Age-Dependent Effects of Voluntary Wheel Running Exercise on Voiding Behavior and Potential Age-Related Molecular Mechanisms in Mice.

BACKGROUND: Older men frequently develop lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). Risk factors for LUTS/BPH include sedentary lifestyle, anxiety/depression, obesity, and frailty, which all increase with age. Although physical exercise may reduce the progression and/or severity of LUTS/BPH, the age-related mechanisms responsible remain unknown. METHODS: Voiding symptoms, body mass, and frailty were assessed after 4-weeks of voluntary wheel running in 2-month (n = 10) and 24-month (n = 8) old C57Bl/6J male mice. In addition, various social and individual behaviors were examined in these cohorts. Finally, cellular and molecular markers of inflammation and mitochondrial protein expression were assessed in prostate tissue and systemically. RESULTS: Despite running less (aged vs young X¯ = 12.3 vs 30.6 km/week; p = .04), aged mice had reduced voiding symptoms (X¯ = 67.3 vs 23.7; p < .0001) after 1 week of exercise, which was sustained through week 4 (X¯ = 67.3 vs 21.5; p < .0001). Exercise did not affect voiding symptoms in young mice. Exercise also increased mobility and decreased anxiety in both young and aged mice (p < .05). Exercise decreased expression of a key mitochondrial protein (PINK1; p < .05) and inflammation within the prostate (CD68; p < .05 and plasminogen activator inhibitor-1; p < .05) and in the serum (p < .05). However, a frailty index (X¯ = 0.17 vs 0.15; p = .46) and grip strength (X¯ = 1.10 vs 1.19; p = .24) were unchanged after 4 weeks of exercise in aged mice. CONCLUSIONS: Voluntary aerobic exercise improves voiding behavior and mobility, and decreases prostatic mitochondrial protein expression and inflammation in aged mice. This promising model could be used to evaluate molecular mechanisms of aerobic exercise as a novel lifestyle intervention for older men with LUTS/BPH.

Aging-Related Mitochondrial Dysfunction is Associated with Fibrosis in Benign Prostatic Hyperplasia.

BACKGROUND: Age is the greatest risk factor for lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). While LUTS/BPH can be managed with pharmacotherapy, treatment failure has been putatively attributed to numerous pathological features of BPH (e.g., prostatic fibrosis, inflammation). Mitochondrial dysfunction is a hallmark of aging, however its impact on the pathological features of BPH remains unknown. METHODS: Publicly available gene array data was analyzed. Immunohistochemistry examined mitochondrial proteins in human prostate. The effect of complex I inhibition (rotenone) on a prostatic cell line was examined using qPCR, immunocytochemistry, and Seahorse assays. Oleic acid was tested as a bypass of complex I inhibition. Aged mice were treated with OA to examine its effects on urinary dysfunction. Voiding was assessed longitudinally, and a critical complex I protein measured. RESULTS: Mitochondrial function and fibrosis genes were altered in BPH. Essential mitochondrial proteins (i.e., VDAC1/2, PINK1 and NDUFS3) were significantly (P<0.05) decreased in BPH. Complex I inhibition in cultured cells resulted in decreased respiration, altered NDUFS3 expression, increased collagen deposition and gene expression. Oleic acid ameliorated these effects. Oleic acid treated aged mice had significantly (P<0.05) improved voiding function and higher prostatic NDUFS3 expression. CONCLUSION: Complex I dysfunction is a potential contributor to fibrosis and lower urinary tract dysfunction in aged mice. Oleic acid partially bypasses complex I inhibition and therefore should be further investigated as a mitochondrial modulator for treatment of LUTS/BPH. Hypotheses generated in this investigation offer a heretofore unexplored cellular target of interest for the management of LUTS/BPH.

Residency Program Directors' Views on Research Conducted During Medical School: A National Survey.

PURPOSE: With the United States Medical Licensing Examination Step 1 transition to pass/fail in 2022, uncertainty exists regarding how other residency application components, including research conducted during medical school, will inform interview and ranking decisions. The authors explore program director (PD) views on medical student research, the importance of disseminating that work, and the translatable skill set of research participation. METHOD: Surveys were distributed to all U.S. residency PDs and remained open from August to November 2021 to query the importance of research participation in assessing applicants, whether certain types of research were more valued, productivity measures that reflect meaningful research participation, and traits for which research serves as a proxy. The survey also queried whether research would be more important without a numeric Step 1 score and the importance of research vs other application components. RESULTS: A total of 885 responses from 393 institutions were received. Ten PDs indicated that research is not considered when reviewing applicants, leaving 875 responses for analysis. Among 873 PDs (2 nonrespondents), 358 (41.0%) replied that meaningful research participation will be more important in offering interviews. A total of 164 of 304 most competitive specialties (53.9%) reported increased research importance compared with 99 of 282 competitive (35.1%) and 95 of 287 least competitive (33.1%) specialties. PDs reported that meaningful research participation demonstrated intellectual curiosity (545 [62.3%]), critical and analytical thinking skills (482 [55.1%]), and self-directed learning skills (455 [52.0%]). PDs from the most competitive specialties were significantly more likely to indicate that they value basic science research vs PDs from the least competitive specialties. CONCLUSIONS: This study demonstrates how PDs value research in their review of applicants, what they perceive research represents in an applicant, and how these views are shifting as the Step 1 exam transitions to pass/fail.

Genetic alterations in CREBRF influence prostate cancer survival and impact prostate tissue homeostasis in mice.

BACKGROUND: Risk factors for prostate cancer include age, environment, race and ethnicity. Genetic variants in cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) gene are frequently observed in Pacific Islanders, a population with elevated prostate cancer incidence. CREBRF has been shown to play a role in other cancers, however its function in prostate homeostasis and tumorigenesis has not been previously explored. We determined the incidence of CREBRF alterations in publicly available databases and examined the impact of CREBRF deletion on the murine prostate in order to determine whether CREBRF impacts prostate physiology or pathophysiology. METHODS: Alterations in CREBRF were identified in prostate cancer patients via in silico analysis of several publicly available datasets through cBioPortal. Male Crebrf knockout and wild-type littermate mice were generated and examined for prostate defects at 4 months of age. Immunohistochemical staining of murine prostate sections was used to determine the impact of Crebrf knockout on proliferation, apoptosis, inflammation and blood vessel density in the prostate. Serum adipokine levels were measured using a Luminex Multiplex Assay. RESULTS: CREBRF alterations were identified in up to 4.05% of prostate tumors and the mutations identified were categorized as likely damaging. Median survival of prostate cancer patients with genetic alterations in CREBRF was 41.23 months, compared to 131 months for patients without these changes. In the murine model, the prostates of Crebrf knockout mice had reduced epithelial proliferation and increased TUNEL+ apoptotic cells. Circulating adipokines PAI-1 and MCP-1 were also altered in Crebrf knockout mice compared to age-matched controls. CONCLUSIONS: Prostate cancer patients with genetic alterations in CREBRF had a significantly decreased overall survival suggesting that wild type CREBRF may play a role in limiting prostate tumorigenesis and progression. The murine knockout model demonstrated that CREBRF could modulate proliferation and apoptosis and macrophage density in the prostate. Serum levels of adipokines PAI-1 and MCP-1 were also altered and may contribute to the phenotypic changes observed in the prostates of Crebrf knockout mice. Future studies focused on populations susceptible to CREBRF mutations and mechanistic studies will be required to fully elucidate the potential role of CREBRF in prostate tumorigenesis.

The SARS-CoV-2 E protein induces Toll-like receptor 2-mediated neonatal lung injury in a model of COVID-19 viremia that is rescued by the glucocorticoid ciclesonide.

SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.

Genomic glucocorticoid action in embryonic mouse neural stem cells.

Prenatal exposure to synthetic glucocorticoids (sGCs) reprograms brain development and predisposes the developing fetus towards potential adverse neurodevelopmental outcomes. Using a mouse model of sGC administration, previous studies show that these changes are accompanied by sexually dimorphic alterations in the transcriptome of neural stem and progenitor cells (NSPCs) derived from the embryonic telencephalon. Because cell type-specific gene expression profiles tightly regulate cell fate decisions and are controlled by a flexible landscape of chromatin domains upon which transcription factors and enhancer elements act, we multiplexed data from four genome-wide assays: RNA-seq, ATAC-seq (assay for transposase accessible chromatin followed by genome wide sequencing), dual cross-linking ChIP-seq (chromatin immunoprecipitation followed by genome wide sequencing), and microarray gene expression to identify novel relationships between gene regulation, chromatin structure, and genomic glucocorticoid receptor (GR) action in NSPCs. These data reveal that GR binds preferentially to predetermined regions of accessible chromatin to influence gene programming and cell fate decisions. In addition, we identify SOX2 as a transcription factor that impacts the genomic response of select GR target genes to sGCs (i.e., dexamethasone) in NSPCs.

Increased COX-1 expression in benign prostate epithelial cells is triggered by mitochondrial dysfunction.

BACKGROUND: Prostatic inflammation is closely linked to the development and progression of benign prostatic hyperplasia (BPH). Clinical studies of non-steroidal anti-inflammatory drugs, which inhibit cyclooxygenase-2 (COX-2), targeting prostate inflammation patients with symptomatic BPH have demonstrated conflicting results, with some studies demonstrating symptom improvement and others showing no impact. Thus, understanding the role of the cyclooxygenases in BPH and prostatic inflammation is important. METHODS: The expression of COX-1 was analyzed in a cohort of donors and BPH patients by immunohistochemistry and compared to previously determined characteristics for this same cohort. The impact of mitochondrial dysfunction on COX-1 and COX-2 was determined in experiments treating human benign prostate epithelial cell lines BPH-1 and RWPE-1 with rotenone and MitoQ. RWPE-1 cells were transfected with small interfering RNA specific to complex 1 gene NDUFS3. RESULTS: COX-1 expression was increased in the epithelial cells of BPH specimens compared to young healthy organ donor and normal prostate adjacent to BPH and frequently co-occurred with COX-2 alteration in BPH patients. COX-1 immunostaining was associated with the presence of CD8+ cytotoxic T-cells, but was not associated with age, prostate size, COX-2 or the presence of CD4+, CD20+ or CD68+ inflammatory cells. In cell line studies, COX protein levels were elevated following treatment with inhibitors of mitochondrial function. MitoQ significantly decreased mitochondrial membrane potential in RWPE-1 cells. Knockdown of NDUFS3 stimulated COX-1 expression. CONCLUSION: Our findings suggest COX-1 is elevated in BPH epithelial cells and is associated with increased presence of CD8+ cytotoxic T-cells. COX-1 can be induced in benign prostate epithelial cells in response to mitochondrial complex I inhibition, and knockdown of the complex 1 protein NDUFS3. COX-1 and mitochondrial dysfunction may play more of a role than previously recognized in the development of age-related benign prostatic disease.

E-cadherin deficiency promotes prostate macrophage inflammation and bladder overactivity in aged male mice.

Decreased E-cadherin immunostaining is frequently observed in benign prostatic hyperplasia (BPH) and was recently correlated with increased inflammation in aging prostate. Homozygous E-cadherin deletion in the murine prostate results in prostate inflammation and bladder overactivity at 6 months of age. However, this model is limited in that while E-cadherin is significantly reduced in BPH, it is not completely lost; BPH is also strongly associated with advanced age and is infrequent in young men. Here, we examined the functional consequences of aging in male mice with prostate luminal epithelial cell-specific E-cadherin heterozygosity. In control mice, aging alone resulted in an increase in prostate inflammation and changes in bladder voiding function indicative of bladder underactivity. At 24 months of age, mice with prostate-specific Cre-mediated heterozygous deletion of E-cadherin induced at 7 weeks of age developed additional prostatic defects, particularly increased macrophage inflammation and stromal proliferation, and bladder overactivity compared to age-matched control mice, which are similar to BPH/LUTS in that the phenotype is slow-progressing and age-dependent. These findings suggest that decreased E-cadherin may promote macrophage inflammation and fibrosis in the prostate and subsequent bladder overactivity in aging men, promoting the development and progression of BPH/LUTS.

Impact of A Required, Longitudinal Scholarly Project in Medical School: A Content Analysis of Medical Students' Reflections.

INTRODUCTION: Medical schools increasingly require students to complete scholarly projects. Scholarly project programs that are required and longitudinal require considerable resources to implement. It is necessary to understand medical students' perspectives on the impact of such programs. Students at the University of Pittsburgh School of Medicine participate in a required, longitudinal research program (LRP) throughout all years of medical school training. Authors studied students' perceptions of this program. METHODS: Fourth-year medical students submit a written report in which they reflect on their experience with the LRP. Qualitative analysis of students' written reflections was performed on 120 reports submitted 2012-2017. Content analysis was performed using an inductive approach in which investigators coded information and searched for emerging themes. RESULTS: Four themes were identified. First, students described engaging in many steps of the research process, with many participating in projects from conception to completion. Second, students reported the LRP provided opportunities for leadership and independence, and many found this to be meaningful. Third, students developed appreciation for the difficulty of the research process through challenges encountered and practiced problem solving. Fourth, students acquired skills useful across multiple career paths, including critical appraisal of scientific literature, teamwork, and communication. DISCUSSION: Through participation in a required, longitudinal research program, medical students reported gaining valuable skills in leadership, problem solving, critical thinking, and communication. Students found that the longitudinal nature of the program enabled meaningful research experiences. These educational impacts may be worth the effort of implementing and maintaining longitudinal research experiences for medical students. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40670-021-01319-6.

Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum.

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1-5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.

Claudin-1 down-regulation in the prostate is associated with aging and increased infiltration of inflammatory cells in BPH.

INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) is an age-related disease that is frequently associated with chronic prostatic inflammation. In previous studies, we detected the presence of PSA protein in the stroma of BPH nodules and down-regulation of junction proteins E-cadherin and claudin-1. Transmission electron microscopy (TEM) imaging showed a decrease in tight junctions suggesting the luminal epithelial barrier in BPH tissues may be compromised. Recent in vitro studies showed that stimulation of benign prostate epithelial cell lines with TGF-ß1 induced a decrease in claudin-1 expression suggesting that inflammation might be associated with alterations in the prostate epithelial barrier. This study explored the potential associations between aging and loss of junction proteins and the presence of inflammatory cells in prostate tissue specimens from young healthy donors and aged BPH patients. METHODS: Immunostaining of serial prostate sections from 13 BPH patients and five healthy young donors was performed for claudin-1, CD4, CD8, CD20 and CD68. H-Scores and the number of inflammatory cells were calculated for the same area in donor, normal adjacent prostate (NAP) to and BPH specimens. Quantification and statistical correlation analyses were performed. RESULTS: Claudin-1 immunostaining was inversely associated with increasing age, and inflammation in prostate specimens. B-cell infiltration increased with age and BPH was associated with an increased infiltration of T-cells and macrophages compared to NAP. CONCLUSIONS: These findings suggest that aging is associated with down-regulation of claudin-1 and claudin-1 is further decreased in BPH. Claudin-1 down-regulation was associated with increased infiltration of inflammatory cells in both NAP and BPH tissues. Claudin-1 down-regulation in the aging prostate could contribute to increased prostatic inflammation, subsequently contributing to BPH pathogenesis.

E-cadherin expression is inversely correlated with aging and inflammation in the prostate.

INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) is a prostatic disease that is significantly associated with aging. However, it is not well understood how aging contributes to BPH pathogenesis. Several factors associated with an increased risk of BPH are also associated with increasing age, including chronic inflammation and declining epithelial barrier function. Thus, this study explored the potential associations between aging, loss of adherens junction protein E-cadherin and the presence of inflammatory mediators in prostate tissue specimens from healthy young donor and BPH patients. METHODS: Serial prostate sections from a cohort of five donors aged 15-26 years and 13 BPH patients aged 50-77 years were immunostained with E-cadherin, COX-2, CD4, CD8, CD20 and CD68. E-cadherin and COX-2 H-Scores and the number of inflammatory cells were calculated for the same area in donor, normal adjacent prostate to BPH (NAP) and BPH specimens. Quantification and statistical correlation analyses were performed for comparisons between groups. RESULTS: E-cadherin was decreased in aged NAP tissues and in BPH compared to young donor tissue. E-cadherin was inversely correlated with age and infiltration of inflammatory cells in NAP compared to young healthy donor prostate. Stromal COX-2 was positively correlated with age and inflammation. E-cadherin was further down-regulated in BPH, while COX-2 H-Scores were not significantly altered in BPH compared to NAP. CONCLUSIONS: These findings suggest that aging is associated with down-regulation of E-cadherin and up-regulation of stromal COX-2 immunostaining in the prostate. E-cadherin immunostaining was inversely associated with age and inflammation, while stromal COX-2 immunostaining was positively associated with age and inflammation in the prostate. These findings suggest that the prostate epithelial barrier is altered and inflammation is increased with age in the prostate. These changes are further exacerbated in BPH, and may be involved in BPH pathogenesis.

Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings.

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Prostate-Specific Deletion of Cdh1 Induces Murine Prostatic Inflammation and Bladder Overactivity.

Benign prostatic hyperplasia (BPH) is an age-related debilitating prostatic disease that is frequently associated with prostatic inflammation and bothersome lower urinary tract symptoms (LUTS). Animal models have shown that formalin- and bacterial-induced prostatic inflammation can induce bladder dysfunction; however, the underlying mechanisms contributing to prostatic inflammation in BPH and bladder dysfunction are not clear. We previously reported that E-cadherin expression in BPH is downregulated in hyperplastic nodules compared with expression in adjacent normal tissues. Here, we explored the potential consequences of prostatic E-cadherin downregulation on the prostate and bladder in vivo using an inducible murine model of prostate luminal epithelial-specific deletion of Cdh1. The prostate-specific antigen (PSA)-CreERT2 transgenic mouse strain expressing tamoxifen-inducible CreERT2 recombinase driven by a 6-kb human PSA promoter/enhancer was crossed with the B6.129-Cdh1tm2Kem/J mouse to generate bigenic PSA-CreERT2/Cdh1-/- mice. Deletion of E-cadherin was induced by transient administration of tamoxifen when mice reached sexual maturity (7 weeks of age). At 21 to 23 weeks of age, the prostate, bladder, and prostatic urethra were examined histologically, and bladder function was assessed using void spot assays and cystometry. Mice with Cdh1 deletion had increased prostatic inflammation, prostatic epithelial hyperplasia, and stromal changes at 21 to 23 weeks of age, as well as changes in bladder voiding function compared with age-matched controls. Thus, loss of E-cadherin in the murine prostate could result in prostatic defects that are characteristic of BPH and LUTS, suggesting that E-cadherin downregulation could be a driving force in human BPH development and progression.

Effects of dutasteride in a rat model of chemically induced prostatic inflammation-Potential role of estrogen receptor ß.

BACKGROUND: Dutasteride administration reportedly improves lower urinary tract symptoms in patient with chronic, histologically-identified prostatic inflammation, potentially through estrogen receptor ß (ERß), activation of which has anti-inflammatory effects in the prostate tissue. Therefore, we investigated the effect of dutasteride on intraprostatic inflammatory responses and bladder activity using a rat model of chemically induced prostatic inflammation. METHODS: Male Sprague-Dawley rats at 10 weeks old were used. Prostatic inflammation was induced by 5% formalin injection into ventral lobes of the prostate and saline was injected in the control group (control, n = 5). Rats with prostatic inflammation were divided into dutasteride therapy (dutasteride, n = 5) and placebo groups (placebo, n = 5). Dutasteride was administrated at a dose of 0.5 mg/kg daily from 2 days before induction of prostatic inflammation whereas placebo rats received vehicle only. Twenty-eight days later, cystometry was performed in a conscious condition to measure non-voiding contractions (NVCs), intercontraction intervals (ICI) and postvoid residual volume (RV). After cystometry, the prostate was excised for analysis of messenger RNA (mRNA) expression levels of ERα, ERß, interleukin-1ß (IL-1ß), and IL-18 by quantitative polymerase chain reaction. RESULTS: The mean number of NVCs was significantly greater in placebo group than that of control group without prostatic inflammation (p < .05), and ICI were significantly decreased in placebo group compared with control group (p < .05). On the contrary, there was no significant change in NVCs or ICI between control and dutasteride groups. RV was not significantly different among three groups. Gene expression levels of ERα, IL-1ß, and IL-18 was significantly increased in placebo rats compared with control rats (p < .05), but not significantly different between control and dutasteride rats. On the other hand, the mRNA expression level of ERß was significantly decreased in placebo rats (p < .05), but not in dutasteride rats, compared with control rats. CONCLUSION: Dutasteride treatment improved not only prostatic inflammation evident as increased gene expression levels in IL-1ß and IL-18, but also bladder overactivity shown by increased NVCs during bladder filling. These therapeutic effects were associated with the restored expression of anti-inflammatory ERß. Therefore, dutasteride might be effective via ERß modulation for the treatment of prostatic inflammation in addition to its previously known, anti-androgenic effects on benign prostatic hyperplasia.

Loss of CREBRF Reduces Anxiety-like Behaviors and Circulating Glucocorticoids in Male and Female Mice.

Glucocorticoid signaling controls many key biological functions ranging from stress responses to affective states. The putative transcriptional coregulator CREB3 regulatory factor (CREBRF) reduces glucocorticoid receptor levels in vitro, suggesting that CREBRF may impact behavioral and physiological outputs. In the present study, we examined adult male and female mice with global loss of CREBRF (CrebrfKO) for anxiety-like behaviors and circulating glucocorticoids in response to various acute stress conditions. Results demonstrate that both male and female CrebrfKO mice have preserved locomotor activity but reduced anxiety-like behaviors during the light-dark box and elevated plus maze. These behavioral phenotypes were associated with lower plasma corticosterone after restraint stress. Further studies using unhandled female mice also demonstrated a loss of the diurnal circulating corticosterone rhythm in CrebrfKO mice. These results suggest that CREBRF impacts anxiety-like behavior and circulating glucocorticoids in response to acute stressors and serves as a basis for future mechanistic studies to define the impact of CREBRF in glucocorticoid-associated behavioral and physiological responses.

Tight junction protein claudin-1 is downregulated by TGF-ß1 via MEK signaling in benign prostatic epithelial cells.

BACKGROUND: Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-ß1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-ß1 in prostatic epithelial cells are not clear. METHODS: The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-ß1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-ß1 treatment using Western blot analysis. RESULTS: Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-ß1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-ß1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. CONCLUSION: Our findings suggest that downregulation of claudin-1 by TGF-ß1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-ß1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.

Differential impact of paired patient-derived BPH and normal adjacent stromal cells on benign prostatic epithelial cell growth in 3D culture.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-related disease characterized by nonmalignant abnormal growth of the prostate, which is also frequently associated with lower urinary tract symptoms. The prostate with BPH exhibits enhanced growth not only in the epithelium but also in the stroma, and stromal-epithelial interactions are thought to play an important role in BPH pathogenesis. However, our understanding of the mechanisms of stromal-epithelial interactions in the development and progression of BPH is very limited. METHODS: Matched pairs of glandular BPH and normal adjacent prostate specimens were obtained from BPH patients undergoing simple prostatectomy for symptomatic BPH. Tissues were divided further into fresh specimens for culture of primary prostatic stromal cells, and specimens were embedded in paraffin for immunohistochemical analyses. Proliferation assays, immunohistochemistry, and immunoblotting were used to characterize the primary prostate stromal cells and tissue sections. Coculture of the primary stromal cells with benign human prostate epithelial cell lines BHPrE1 or BPH-1 was performed in three-dimensional (3D) Matrigel to determine the impact of primary stromal cells derived from BPH on epithelial proliferation. The effect of stromal-conditioned medium (CM) on BHPrE1 and BPH-1 cell growth was tested in 3D Matrigel as well. RESULTS: BPH stromal cells expressed less smooth muscle actin and calponin and increased vimentin, exhibiting a more fibroblast and myofibroblast phenotype compared with normal adjacent stromal cells both in culture and in corresponding paraffin sections. Epithelial spheroids formed in 3D cocultures with primary BPH stromal cells were larger than those formed in coculture with primary normal stromal cells. Furthermore, CM from BPH stromal cells stimulated epithelial cell growth while CM from normal primary stromal cells did not in 3D culture. CONCLUSIONS: These findings suggest that the stromal cells in BPH tissues are different from normal adjacent stromal cells and could promote epithelial cell proliferation, potentially contributing to the development and progression of BPH.

Transforming growth factor beta 1 impairs benign prostatic luminal epithelial cell monolayer barrier function.

Our recent studies identifying the presence of luminal secretory protein PSA in the stroma, decreased E-cadherin expression, and reduced number of tight junction kiss points in benign prostatic hyperplasia (BPH) tissues suggest that epithelial barrier permeability is increased in BPH. However, the cause of increased epithelial permeability in BPH is unclear. Transforming growth factor beta 1 (TGF-ß1) has been reported to be up-regulated in clinical BPH specimens and TGF-ß1 overexpression induced fibrosis and inflammation in a murine model. TGF-ß1 was reported to repress the expression of E-cadherin in benign prostatic cells. However, whether and how TGF-ß1 up-regulation affects epithelial barrier permeability is unknown. Here, in vitro benign prostatic epithelial cell lines BHPrE1 and BPH-1 were utilized to determine the impact of TGF-ß1 treatment on epithelial barrier, tight junctions, and expression of E-cadherin and claudin 1 by transepithelial electrical resistance (TEER) measurement, FITC-dextran trans-well diffusion assays, qPCR, as well as transmission electron microscopy (TEM) observation. Laser capture micro-dissection (LCM) combined with reverse transcription-polymerase chain reaction (qPCR) were utilized to determine the expression of E-cadherin and claudin 1 in BPH patient specimens. TGF-ß1 treatment decreased TEER, increased FITC-dextran diffusion, and reduced the mRNA expression of junction protein claudin 1 in cultured cell monolayers. Claudin 1 mRNA but not E-cadherin mRNA was down-regulated in the luminal epithelial cells in BPH nodules compared to normal prostate tissues. Our studies suggest that TGF-ß1 could increase the permeability through decreasing the expression of claudin 1 and inhibiting the formation of tight junctions in BHPrE1 and BPH-1 monolayers. These results suggest that TGF-ß1 might play an important role in BPH pathogenesis through increasing the permeability of luminal epithelial barrier in the prostate.

Prenatal drug exposure and neurodevelopmental programming of glucocorticoid signalling.

Prenatal neurodevelopment is dependent on precise functioning of multiple signalling pathways in the brain, including those mobilised by glucocorticoids (GC) and endocannabinoids (eCBs). Prenatal exposure to drugs of abuse, including opioids, alcohol, cocaine and cannabis, has been shown to not only impact GC signalling, but also alter functioning of the hypothalamic-pituitary-adrenal (HPA) axis. Such exposures can have long-lasting neurobehavioural consequences, including alterations in the stress response in the offspring. Furthermore, cannabis contains cannabinoids that signal via the eCB pathway, which is linked to some components of GC signalling in the adult brain. Given that GCs are frequently used in pregnancy to prevent complications of prematurity, and also that rates of cannabis use in pregnancy are increasing, the likelihood of foetal co-exposure to these compounds is high and may have additional implications for long-term neurodevelopment. Here, we present a discussion of GC signalling and the HPA axis, as well as the effects of prenatal drug exposure on these pathways and the stress response, and we explore the interactions between GC and EC signalling in the developing brain and potential for neurodevelopmental consequences.