Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

Steroid hormone imbalance drives macrophage infiltration and Spp1/osteopontin+ foam cell differentiation in the prostate.

Benign prostatic hyperplasia (BPH) occurs progressively with aging in men and drives deteriorating symptoms collectively known as lower urinary tract symptoms (LUTS). Age-associated changes in circulating steroid hormones, and prostate inflammation have been postulated in the etiology of BPH/LUTS. The link between hormones and inflammation in the development of BPH/LUTS is conflicting because they may occur independently or as sequential steps in disease pathogenesis. This study aimed to decipher the prostatic immune landscape in a mouse model of lower urinary tract dysfunction (LUTD). Steroid hormone imbalance was generated by the surgical implantation of testosterone (T) and estradiol (E2) pellets into male C57BL/6J mice and gene expression analysis was performed on ventral prostates (VPs). These experiments identified an increase in the expression of macrophage markers and Spp1/osteopontin (OPN). Localization studies of OPN pinpointed that OPN+ macrophages travel to the prostate lumen and transition into lipid-accumulating foam cells. We also observed a significant increase in the number of tissue macrophages in the VP which was prevented in OPN-knockout (OPN-KO) mice. In contrast, mast cells, but not macrophages, were significantly elevated in the dorsal prostate of T + E2-treated mice which was diminished in OPN-KO mice. Steroid hormone implantation progressively increased urinary frequency, which was ameliorated in OPN-KO mice. Our study underscores the role of age-associated steroid hormone imbalances as a mechanism of expanding the prostatic macrophage population, their luminal translocation, and foam cell differentiation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.

Progenitors in prostate development and disease.

The prostate develops by epithelial budding and branching processes that occur during fetal and postnatal stages. The adult prostate demonstrates remarkable regenerative capacity, with the ability to regrow to its original size over multiple cycles of castration and androgen administration. This capacity for controlled regeneration prompted the search for an androgen-independent epithelial progenitor in benign prostatic hyperplasia (BPH) and prostate cancer (PCa). BPH is hypothesized to be a reawakening of ductal branching, resulting in the formation of new proximal glands, all while androgen levels are decreasing in the aging male. Advanced prostate cancer can be slowed with androgen deprivation, but resistance eventually occurs, suggesting the existence of an androgen-independent progenitor. Recent studies indicate that there are multiple castration-insensitive epithelial cell types in the proximal area of the prostate, but not all act as progenitors during prostate development or regeneration. This review highlights how recent cellular and anatomical studies are changing our perspective on the identity of the prostate progenitor.

Single-cell analysis of mouse and human prostate reveals novel fibroblasts with specialized distribution and microenvironment interactions.

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A uropathogenic E. coli UTI89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate.

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation using transurethral instillations of Escherichia coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributed exclusively to the bladder, 100- and 200-µL volumes distributed to the bladder and prostate, and a 500-µL volume distributed to the bladder, prostate, and ureter. A threshold optical density of 0.4 E. coli UTI89 in the instillation fluid was necessary for significant (P < 0.05) prostate colonization. E. coli UTI89 infection resulted in a low frequency, high volume spontaneous voiding pattern. This phenotype was due to exposure to E. coli UTI89, not catheterization alone, and was minimally altered by a 50-µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation was isolated to the dorsal prostate and was accompanied by increased collagen density. This was partnered with increased density of protein tyrosine phosphatase receptor type C+, procollagen type I-α1+ copositive cells and decreased density of α2-smooth muscle actin+, procollagen type I-α1+ copositive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Prostate epithelial-specific expression of activated PI3K drives stromal collagen production and accumulation.

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3KGOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3KGOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-ß pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3KGOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-ß signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In vivo replacement of damaged bladder urothelium by Wolffian duct epithelial cells.

The bladder's remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Spatiotemporal Proteomics Reveals the Molecular Consequences of Hormone Treatment in a Mouse Model of Lower Urinary Tract Dysfunction.

Benign prostatic hyperplasia and related lower urinary tract symptoms remain common, costly, and impactful issues for aging males. The etiology and pathogenesis are multifactorial and include steroid hormone changes and inflammation. Noninvasive markers could one day inform personalized medicine, but interindividual variation and lack of healthy age-matched controls hamper research. Experimental models are appealing for insight into disease mechanisms. Here, we present a spatiotemporal proteomics study in a mouse model of hormone-induced urinary dysfunction. Urine samples were collected noninvasively across time: before, during, and after disease onset. A microcomputed tomography analysis implicated the prostate as a spatially relevant contributor to bladder outlet obstruction. Prostates were collected after disease onset and compared with control mice. Notable changes in urine include proteins representing oxidative stress defense and acute phase inflammatory response processes. In the prostate, hormone treatment led to perturbations related to an oxidative stress response and H2O2 metabolism. Several protein changes coincided in both urine and the prostate tissue, including glutathione peroxidase 3, glutathione hydrolase 1 proenzyme, and vitamin D-binding protein. This study supports the concept of noninvasive urinary biomarkers for prostate disease diagnostics. Oxidative stress and acute phase inflammatory processes were identified as key consequences of hormone-induced bladder outlet obstruction. Future research into antioxidants and anti-inflammatories in prostate diseases appears promising.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds.

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.

Impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology: urethral histology.

The National Institutes of Health leveled new focus on sex as a biological variable with the goal of understanding sex-specific differences in health and physiology. We previously published a functional assessment of the impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology (Ruetten H, Wegner KA, Zhang HL, Wang P, Sandhu J, Sandhu S, Mueller B, Wang Z, Macoska J, Peterson RE, Bjorling DE, Ricke WA, Marker PC, Vezina CM. Am J Physiol Renal Physiol 317: F996-F1009, 2019). Here, we measured and compared five characteristics of urethral histology (urethral lumen diameter and area, epithelial cell count, epithelial and rhabdosphincter thickness, epithelial cell area, and total urethral area) in male and female 9-wk-old C57BL/6J mice using hematoxylin and eosin staining. We also compared male mice with castrated male mice, male and female mice treated with the steroid 5α-reductase inhibitor finasteride or testosterone, or male mice harboring alleles (Pbsn4cre/+; R26RDta/+) that reduce prostate lobe mass. The three methods used to reduce prostate mass (castration, finasteride, and Pbsn4cre/+; R26RDta/+) changed urethral histology, but none feminized male urethral histology (increased urethral epithelial area). Exogenous testosterone caused increased epithelial cell count in intact females but did not masculinize female urethral histology (decrease epithelial area). Our results lay a critical foundation for future studies as we begin to parse out the influence of hormones and cellular morphology on male and female urinary function.

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

Urethral luminal epithelia are castration-insensitive cells of the proximal prostate.

BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.

Synthesis and biological evaluation of FICZ analogues as agonists of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor involved in multiple biological processes including immune cell differentiation, intestinal function and inflammation. Based on the scaffold of naturally occurring AhR ligand 6-formylindolo (3,2-b) carbazole (FICZ, 2), a series of analogues has been designed, synthesized and evaluated by cell-based assays. The structure-activity relationships study has successfully led to the discovery of compound 11e with extremely potent activity.

Quantitative proteomic analysis of a genetically induced prostate inflammation mouse model via custom 4-plex DiLeu isobaric labeling.

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1ß expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N,N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

Impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology: functional assessment.

Laboratory mice are used to identify causes of urinary dysfunction including prostate-related mechanisms of lower urinary tract symptoms. Effective use of mice for this purpose requires a clear understanding of molecular, cellular, anatomic, and endocrine contributions to voiding function. Whether the prostate influences baseline voiding function has not been specifically evaluated, in part because most methods that alter prostate mass also change circulating testosterone concentrations. We performed void spot assay and cystometry to establish a multiparameter "baseline" of voiding function in intact male and female 9-wk-old (adult) C57BL/6J mice. We then compared voiding function in intact male mice to that of castrated male mice, male (and female) mice treated with the steroid 5α-reductase inhibitor finasteride, or male mice harboring alleles (Pbsn4cre/+; R26RDta/+) that significantly reduce prostate lobe mass by depleting prostatic luminal epithelial cells. We evaluated aging-related changes in male urinary voiding. We also treated intact male, castrate male, and female mice with exogenous testosterone to determine the influence of androgen on voiding function. The three methods used to reduce prostate mass (castration, finasteride, and Pbsn4cre/+; R26RDta/+) changed voiding function from baseline but in a nonuniform manner. Castration feminized some aspects of male urinary physiology (making them more like intact female mice) while exogenous testosterone masculinized some aspects of female urinary physiology (making them more like intact male mice). Our results provide evidence that circulating testosterone is responsible in part for baseline sex differences in C57BL/6J mouse voiding function while prostate lobe mass in young, healthy adult mice has a lesser influence.

A folic acid-enriched diet attenuates prostate involution in response to androgen deprivation.

BACKGROUND: Serum folate concentrations in the United States have risen since dietary folic acid fortification was first mandated in 1998. Although maternal folic acid offers protection against neural tube defects in conceptuses, its impact on other organ systems and life stages have not been fully examined. Here, we used a mouse model to investigate the impact of a Folic acid (FA) enriched diet on prostate homeostasis and response to androgen deprivation. METHODS: Male mice were fed a control diet (4 mg FA/kg feed) or a folic acid supplemented diet (24 mg FA/kg feed) beginning at conception and continuing through early adulthood, when mice were castrated. RESULTS: We made the surprising observation that dietary FA supplementation confers partial resistance to castration-mediated prostate involution. At 3, 10, and 14 days post-castration, FA enriched diet fed mice had larger prostates as assessed by wet weight, taller prostatic luminal epithelial cells, and more abundant RNAs encoding prostate secretory proteins than castrated control diet fed mice. Diet did not significantly affect prostate weights of intact mice or serum testosterone concentrations of castrated mice. RNA-Seq analysis revealed that the FA enriched diet was associated with a unique prostate gene expression signature, affecting several signaling and metabolic pathways. CONCLUSIONS: Continuous exposure to a FA enriched diet slows prostate involution in response to androgen deprivation. Prostates from FA diet mice have increased secretory gene expression and increased luminal cell heights. The influence of dietary FA supplementation on the prostate response to androgen deprivation raises a future need to consider how dietary folic acid supplementation affects efficacy of androgen-reducing therapies for treating prostate disease.