT controls (Fig. 2D, 2E, 23.2% ± 10.9% vs. 14.5% ± 7.0% for WT and MAOA/B-KO respectively), which is correlated with the findings for CK5+ cells. Additionally, we demonstrated a 38% decrease of Sca-1 expression, a stem cell marker [34], in p63+ basal cells in the MAOA/B-KO ventral and dorsolateral prostates relative to the WT prostates (Fig. 2F, 2G). Figure 2 Open in new tabDownload slide MAOA/B ablation reduces basal and progenitor cells in the prostate. (A): IHC analysis of luminal cell marker CK8 (green) and basal cell marker CK5 (red) in WT and MAOA/B-KO mouse prostates. Scale bars represent 20 µm. (B): The percentage of CK8+ and CK5+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). (C): Average cell-based staining intensity counts for CK8 and CK5 in ventral and dorsolateral mouse prostates (n = 5 mice/group) as determined by inForm software. (D): IHC analysis of basal cell marker p63 in mouse prostates. Red arrows point to p63+ cells in enlarged fields. Scale bars represent 20 µm. (E): The percentage of p63+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). (F): IHC analysis of p63 (red) and stem cell marker Sca-1 (green) in mouse prostates. White arrows point to p63+/Sca-1+ cells. Scale bars represent 20 µm. (G): Average cell-based staining intensity counts for Sca-1 in p63+ basal cells of ventral and dorsolateral mouse prostates (n = 5 mice/group) as determined by inForm software. Representative images are shown. Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. Figure 2 Open in new tabDownload slide MAOA/B ablation reduces basal and progenitor cells in the prostate. (A): IHC analysis of luminal cell marker CK8 (green) and basal cell marker CK5 (red) in WT and MAOA/B-KO mouse prostates. Scale bars represent 20 µm. (B): The percentage of CK8+ and CK5+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). (C): Average cell-based staining intensity counts for CK8 and CK5 in ventral and dorsolateral mouse prostates (n = 5 mice/group) as determined by inForm software. (D): IHC analysis of basal cell marker p63 in mouse prostates. Red arrows point to p63+ cells in enlarged fields. Scale bars represent 20 µm. (E): The percentage of p63+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). (F): IHC analysis of p63 (red) and stem cell marker Sca-1 (green) in mouse prostates. White arrows point to p63+/Sca-1+ cells. Scale bars represent 20 µm. (G): Average cell-based staining intensity counts for Sca-1 in p63+ basal cells of ventral and dorsolateral mouse prostates (n = 5 mice/group) as determined by inForm software. Representative images are shown. Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. We further characterized mouse prostates for expression of several biomarkers. We showed 26% lower αSMA expression, a stromal cell marker, implying a less stromal reactive phenotype, in MAOA/B-KO prostates compared with the WT controls (Supporting Information Fig. S2A, S2B). By examining both the percentage of AR+ cells and AR staining intensity per cell, we found intact AR expression in MAOA/B-KO prostates compared with the WT prostates (Supporting Information Fig. S2A, S2C, S2D). We also demonstrated reduced NF-H staining, which marks mature nerve fibers, in MAOA/B-KO prostates in comparison with the WT controls (Supporting Information Fig. S2A). We observed a threefold increase of serotonin secretion, a neurotransmitter metabolized by MAOs, in mouse prostates in response to MAOA/B ablation (Supporting Information Fig. S2A, S2E). Collectively, these results suggest that MAOs support basal maintenance of structural morphology and stemness activity in the prostate, which may involve communication between prostate epithelial cells and other surrounding cell types. Decreased Cell Proliferation in MAOA/B-KO Mouse Prostates We sought to determine whether deregulation of cell proliferation accounts for the reduced mass in MAOA/B-KO prostates by measuring Ki-67 expression in prostatic epithelial cells along the proximal-distal ductal axis. We observed Ki-67+ proliferating cells in both the basal and luminal compositions of prostate glands (Fig. 3A, yellow and white arrows indicating Ki-67+ basal and luminal cells respectively). We also found a significantly decreased percentage of Ki-67+ proliferating cells in MAOA/B-KO ventral and dorsolateral prostates compared with the WT controls (Fig. 3B, 2.7% ± 1.7% vs. 1.0% ± 1.2% for WT and MAOA/B-KO, respectively), which is consistent with the observation of reduced prostate mass in MAOA/B-KO mice. In addition, we found minimal cell apoptosis with negative expression of cleaved caspase-3 in the prostatic epithelia of both WT and MAOA/B-KO mice (Supporting Information Fig. S3). Figure 3 Open in new tabDownload slide MAOA/B ablation decreases cell proliferation in the prostate. (A): IHC analysis of Ki-67 expression in WT and MAOA/B-KO mouse prostates. White and yellow arrows point to Ki-67+ luminal and basal cells, respectively. Representative images are shown. Scale bars represent 20 µm. (B): The percentage of Ki-67+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). Data represent the mean ± SEM. **, p < .01. Figure 3 Open in new tabDownload slide MAOA/B ablation decreases cell proliferation in the prostate. (A): IHC analysis of Ki-67 expression in WT and MAOA/B-KO mouse prostates. White and yellow arrows point to Ki-67+ luminal and basal cells, respectively. Representative images are shown. Scale bars represent 20 µm. (B): The percentage of Ki-67+ cells in ventral and dorsolateral mouse prostates (n = 5 mice/group). Data represent the mean ± SEM. **, p < .01. MAOA/B Silencing Reduces Prostate Stemness in Human Prostate Epithelial Cells Given that MAOA/B ablation reduces the stem cell marker Sca-1 expression in p63+ basal cells in mouse prostates (Fig. 2F, 2G), we attempted to investigate MAOs’ effect on prostate stem/progenitor cell activity in cultured prostate epithelial cells. We used shRNAs to sequentially knock down MAOA and MAOB in normal human prostate epithelial RWPE-1 cells, which co-express both luminal and basal cytokeratins [35]. shRNA-mediated KD of MAOs effectively inhibited MAOA and MAOB enzymatic activities by 68% and 58%, respectively (Fig. 4A). To determine whether MAO silencing affects the regenerative capacity of prostate cells, we determined the ability of cells to form clonal spheres that can be serially passaged, a proven approach testing the self-renewal capacity of prostate progenitor cells [36, 37]. MAOA/B KD significantly lowered the percentage of sphere-forming cells for both primary and secondary spheres compared with the control cells (Fig. 4B, 4C, 2.8% ± 0.2% vs. 1.9% ± 0.4% for control and MAOA/B-KD primary spheres respectively; 8.2% ± 0.7% vs. 4.0% ± 0.8% for control and MAOA/B-KD secondary spheres respectively). We also noticed a 35% decrease of sphere diameter in the MAOA/B-KD group relative to the control group (Fig. 4B, 4D, 139.2 ± 32.5 µm vs. 90.3 ± 32.7 µm for control and MAOA/B-KD spheres respectively). Moreover, MAOA/B KD resulted in a smaller CD133+/CD44+/CD24– stem cell population in RWPE-1 cells (Fig. 4E, 3.5% ± 1.1% vs. 1.4% ± 0.4% for control and MAOA/B-KD cells, respectively) as well as 39% lower proliferation of RWPE-1 sphere-derived CD133+/CD44+/CD24– stem cells (Fig. 4F) compared with the controls. Previous studies have demonstrated a combinational use of these three markers for successfully isolating subpopulations exhibiting stem-like growth characteristics in human prostate epithelial cells [38-40]. Furthermore, we found a prevalent reduction by up to ∼60% in select stem cell marker expression, including ALDH1A1 [41], TROP2 [42], and CD166 [43], in MAOA/B-KD cells relative to the control cells (Fig. 4G). We also demonstrated significantly reduced CK5 expression at both protein and mRNA levels in MAOA/B-KD spheres compared with the control spheres. Meanwhile, a marginal change in CK8 expression was seen between the two groups of spheres (Fig. 4H, 4I). These results reinforce our findings that MAOA/B ablation suppresses basal progenitor cells in mice. Figure 4 Open in new tabDownload slide shRNA-mediated silencing of MAOA and MAOB reduced prostate epithelial progenitor cell activity. (A): MAOA and MAOB enzymatic activities in control (shCon) and MAOA/B-KD (shMAOA/B) RWPE-1 cells (n = 3). (B): Representative images of prostate spheres formed by control and MAOA/B-KD RWPE-1 cells. Scale bars represent 200 µm. (C): The percentage of sphere-forming cells from first and second generation prostate sphere cultures (n = 3). (D): Quantitative analysis of sphere diameters in first generation sphere cultures. (E): Flow cytometric analysis of the percentage of CD133+/CD44+/CD24– cell population in control and MAOA/B-KD RWPE-1 cells (n = 3). (F): Cell counting analysis of CD133+/CD44+/CD24– cells isolated from control and MAOA/B-KD RWPE-1 spheres over a 5-day observing period (n = 3). (G): RT-qPCR analysis of mRNA expression of select stem cell marker genes in control and MAOA/B-KD RWPE-1 cells (n = 3). (H): Western blotting analysis of MAOA, MAOB, CK8, and CK5 expression in control and MAOA/B-KD RWPE-1 spheres. Protein expression as normalized to β-Actin in control cells was set as 1. (I): RT-qPCR analysis of CK8 and CK5 mRNA levels in control and MAOA/B-KD RWPE-1 spheres (n = 3). Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. Figure 4 Open in new tabDownload slide shRNA-mediated silencing of MAOA and MAOB reduced prostate epithelial progenitor cell activity. (A): MAOA and MAOB enzymatic activities in control (shCon) and MAOA/B-KD (shMAOA/B) RWPE-1 cells (n = 3). (B): Representative images of prostate spheres formed by control and MAOA/B-KD RWPE-1 cells. Scale bars represent 200 µm. (C): The percentage of sphere-forming cells from first and second generation prostate sphere cultures (n = 3). (D): Quantitative analysis of sphere diameters in first generation sphere cultures. (E): Flow cytometric analysis of the percentage of CD133+/CD44+/CD24– cell population in control and MAOA/B-KD RWPE-1 cells (n = 3). (F): Cell counting analysis of CD133+/CD44+/CD24– cells isolated from control and MAOA/B-KD RWPE-1 spheres over a 5-day observing period (n = 3). (G): RT-qPCR analysis of mRNA expression of select stem cell marker genes in control and MAOA/B-KD RWPE-1 cells (n = 3). (H): Western blotting analysis of MAOA, MAOB, CK8, and CK5 expression in control and MAOA/B-KD RWPE-1 spheres. Protein expression as normalized to β-Actin in control cells was set as 1. (I): RT-qPCR analysis of CK8 and CK5 mRNA levels in control and MAOA/B-KD RWPE-1 spheres (n = 3). Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. Pharmacological Inhibition of MAOA/B Impairs the Capacity of Prostate Progenitor Cells to Form Spheres In Vitro We used alternative pharmacological approaches to further determine MAOs’ role in regulating prostate progenitor cell activity. Treatment of RWPE-1 cells with clorgyline and deprenyl, two potent small molecular inhibitors for MAOA and MAOB, respectively [1], significantly decreased the percentage of sphere-forming cells in both the first and second passages (Fig. 5A, 5B, 2.7% ± 0.5% vs. 1.8% ± 0.2% for control and treatment groups respectively in the first passage; 6.1% ± 1.3% vs. 2.9% ± 0.9% for control and treatment groups respectively in the second passage). Pharmacological inhibition of MAOA and MAOB also resulted in smaller spheres with a 36% decrease in diameter (Fig. 5A, 5C, 167.0 ± 71.2 µm vs. 106.3 ± 36.3 µm for control and treatment groups respectively). Consistent with the shRNA-mediated KD approach, we found a smaller CD133+/CD44+/CD24– stem cell population (Fig. 5D, 4.2% ± 1.4% vs. 1.7% ± 0.5% for control and treatment groups respectively) in MAO inhibitor-treated RWPE-1 cells as well as less CK5 expression (Fig. 5E) in MAO inhibitor-treated RWPE-1 spheres compared with the controls. These observations support the concept that MAOs regulate prostate progenitor cells through enzymatic function. Figure 5 Open in new tabDownload slide Pharmacological inhibition of MAOA and MAOB lowered the progenitor cell properties of prostate epithelial cells. (A): Representative images of prostate spheres formed by RWPE-1 cells treated with PBS (Veh) or a combination of Clg (1 µM), and Dpn (1 µM). Scale bars represent 200 µm. (B): The percentage of sphere-forming cells from first and second generation prostate sphere cultures (n = 3). (C): Quantitative analysis of sphere diameters in first generation sphere cultures. (D): Flow cytometric analysis of the percentage of CD133+/CD44+/CD24– cell population in vehicle and Clg/Dpn-treated (1 µM each, 72 hours) RWPE-1 cells (n = 3). (E): RT-qPCR analysis of CK8 and CK5 mRNA levels in vehicle and Clg/Dpn-treated (1 µM each) RWPE-1 spheres (n = 3). Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. Abbreviations: Clg, clorgyline; Dpn, deprenyl; Veh, vehicle. Figure 5 Open in new tabDownload slide Pharmacological inhibition of MAOA and MAOB lowered the progenitor cell properties of prostate epithelial cells. (A): Representative images of prostate spheres formed by RWPE-1 cells treated with PBS (Veh) or a combination of Clg (1 µM), and Dpn (1 µM). Scale bars represent 200 µm. (B): The percentage of sphere-forming cells from first and second generation prostate sphere cultures (n = 3). (C): Quantitative analysis of sphere diameters in first generation sphere cultures. (D): Flow cytometric analysis of the percentage of CD133+/CD44+/CD24– cell population in vehicle and Clg/Dpn-treated (1 µM each, 72 hours) RWPE-1 cells (n = 3). (E): RT-qPCR analysis of CK8 and CK5 mRNA levels in vehicle and Clg/Dpn-treated (1 µM each) RWPE-1 spheres (n = 3). Data represent the mean ± SEM. *, p < .05; **, p < .01; ns, not significant. Abbreviations: Clg, clorgyline; Dpn, deprenyl; Veh, vehicle. Elevated MAOA and MAOB Expression in Prostatic Hyperplasia Compared with Normal Prostates Since the relation of MAO loss to development of prostate atrophy may have translational relevance for benign prostatic hyperplasia (BPH), a prostatic disease characterized by enlarged prostates with increased cell proliferation and commonly found in aged males [44], we attempted to evaluate MAO expression patterns in human prostatic hyperplasia and normal prostate tissue microarrays. MAOA and MAOB are expressed in both epithelial and stromal compartments of normal prostates, with MAOA expression more abundant in prostate epithelia than stroma and MAOB expression slightly higher in prostate stroma relative to epithelia. MAOA expression is elevated by 2.7-fold in prostatic hyperplasia epithelia compared with normal prostate epithelia. No significant changes were seen in epithelial MAOB expression between prostatic hyperplasia and normal prostates. Moreover, both MAOA and MAOB expression are increased in the stroma compartment of prostatic hyperplasia by 2.5-fold and 4-fold, respectively, compared with normal prostate stroma (Fig. 6A, 6B). These data may have future implications for the clinical management of BPH by MAO interference. Figure 6 Open in new tabDownload slide MAOA and MAOB expression is elevated in human prostate hyperplasia. (A): Representative IHC images of MAOA and MAOB expression in tissue microarrays composed of human prostate hyperplasia (n = 70) and normal prostate (n = 24) tissues. Scale bars represent 20 µm. (B): Average cell-based staining intensity counts for MAOA and MAOB in prostate epithelial or stromal compartments of tissue microarrays as determined by inForm software. *, p < .05; **, p < .01; ns, not significant. Figure 6 Open in new tabDownload slide MAOA and MAOB expression is elevated in human prostate hyperplasia. (A): Representative IHC images of MAOA and MAOB expression in tissue microarrays composed of human prostate hyperplasia (n = 70) and normal prostate (n = 24) tissues. Scale bars represent 20 µm. (B): Average cell-based staining intensity counts for MAOA and MAOB in prostate epithelial or stromal compartments of tissue microarrays as determined by inForm software. *, p < .05; **, p < .01; ns, not significant. Discussion The development and maintenance of the prostate is regulated by a combination of cell autonomous factors and extracellular signals in a complex, age-dependent cellular and molecular environment. Our findings suggest that MAOs, the major enzymes involved in monoamine metabolism, influence the structural morphology of the prostate and prostate basal progenitor cells in mice. Adult MAOA/B-KO mice exhibit smaller prostate mass along with an atrophic phenotype in the ventral and dorsolateral prostates, which is evident in the basal cell layer but not the luminal counterpart. These findings reveal a novel role for MAOs in the regulation of prostate gland maintenance and the modulation of prostate progenitor cell activity. Ample evidence has suggested that prostate innervation is a contributing mechanism for the structural morphology and maintenance of the prostate. Chemical sympathectomy with 6-hydroxydopamine or an alternative neurotoxin promoted atrophy of the epithelium in all prostate lobes of rats [45, 46]. In addition, denervation surgery of prostate innervating nerves caused prostate atrophy in dogs [47]. The prostate gland is supplied with a wide range of neurotransmitters or catecholamines diffused in prostate cells, such as serotonin and norepinephrine, which are released from neuroendocrine cells and sympathetic adrenergic nerves in the prostate [48, 49]. The lack of MAOs could result in significantly elevated levels of these monoamines by an up to sevenfold increase in individual monoamine levels in mice, which is greater than that found in either MAOA or MAOB single KO mice [29]. These extremely high levels of monoamines could be neurotoxic, deregulating neurotransmission and impairing nerve function in the prostate [50]. Indeed, we demonstrated that MAOA/B-KO prostates were associated with a threefold increase in serotonin level as well as fewer nerve fibers compared with WT controls (Supporting Information Fig. S2). Whether direct interaction of these monoamines with the prostate gland exists and contributes to the development of prostate atrophy merits future investigations. The roles of MAOs as regulators of prostate epithelium and in prostate cancer have recently drawn interest. MAOA was found to be expressed exclusively in the basal epithelial cells but not in the luminal secretory epithelial cells, as confirmed by co-expression with p63 [12]. This is consistent with our findings, where MAOA/B ablation uniquely affected the basal cell layer but not the luminal cell layer. MAOA was shown to prevent differentiation of basal epithelial cells into secretory epithelial cells using primary cultures of normal human prostatic epithelial cells [12]. There is a widely accepted theory that cells residing in the basal cell layer differentiate into secretory cells to maintain tissue homeostasis [51-53], while in mouse prostates it appears that secretory cells can develop independent of basal cells [54]. In parallel with reduced basal cells, we found negligible luminal cell differentiation evidenced by intact CK8+ cell percentage and CK8 staining in MAOA/B-KO mouse prostates. This is supported by unchanged AR expression, a hallmark of secretory epithelial cells, in MAOA/B-KO prostates since androgen signaling is a key regulator of the differentiated phenotype [55]. Given the potential differences in prostate cell lineage differentiation between humans and mice, future studies in a human prostate cell differentiation model would be needed to more comprehensively understand MAOA's regulatory role in cell lineage differentiation in the prostate. Notably, MAOA ablation by itself is not sufficient to develop prostate atrophy. We did not observe atrophic glands in MAOA single KO mouse prostates (Supporting Information Fig. S4), which suggests that cooperative action between the two MAO isoforms is involved in prostate maintenance. Although MAOA and MAOB exhibit ∼70% amino acid sequence identity and share a number of structural and mechanistic features, these isoforms differ in several aspects, including substrate preferences and inhibitor sensitivities [1]. These differences could be a determinant for each individual isoform's explicit role in the development and maintenance of the prostate. Our study demonstrates MAOs’ role in regulating prostate basal progenitor cells. MAOA/B-KO mice showed a lower percentage of p63+ prostate basal cells and less Sca-1 expression in p63+ basal cells, which is consistent with a previous report that mice lacking MAOA and MAOB exhibit diminished proliferation of neural stem cells in the developing telencephalon of brain [56]. Recent evidence has indicated that prostate basal cells functionally exhibit intrinsic stem-like and neurogenic properties, which coincidentally links neuronal genes, such as MAOs, with molecular profiling of prostate cell stemness [57]. One prominent mechanistic feature of MAOs we have deciphered so far is the ability to affect a number of transcription factors, such as Twist1 and forkhead box proteins [15, 16, 58]. An induced stem-like transcriptional program might be an underlying mechanism for MAOs’ effects on the regulation of prostate basal progenitor cells. Conclusion In summary, our data suggest that MAO deficiency causes prostate atrophy with reduced basal cells and stem/progenitor cell activity, which provides new insight into the maintenance of prostate structure and function. The promotion of atrophic prostates by MAO abrogation has several implications relevant to prostate disease and prostate cancer. Increased intrinsic expression of both MAO isoforms in prostatic hyperplasia, which coincides with our findings on MAO loss-related prostate atrophy, provides a rationale for extending the application of MAO inhibitors to BPH treatment. Prostate gland atrophy is another commonly occurring phenomenon with increasing age in prostate biopsy specimens. Prostate atrophy associated with chronic inflammation, especially the phenotype of proliferative inflammatory atrophy (PIA), has been linked to high-grade prostatic intraepithelial neoplasia and/or prostate carcinoma [59]. On the other hand, there is some limited evidence that baseline prostate atrophy (PA) is independently associated with a lower risk of prostate cancer [60, 61]. MAOA/B-KO mice showed a lack of inflammation in atrophic prostates with reduced cell proliferation, which could be due in part to lessened oxidative stress in the MAOA/B-deficient prostate environment. These histological observations suggest that MAO-regulated prostate atrophy belongs to the PA but not PIA phenotype. In line with this histological evidence, our study also supports the idea of targeting MAOs as a new approach for prostate cancer prevention via suppression of prostate basal progenitor cell activity to block the cells-of-origin in prostate cancer. Acknowledgments We thank Dr. Jean C. 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TI - Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity
JF - Stem Cells
DO - 10.1002/stem.2831
DA - 2018-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/monoamine-oxidase-deficiency-causes-prostate-atrophy-and-reduces-TXngv049WO
SP - 1249
EP - 1258
VL - 36
IS - 8
DP - DeepDyve
ER -