Prostatic Smooth Muscle Cells Research Articles

Elsholtzia ciliata Essential Oil Exhibits a Smooth Muscle Relaxant Effect.

A recent in vivo study in pigs demonstrated the hypotensive properties of essential oil extracted from the blossoming plant Elsholtzia ciliata. This study was designed to examine the effect of E. ciliata essential oil (EO) on smooth muscle contraction. Tension measurements were performed on prostate strips and intact aortic rings isolated from rats. Results showed that EO caused a concentration-dependent reduction in phenylephrine-induced contraction of both the prostate and aorta, with a more pronounced inhibitory effect in the prostate. The IC50 of EO for the prostate was 0.24 ± 0.03 µL/mL (n = 10) and for the aorta was 0.72 ± 0.11 µL/mL (n = 4, p < 0.05 vs. prostate). The chromatographic analysis identified elsholtzia ketone (10.64%) and dehydroelsholtzia ketone (86.23%) as the predominant compounds in the tested EO. Since both compounds feature a furan ring within their molecular structure, other furan ring-containing compounds, 2-acetylfuran (2AF) and 5-methylfurfural (5MFF), were examined. For the first time, our study demonstrated the relaxant effects of 2AF and 5MFF on smooth muscles. Further, results showed that EO, 2AF, and 5MFF altered the responsiveness of prostate smooth muscle cells to phenylephrine. Under control conditions, the EC50 of phenylephrine was 0.18 ± 0.03 µM (n = 5), while in the presence of EO, 2AF, or 5MFF, the EC50 values were 0.81 ± 0.3 µM (n = 5), 0.89 ± 0.11 µM (n = 5), and 0.69 ± 0.23 µM (n = 4), respectively, p < 0.05 vs. control. Analysis of the affinity of EO for α1-adrenergic receptors in the prostate suggested that EO at a certain range of concentrations has a competitive antagonistic effect on α1-adrenergic receptors. In conclusion, EO elicits a relaxant effect on smooth muscles which may be related to the inhibition of α1-adrenoreceptors.

The Effect of Tamsulosin, Dutasteride Monotherapy and Tamsulosin-Dutasteride Combination on Prostate Smooth Muscle Contractility in BPH Model Wistar Strain Rattus Novergicus.

Following the c In the management of BPH, Tamsulosin is an example of a-adrenergic receptor blocker drug that is usually used. In addition, dutasteride is also a BPH drug that works as a group of 5 a reductase inhibitor. However, the weakness of long-term administration of a1-adrenergic receptor antagonists can result in upregulation of prostate smooth muscle cell contractility and expression of a-adrenergic mRNA receptors, resulting in hyperactivity and supersensitivity to a-agonists. Our study aimed to determine the effect of long-term administration of tamsulosin, dutasteride and tamsulosin-dutasteride combination on the contractility of prostate smooth muscle cells in BPH model rats. This study was designed using an experimental post test only method, control group design. It measured the contractility of prostate smooth muscle cells from samples obtained from the prostatic stroma of experimental animals adult male Rattus norvegicus Wistar strain induced BPH and administered tamsulosin 1 mg/kg/day, dutasteride 0.5 mg/kg/day, and a combination of continuous administration for 1, 6 and 12 consecutive days. Data were analyzed using one way ANOVA if the data distribution was normal or Kruskall Walis if the data distribution was abnormal. The effect of tamsulosin, dutasteride and the combination of tamsulosin with dutasteride on prostate smooth muscle cell contractility in experimental animals Rattus norvegicus Wistar strain showed that tamsulosin administration for six days, twelve days, and the combination of tamsulosin dutasteride for one day got statistically significant different result (p=0.016; p=0.006; p=0.029) compared to the negative control group. In addition, there was a difference between the tamsulosin and dutasteride combination group for 12 days compared to tamsulosin monotherapy for 6 days and 12 days (p=0.160; p=0.010). Continuous administration of monotherapy tamsulosin has an upregulation effect on the sixth to twelfth day. Decreased contractility of prostate smooth muscle cells occurs on the first day but will increase on the sixth to twelfth day. On the other hand, the results of our study also showed that the combination of tamsulosin and dutasteride gave the effect of reducing contractility and was most effective on day 12.

A0600 - Post-receptor signaling by G protein-coupled, contractile receptors is a prerequisite for cytoskeletal organization and cell proliferation in human prostate smooth muscle cells

A0600 - Post-receptor signaling by G protein-coupled, contractile receptors is a prerequisite for cytoskeletal organization and cell proliferation in human prostate smooth muscle cells

Fibrin and Transforming Growth Factor Alpha Affect Prostatic Smooth Muscle Cell's Phenotype and Motility.

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFβ1 or TGFβ3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

Effects of shRNA‑mediated silencing of PDE5A3 on intracellular cGMP and free Ca2+ levels and human prostate smooth muscle cell proliferation from benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) with lower urinary tract symptoms (LUTS) is a common disease among elderly men, for which safe and effective treatment strategies remain limited. The aim of the present study was to explore the potential effects of phosphodiesterase 5A3 (PDE5A3) silencing on human prostate smooth muscle cells (HPSMCs). HPSMCs were initially obtained from patients with BPH/LUTS. Short hairpin RNA (shRNA) targeting the PDE5A3 gene was subsequently transfected into cultured HPSMCs. The expression of PDE5A3 was measured using reverse transcription-quantitative PCR and western blotting. cGMP levels were then measured using western blotting and immunocytochemical staining and intracellular Ca2+ concentration was measured using rhod2-AM in HPSMCs after transfection. HPSMC proliferation was also observed within 4 days. Cells transfected with PDE5A3-shRNA2 exhibited the most notable decline in PDE5A3 expression compared with that in the Control or NC groups. cGMP levels in HPSMCs transfected with PDE5A3-shRNA2 was significantly increased compared with those in the Control or NC groups, whereas intracellular Ca2+ concentrations in cells in the PDE5A3-shRNA2 group were decreased compared with that in the Control or NC groups. The proliferation of HPSMCs in the PDE5A3-shRNA2 group was also inhibited compared with that in the Control or NC groups after 72 h of culture. In conclusion, shRNA-mediated silencing of PDE5A3 was able to increase the levels of cGMP whilst reducing the concentration of Ca2+ in HPSMCs, in turn suppressing their proliferation. These findings may potentially provide a novel therapeutic target for treating BPH/LUTS.

Polarization, migration, and homotypical interactions among prostatic smooth muscle cells in a laminin 111-rich extracellular matrix.

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.

Inhibition of neurogenic and thromboxane A2 -induced human prostate smooth muscle contraction by the integrin α2β1 inhibitor BTT-3033 and the integrin-linked kinase inhibitor Cpd22.

Prostate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin-linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real-time polymerase chain reaction (RT-PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide), the integrin α2β1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide), or the integrin α4β1/α9β1 inhibitor BOP (N-(benzenesulfonyl)- l-prolyl- l-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt). Western blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, β1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT-PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate-specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and β1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency-dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT-3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT-3033 at 32 Hz). The thromboxane A2 analog U46619-induced concentration-dependent contractions, which were inhibited by Cpd22 and BTT-3033 (around 67% by Cpd22 and 39% by BTT-3033 at 30 µM U46619). Endothelin-1 induced concentration-dependent contractions, which were not affected by Cpd22 or BTT-3033. Noradrenaline and the α1 -adrenergic agonists methoxamine and phenylephrine-induced concentration-dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT-3033. BOP did not change EFS- or agonist-induced contraction. Integrin α2β1 and ILK inhibitors inhibit neurogenic and thromboxane A2 -induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.

MP06-10 INHIBITION OF HUMAN PROSTATE SMOOTH MUSCLE CONTRACTION BY THE INTEGRIN α2β1-SELECTIVE INHIBITOR BTT3033 AND THE INTEGRIN-LINKED KINASE INHIBITOR CPD22

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-10 INHIBITION OF HUMAN PROSTATE SMOOTH MUSCLE CONTRACTION BY THE INTEGRIN α2β1-SELECTIVE INHIBITOR BTT3033 AND THE INTEGRIN-LINKED KINASE INHIBITOR CPD22 Bingsheng Li*, Xiaolong Wang, Beata Rutz, Anna Ciotkowska, Christian Gratzke, Alexander Tamalunas, Raphaela Waidelich, Christian Stief, and Martin Hennenberg Bingsheng Li*Bingsheng Li* More articles by this author , Xiaolong WangXiaolong Wang More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.010AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Integrins connect the cytoskeleton of cells to membranes and extracellular matrix, what is essential for force generation by smooth muscle contraction. Suprisingly, their role for contraction has not been tested yet in any organ. In the prostate, smooth muscle contraction is critical for lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins are composed by different subunits in different organs, so that subunit-specific inhibitors may be attractive to attain prostate-specific inhibition of smooth muscle contraction. Here we examined effects of the integrin α2β1 inhibitor BTT3033 and the integrin-linked kinase (ILK) inhibitor Cpd22 on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy (n=56 patients). Contraction of prostate strips was studied in an organ bath. Integrins and ILK were detected by Western blot and fluorescence staining. RESULTS: Electric field stimulation (EFS) induced frequency-dependent contractions of prostate tissues, which were inhibited by BTT3033 (1 µM) (55% inhibition at 32 Hz; p<0.004 for whole frequency-response curves; n=5) and Cpd22 (3 µM) (47% inhibition at 32 Hz, p<0.02, n=5). The α1-adrenoceptor agonists noradrenaline and methoxamine induced concentration-dependent, which were inhibited BTT3033 (1 µM) (p<0.004 for noradrenaline, n=5; p<0.03 for methoxamine, n=6), and by Cpd22 (3 µM) (p<0.008 for noradrenaline, n=6; p<0.02 for methoxamine, n=5). The thromboxane A2 analog U46619 induced concentration-dependent contractions, which were inhibited by BTT3033 (1 µM) (p<0.03, n=5), and by Cpd22 (p<0.001, n=5). Western blot analyses of prostate tissues revealed bands with sizes matching the expected sizes for integrin α2b, integrin β1 and ILK, which were observed in each included sample (n=9). Double fluorescence staining of prostate sections (n=5) resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. CONCLUSIONS: Integrin and ILK inhibitors inhibit agonist-induced and neurogenic prostate smooth muscle contraction in BPH. As integrins show organ-specific composition, targeting the prostate-specific integrin code by subunit-specific inhibitors may offer a promising approach to attain inhibition in the prostate, without side effects in the cardiovascular system. Source of Funding: Deutsche Forschungsgemeinschaft (DFG), Chinese Scholarship Council (CSC) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e54-e55 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bingsheng Li* More articles by this author Xiaolong Wang More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Christian Gratzke More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Efficacy, potency and kinetics of activation process of Kv7.4 and Kv7.5 channel with novel pharmacological activators targeted for Lower Urinary Tract Syndrome (LUTS)

Among several types of Kv7 (KCNQ) voltage gated potassium channel family, Kv7.4 (KCNQ4) and Kv7.5 (KCNQ5) channels show prominent expression in smooth muscle, cardiac tissues and vasculature. In general, activation of the channel would mediate strong efflux of potassium and lead membrane potential to hyperpolarization, though the extent may depend on established potassium concentration gradient and membrane potential. Once membrane potential is hyperpolarized, it would decrease the excitability of excitable cells. Decreased excitability may reduce calcium spikes and Ca‐CaM mediated activation of myosin light chain kinase (MLCK) in smooth muscle cells. Recently, it was reported that Kv7.4 channels are highly expressed in corpus cavernosum smooth muscle cells and Kv7.5 channels in prostate smooth muscle cells, respectively. Since smooth muscle relaxation of corpus cavernosum and smooth muscle tension in prostate are in the center of pathophysiology of erectile dysfunction and LUTS, we examined the effect of novel Kv7.4 and Kv7.5 activators to the channel activation process.In HEK293 cells expressing human Kv7.4 or Kv7.5 channels, we performed whole‐cell patch clamp technique with two different voltage protocols in order to measure specific activity of the channel. For kinetic analysis of the activation process, voltage steps from –100 mV to +100 mV with 10 mV of step interval was applied. For each step, pulse duration was 3 seconds for activation process of Kv7.4 and Kv7.5 channels is rather slow. Intersweep duration was 10 seconds and holding potential was –80 mV. For measurement of time‐dependent action of the drug, 10 seconds single step with +50 mV was applied in every 30s with holding potential of –80 mV. The composition of internal solution is as following; 120 KCl, 5.37 CaCl2, 1.75 MgCl2, 10 EGTA, 10 HEPES, 4 ATP, 0.2 GTP. pH was adjusted to 7.3 using KOH and [Ca2+]free = 100 nM. Normal tyrode solution was used for external solution and drugs stocked with DMSO was diluted to corresponding concentrations with the Normal tyrode solution.We have found that URO‐K10, a novel activator, increases not only maximum conductance of the channel but also time constant of activation gate. In comparison to positive control, ML‐213, a well‐known Kv7.4 channel activator, URO‐K10 showed significant supremacy. EC50 of URO‐K10 was 0.304 ± 0.058 μM for Kv7.4 channel which is smaller than EC50 of ML‐213 (0.8 ± 0.3 μM). URO‐K10 not only increased the maximum conductance of the channel but also left‐shifted activation curve in terms of Boltzmann distribution. Half‐maximal voltage (V1/2) of Kv7.4 channel without any of the drugs was −43.03 mV whereas V1/2 of URO‐K10 activated channel was −67.65 mV, which is near the resting membrane potential of smooth muscle cells. ML‐213 activated channels showed V1/2 of −51.91 mV.Support or Funding InformationThis work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information &amp; Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Jung Eun Lee was supported by the BK plus program from the MSIP.

Bimolecular modeling and simulation of antagonist binding to the alpha‐1A adrenergic receptor

Benign prostatic hyperplasia (BPH), which is a noncancerous enlargement of the prostate, currently affects 70% of men 60–69 years old, and 80% of men 70 years and older. By 2030, BPH incidence is predicted to increase based on the aging US population, and estimates suggest 20 million men 65 years and older will be diagnosed with BPH. BPH symptoms include frequent urination, difficulty initiating, maintaining, or a weak stream during urination, as well as inability to urinate or loss of bladder control. α1‐Adrenergic receptors (α1‐ARs), and particularly the α1A‐subtype, are highly expressed in prostate smooth muscle cells and mediate contraction. Clinically, α1‐AR antagonists, or α1‐blockers, are the primary treatment BPH with lower urinary tract symptoms (LUTS), including tamsulosin, touted as an α1A‐subtype selective antagonist. Here, our goal was to understand the molecular forces governing α1‐blocker binding to α1‐ARs to better understand α1‐blocker pharmacology. We have developed a homology model of α1‐antagonist binding to the α1A‐AR to identify essential molecular parameters that can be exploited for structure‐based drug design. Through homology modeling, docking, and molecular dynamics simulation, our preliminary model complex suggests for the first time 1) the burial of the terminal aromatic core within the hydrophobic pocket α1A‐AR and 2) the salt bridge interaction between its charged ammonium group with ionized aspartate are essential for ligand binding. This finding provides invaluable insight into design of the next generation, subtype‐specific of novel a1‐blockers.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protease-activated receptor 2 activates CRAC-mediated Ca2+ influx to cause prostate smooth muscle contraction.

Protease‐activated receptor 2 (PAR2) is a G‐protein‐coupled receptor that contributes to prostate fibrosis and lower urinary tract symptoms (LUTS). In addition to fibrosis, aberrant smooth muscle tone in the prostate has been hypothesized to play a role. We therefore examined PAR2 expression in primary human prostate smooth muscle cells (PSMC) and studied the downstream signaling effects of PAR2 activation. Signaling pathways involved in the process were assessed using the PAR2 activating peptide SLIGKV‐NH2. We show that PAR2 is expressed in PSMC and that PAR2 activation mediates a biphasic elevation in intracellular Ca2+ and phosphorylation of myosin light chain 20 (MLC20), causing cellular contraction as assessed in a gel contraction assay. Intracellular Ca2+ flux was inhibited by a phosphoinositide hydrolysis inhibitor, U73122, showing a requirement for phospholipase C β (PLCβ) activation. PSMC expressed mRNA for L‐type voltage dependent Ca2+ channels (VDCC) as well as Ca2+ release activated channels (CRAC), a hitherto unreported finding. Secondary intracellular Ca2+ oscillations were abrogated only by BTP2, the CRAC channel inhibitor, but not by nifedipine, an inhibitor of VDCC. These data suggest that, PAR2 activation and subsequent Ca2+ entry through CRAC channels are important mechanisms in prostate smooth muscle contraction.

291 Gray-scale ultrasound to assess persistent erectile dysfunction in young men

Young men exposed to 5 alpha reductase inhibitor (5ARI) finasteride for androgenic alopecia have reported persistent erectile dysfunction (PED). We are unaware of studies assessing their PED pathophysiology. Animals exposed to 5ARI have revealed both prostate smooth muscle and penile smooth muscle cell atrophy and apoptosis. High resolution gray-scale ultrasound (GSU) imaging is an efficient, non-invasive technique to assess cavernosal erectile tissue fibrosis (CETF) in men with ED. In selected men with PED from finasteride, high resolution GSU during maximal pharmacologic smooth muscle relaxation was used to better understand the pathophysiology. A renowned penile ultrasound expert (MB) blindly reviewed high resolution GSU images. Under IRB approval, charts of 27 men with PED (mean age 31) met inclusion/exclu-sion: potent prior to finasteride use; <40 years old with no obvious cardiovascular risk factors; underwent high resolution GSU/color duplex Doppler ultrasound with a high frequency probe (Aixplorer 15.4 MHz transducer) during maximal pharmacologic erection. GSU was performed in axial B-mode at multiple gains, with various dynamic ranges, in multiple penile shaft locations, to determine presence/absence of erectile tissue homogeneity or observation of heterogeneous regions. Color Doppler parameters of peak systolic velocity (PSV) and end-diastolic velocity (EDV) were also assessed. Ten control subjects with ED not exposed to finasteride who had erectile tissue homogeneity on high resolution GSU were also reviewed blindly by the ultrasound expert.

Inhibition of Prostate Smooth Muscle Contraction by Inhibitors of Polo-Like Kinases.

Background: Prostate smooth muscle contraction plays an important role for pathophysiology and treatment of male lower urinary tract symptoms (LUTS) but is incompletely understood. Because the efficacy of available medication is limited, novel options and improved understanding of prostate smooth muscle contraction are of high demand. Recently, a possible role of polo-like kinase 1 (PLK1) has been suggested for smooth muscle contraction outside the lower urinary tract. Here, we examined effects of PLK inhibitors on contraction of human prostate tissue.Methods: Prostate tissues were obtained from radical prostatectomy. RT-PCR, Western blot and immunofluorescence were performed to detect PLK expression and phosphorylated PLK. Smooth muscle contractions were induced by electric field stimulation (EFS), α1-agonists, endothelin-1, or the thromboxane A2 analog U46619 in organ bath.Results: RT-PCR, Western blot, and immunofluorescence suggested expression of PLK1 in the human prostate, which may be located and active in smooth muscle cells. EFS-induced contractions of prostate strips were reduced by SBE 13 (1 μM), cyclapolin 9 (3 μM), TAK 960 (100 nM), and Ro 3280 (100 nM). SBE 13 and cyclapolin 9 inhibited contractions by the α1-agonists methoxamine, phenylephrine, and noradrenaline. In contrast, no effects of SBE 13 or cyclapolin 9 on endothelin-1- or U46619-induced contractions were observed.Conclusion: Alpha1-adrenergic smooth muscle contraction in the human prostate can be inhibited by PLK inhibitors. PLK-dependent signaling may be a new pathway, which promotes α1-adrenergic contraction of prostate smooth muscle cells. As contractions by endothelin and U46619 are not susceptible to PLK inhibition, this reflects divergent regulation of adrenergic and non-adrenergic prostate smooth muscle contraction.

The Response of Prostate Smooth Muscle Cells to Testosterone Is Determined by the Subcellular Distribution of the Androgen Receptor.

Androgen signaling in prostate smooth muscle cells (pSMCs) is critical for the maintenance of prostate homeostasis, the alterations of which are a central aspect in the development of pathological conditions. Testosterone can act through the classic androgen receptor (AR) in the cytoplasm, eliciting genomic signaling, or through different types of receptors located at the plasma membrane for nongenomic signaling. We aimed to find evidence of nongenomic testosterone-signaling mechanisms in pSMCs and their participation in cell proliferation, differentiation, and the modulation of the response to lipopolysaccharide. We demonstrated that pSMCs can respond to testosterone by a rapid activation of ERK1/2 and Akt. Furthermore, a pool of ARs localized at the cell surface of pSMCs is responsible for a nongenomic testosterone-induced increase in cell proliferation. Through membrane receptor stimulation, testosterone favors a muscle phenotype, indicated by an increase in smooth muscle markers. We also showed that the anti-inflammatory effects of testosterone, capable of attenuating lipopolysaccharide-induced proinflammatory actions, are promoted only by receptors located inside the cell. We postulate that testosterone might perform prohomeostatic effects through intracellular-initiated mechanisms by modulating cell proliferation and inflammation, whereas some pathological, hyperproliferative actions would be induced by membrane-initiated nongenomic signaling in pSMCs.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Testosterone Rescues the De-Differentiation of Smooth Muscle Cells Through Serum Response Factor/Myocardin.

Prostatic smooth muscle cells (pSMCs) differentiation is a key factor for prostatic homeostasis, with androgens exerting multiple effects on these cells. Here, we demonstrated that the myodifferentiator complex Srf/Myocd is up-regulated by testosterone in a dose-dependent manner in primary cultures of rat pSMCs, which was associated to the increase in Acta2, Cnn1, and Lmod1 expressions. Blocking Srf or Myocd by siRNAs inhibited the myodifferentiator effect of testosterone. While LPS led to a dedifferentiated phenotype in pSMCs, characterized by down-regulation of Srf/Myocd and smooth muscle cell (SMC)-restricted genes, endotoxin treatment on Myocd-overexpressing cells did not result in phenotypic alterations. Testosterone at a physiological dose was able to restore the muscular phenotype by normalizing Srf/Myocd expression in inflammation-induced dedifferentiated pSMCs. Moreover, the androgen reestablished the proliferation rate and IL-6 secretion increased by LPS. These results provide novel evidence regarding the myodifferentiating role of testosterone on SMCs by modulating Srf/Myocd. Thus, androgens preserve prostatic SMC phenotype, which is essential to maintain the normal structure and function of the prostate. J. Cell. Physiol. 232: 2806-2817, 2017. © 2016 Wiley Periodicals, Inc.

The regulation of transient receptor potential canonical 4 (TRPC4) channel by phosphodiesterase 5 inhibitor via the cyclic guanosine 3'5'-monophosphate.

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to the fruit fly Drosophila TRP. The TRP superfamily is distinct from other groups of ion channels in displaying a large diversity in ion selectivity, modes of activation, and physiological functions. Classical TRP (transient receptor potential canonical (TRPC)) channels are activated by stimulation of Gq-PLC-coupled receptors and modulated by phosphorylation. The cyclic guanosine monophosphate (cGMP)-PKG pathway is involved in the regulation of TRPC3 and TRPC6 channels. Phosphodiesterase (PDE) 5 inhibitor induced muscle relaxation in corporal smooth muscle cells and was used to treat erectile dysfunction by inhibiting cGMP degradation. Here, we report the functional relationship between TRPC4 and cGMP. In human embryonic kidney (HEK) 293 cells overexpressing TRPC4, cGMP selectively activated TRPC4 channels and increased cytosolic calcium level through TRPC4 channel. We investigated phosphorylation sites in TRPC4 channels and identified S688 as an important phosphorylation site for the cGMP-PKG pathway. Cyclic GMP also activated TRPC4-like current with doubly rectifying current-voltage relationship in prostate smooth muscle cell lines. Taken together, these results show that TRPC4 is phosphorylated by the cGMP-PKG pathway and might be an important target for modulating prostate function by PDE5 inhibitors.

MP61-18 ELK-1 PROMOTES PROSTATE CANCER PROGRESSION AND SILODOSIN THAT INACTIVATES ELK-1 IN PROSTATE CANCER CELLS INCREASES SENSITIVITY TO GEMCITABINE

INTRODUCTION AND OBJECTIVES: LK-1 is a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation. However, little is known about biological significance of ELK1 in prostate cancer. In addition, silodosin, a selective a1-adrenergic blocker used for the symptomatic treatment of benign prostatic hyperplasia, has been shown to decrease ELK-1 expression in human prostate smooth muscle cells. In this study, we aim to investigate the functions of ELK-1 in prostate cancer growth and the efficacy of silodosin, in combination with chemotherapeutic drugs, in cell proliferation. METHODS: We first immunohistochemically determined the levels of phospho-ELK-1 (pELK-1) expression in radical prostatectomy specimens. We then assessed the effects of ELK-1 knockdown via short hairpin RNA on cell proliferation, migration, and invasion as well as those of silodosin on ELK-1 expression/activity in prostate cancer lines and their viability. RESULTS: Immunohistochemistry showed that the levels of pELK-1 expression were significantly higher in carcinoma (positivity & score, P<0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (positivity & score, P<0.001) than in benign as well as in carcinoma than in HGPIN (positivity, P1⁄40.330; score, P1⁄40.002). Although none of the 7 patients with pELK-1-negative carcinoma had biochemical recurrence after radical prostatectomy, Kaplan-Meier and log-rank tests revealed no significant correlations of pELK-1 levels with patient outcomes. In PC3 and DU145 cells expressing ELK-1 (mRNA, protein), ELK-1 silencing resulted in decreases in the expression of cfos, a downstream target of ELK-1 signals, as well as in cell viability/ colony formation, cell migration/invasion, and the expression of matrix metalloproteinase (MMP)-2 and MMP-9. In these cells, silodosin reduced the expression of a1-adrenergic receptor, ELK-1, and c-fos as well as the transcriptional activity of ELK-1. However, silodosin alone (up to 10 mM) only marginally inhibited PC3/DU145 cell growth. Interestingly, silodosin treatment in the prostate cancer lines increased sensitivity to gemcitabine, but not to cisplatin or docetaxel. CONCLUSIONS: ELK-1 is likely activated in prostate cancer cells. Our results also suggest that ELK-1 plays an important role in prostate cancer progression. Thus, ELK-1 inhibition has the potential of being a therapeutic approach for prostate cancer. Furthermore, silodosin that inactivates ELK-1 in prostate cancer cells enhances the cytotoxic activity of gemcitabine.

MP61-19 A NOVEL STRATEGY TO OPTIMISE TREATMENT OF DOCETAXEL-RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: LK-1 is a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation. However, little is known about biological significance of ELK1 in prostate cancer. In addition, silodosin, a selective a1-adrenergic blocker used for the symptomatic treatment of benign prostatic hyperplasia, has been shown to decrease ELK-1 expression in human prostate smooth muscle cells. In this study, we aim to investigate the functions of ELK-1 in prostate cancer growth and the efficacy of silodosin, in combination with chemotherapeutic drugs, in cell proliferation. METHODS: We first immunohistochemically determined the levels of phospho-ELK-1 (pELK-1) expression in radical prostatectomy specimens. We then assessed the effects of ELK-1 knockdown via short hairpin RNA on cell proliferation, migration, and invasion as well as those of silodosin on ELK-1 expression/activity in prostate cancer lines and their viability. RESULTS: Immunohistochemistry showed that the levels of pELK-1 expression were significantly higher in carcinoma (positivity & score, P<0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (positivity & score, P<0.001) than in benign as well as in carcinoma than in HGPIN (positivity, P1⁄40.330; score, P1⁄40.002). Although none of the 7 patients with pELK-1-negative carcinoma had biochemical recurrence after radical prostatectomy, Kaplan-Meier and log-rank tests revealed no significant correlations of pELK-1 levels with patient outcomes. In PC3 and DU145 cells expressing ELK-1 (mRNA, protein), ELK-1 silencing resulted in decreases in the expression of cfos, a downstream target of ELK-1 signals, as well as in cell viability/ colony formation, cell migration/invasion, and the expression of matrix metalloproteinase (MMP)-2 and MMP-9. In these cells, silodosin reduced the expression of a1-adrenergic receptor, ELK-1, and c-fos as well as the transcriptional activity of ELK-1. However, silodosin alone (up to 10 mM) only marginally inhibited PC3/DU145 cell growth. Interestingly, silodosin treatment in the prostate cancer lines increased sensitivity to gemcitabine, but not to cisplatin or docetaxel. CONCLUSIONS: ELK-1 is likely activated in prostate cancer cells. Our results also suggest that ELK-1 plays an important role in prostate cancer progression. Thus, ELK-1 inhibition has the potential of being a therapeutic approach for prostate cancer. Furthermore, silodosin that inactivates ELK-1 in prostate cancer cells enhances the cytotoxic activity of gemcitabine.

Structural modifications of the prostate in hypoxia, oxidative stress, and chronic ischemia.

PurposeClinical studies have reported a correlation between pelvic ischemia and voiding dysfunction in elderly men. The aim of this study was to identify and compare prostate structural modifications in cultured cells and in a rabbit model after exposure to hypoxia, oxidative stress, and chronic ischemia.Materials and MethodsCultured human prostate smooth muscle cells (SMCs), epithelial cells (ECs), and stromal cells (SCs) were incubated under normoxia, hypoxia, and oxidative stress conditions by use of a computerized oxycycler system. We developed a rabbit model of chronic prostate ischemia by creating aorto-iliac arterial atherosclerosis. Markers of oxidative stress were examined by using fluorometric analysis and enzyme immunoassay. Prostate structure was examined by using Masson's trichrome staining and transmission electron microscopy (TEM).ResultsLipid peroxidation was found in SMCs exposed to hypoxia and in all cell types exposed to oxidative stress. We identified protein oxidation in ECs exposed to hypoxia and in all cell types exposed to oxidative stress. Markers indicating oxidative damage were present in chronically ischemic rabbit prostate tissue. These reactions were associated with DNA damage. Prostate ischemia resulted in epithelial atrophy, loss of smooth muscle, and diffuse fibrosis. TEM showed swollen mitochondria with degraded cristae, loss of membrane, loss of Golgi bodies, degenerated nerves, and disrupted cell-to-cell junctions.ConclusionsHuman prostate cells exhibited differential reactions to hypoxia and oxidative stress with widespread DNA damage. Structural modifications in ischemic prostate tissue were similar to those in cells exposed to oxidative stress. Structural changes due to ischemia and oxidative stress may contribute to prostatic noncompliance in aging men.