Corneal acetylcholine regulates sensory nerve activity via nicotinic receptors.

PURPOSE: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea. METHODS: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs. RESULTS: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist. CONCLUSIONS: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern.

How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [3 H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [3 H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [3 H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [3 H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain.

Role of Muscarinic Acetylcholine Receptors in Intestinal Epithelial Homeostasis: Insights for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

Expression of Chrna9 is regulated by Tbx3 in undifferentiated pluripotent stem cells.

It was reported that nicotinic acetylcholine receptor (nAChR)-mediated signaling pathways affect the proliferation and differentiation of pluripotent stem cells. However, detail expression profiles of nAChR genes were unrevealed in these cells. In this study, we comprehensively investigated the gene expression of α subunit of nAChRs (Chrna) during differentiation and induction of pluripotent stem cells. Mouse embryonic stem (ES) cells expressed multiple Chrna genes (Chrna3-5, 7 and 9) in undifferentiated status. Among them, Chrna9 was markedly down-regulated upon the differentiation into mesenchymal cell lineage. In mouse tissues and cells, Chrna9 was mainly expressed in testes, ES cells and embryonal F9 teratocarcinoma stem cells. Expression of Chrna9 gene was acutely reduced during differentiation of ES and F9 cells within 24 h. In contrast, Chrna9 expression was increased in induced pluripotent stem cells established from mouse embryonic fibroblast. It was shown by the reporter assays that T element-like sequence in the promoter region of Chrna9 gene is important for its activities in ES cells. Chrna9 was markedly reduced by siRNA-mediated knockdown of Tbx3, a pluripotency-related transcription factor of the T-box gene family. These results indicate that Chrna9 is a nAChR gene that are transcriptionally regulated by Tbx3 in undifferentiated pluripotent cells.

Voluntary wheel-running activities ameliorate depressive-like behaviors in mouse dry eye models.

Recent clinical studies indicate that dry eye is closely associated with psychiatric disorders such as depression and anxiety. Here, we investigated whether two types of mouse dry eye models showed depressive-like behavior in forced swim and sucrose preference tests, and whether voluntary wheel-running helped ameliorate depressive states. To reproduce the dry eye models, the exorbital lacrimal glands (ELG) or exorbital and intraorbital lacrimal glands (ELG+ILG) were bilaterally excised from male C57BL/6J mice. Tear volume was persistently reduced in both models, but the ELG+ILG excision mice exhibited more severe corneal damage than the ELG excision mice. In the forced swim and sucrose preference tests, the gland excision mice showed longer immobility and shorter climbing times, and lower sucrose preference than sham-operated mice, respectively, which appeared earlier in the ELG+ILG excision mice. Wheel-running activities were significantly lower in the ELG+ILG excision mice, but not in the ELG excision mice. After short-period wheel-running, the longer immobility times and the shorter climbing times in the forced swim completely disappeared in both models. Our results suggest that dry eyes might directly cause a depressive disorder that depends on the severity and duration of the ocular surface damage, and that voluntary motor activity could help recovery from a depressive state induced by dry eye.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3.

17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

Pleiotropic effects of probenecid on three-dimensional cultures of prostate cancer cells.

AIMS: Chemoresistance remains a persistent challenge in advanced prostate cancer therapy. Probenecid reportedly inhibits multiple drug-efflux transporters; hence, it can be employed as a potential sensitizer for chemotherapy. In the present study, we evaluated the effects of probenecid on three-dimensional (3D)-cultures of prostate cancer cells. MAIN METHODS: Prostate cancer cell lines, 22Rv1 and PC-3 were cultured as multicellular tumor spheroids. The effects of probenecid were evaluated using the MTT assay for viability, microscopy for spheroid size, and soft agar colony formation assay for anchorage-independent growth. KEY FINDINGS: The 3D-cultured 22Rv1 cells were less sensitive to cisplatin and doxorubicin than two-dimensional (2D) cell culture. Co-administration of probenecid at a low (100 or 300 µM), but not high (500 µM), concentration increased the sensitivity to cisplatin or doxorubicin in 22Rv1 spheroids. Probenecid increased the expression of ABCG2, a multidrug resistance transporter, in a dose-dependent manner. Furthermore, treatment with probenecid alone reduced the growth of 22Rv1 spheroids. Conversely, probenecid inhibited spheroid compaction rather than growth inhibition in 3D-cultured PC-3 cells. Moreover, probenecid inhibited colony formation of 22Rv1 and PC-3 cells in soft agar, as well as downregulated focal adhesion kinase (FAK), a crucial factor in anchorage-independent growth. SIGNIFICANCE: In 3D-cultured prostate cancer cells, probenecid demonstrated pleiotropic effects such as chemosensitization, growth suppression, inhibition of spheroid compaction, and suppression of anchorage-independent growth. Elucidating the detailed mechanism underlying these probenecid actions could result in the identification of novel therapeutic targets toward the advanced prostate cancer.

Profiles of 5α-Reduced Androgens in Humans and Eels: 5α-Dihydrotestosterone and 11-Ketodihydrotestosterone Are Active Androgens Produced in Eel Gonads.

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes (srd5a1, srd5a2a and srd5a2b) were cloned from eel gonads. All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arß were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17ß-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11ß-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

PNU-120596, a positive allosteric modulator of α7 nicotinic acetylcholine receptor, directly inhibits p38 MAPK.

PNU-120596 is a classical positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (α7 nAChR) and widely used to investigate the effect of α7 nAChR activation on several inflammation-associated diseases including rheumatoid arthritis, inflammatory bowel disease and cerebral ischemia. In this study, we report that PNU-120596 directly inhibits p38 mitogen-activated protein kinase (MAPK) activity. In 293A cells, p38 MAPK phosphorylation by several factors (oxidative stress, osmotic stress, TNF-α, or muscarinic stimulation) was inhibited by PNU-120596 as well as p38 MAPK inhibitor BIRB-796. Inhibition of p38 MAPK phosphorylation by PNU-120596 was not affected by α7 nAChR antagonist, methyllycaconitine (MLA). In vitro kinase assay revealed that PNU-120596 directly inhibits p38α MAPK-induced activating transcription factor 2 (ATF2) phosphorylation. MKK6-induced phosphorylation of p38α MAPK was also inhibited by PNU-120596. Real-time monitoring of binding to p38α MAPK using fluoroprobe SKF-86002 showed quite rapid binding of PNU-120596 compared to BIRB-796 which is known as a slow binder. Finally, we showed that PNU-120596 suppressed LPS-induced phosphorylation of p38 MAPK and expression of inflammatory factors including TNF-α, IL-6 and COX-2, independent on α7 nAChR activity in microglial cell BV-2. Thus, PNU-120596 might exert an anti-inflammatory effect through not only α7 nAChR potentiation but also direct inhibition of p38 MAPK.

AR420626, a selective agonist of GPR41/FFA3, suppresses growth of hepatocellular carcinoma cells by inducing apoptosis via HDAC inhibition.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide and establishment of new chemotherapies for HCC is urgently needed. GPR41 [free fatty acid receptor 3 (FFA3)] is a G protein-coupled receptor for short chain fatty acids, including acetate, propionate, and butyrate. In our previous study, we showed that propionate enhances the cytotoxic effect of cisplatin in HCC cells and that this mechanism is dependent on inhibition of histone deacetylases (HDACs) via GPR41/FFA3. However, the antitumor action of GPR41/FFA3 has not been elucidated. METHODS: In this study, we examined AR420626 as a GPR41-selective agonist in HepG2 and HLE cells. Nude mice were used for HepG2 xenograft studies. The apoptotic effect of AR420626 was evaluated using flow cytometry analysis. Expression of apoptosis-related proteins and HDACs was evaluated by Western immunoblot. Gene silencing of HDAC 3/5/7 and GPR41 was performed using small interfering RNA. Expression of TNF-α mRNA was evaluated by TaqMan real-time polymerase chain reaction. RESULTS: We found that AR420626, a selective GPR41/FFA3 agonist, suppressed growth of HepG2 xenografts and inhibited proliferation of HCC cells by inducing apoptosis. AR420626 induced proteasome activation through mTOR phosphorylation, which reduced HDAC proteins, and then increased expression of TNF-α. CONCLUSION: AR420626, a selective GPR41/FFA3 agonist, may be a candidate as a therapeutic agent for HCC.

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways.

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2-/- and FFA3-/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.

Evaluation of 17ß-hydroxysteroid dehydrogenase activity using androgen receptor-mediated transactivation.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17ß-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17ß-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17ß-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17ß-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.

ß-Hydroxybutyrate enhances the cytotoxic effect of cisplatin via the inhibition of HDAC/survivin axis in human hepatocellular carcinoma cells.

Ketone bodies, including acetoacetate and ß-hydroxybutyrate (ßOHB), are produced from acetyl coenzyme A in the liver and then secreted into the blood. These molecules are a source of energy for peripheral tissues during exercise or fasting. ßOHB has been reported to inhibit histone deacetylases (HDACs) 1, 3, and 4 in human embryonic kidney 293 cells. Thus, ßOHB may regulate epigenetics by modulating HDACs. There have been several reports that the administration of ßOHB or induction of a physiological state of ketosis has an antitumor effect; however, the mechanism remains unclear. The aim of this study was to investigate whether ßOHB enhances cisplatin-induced apoptosis in hepatocellular carcinoma (HCC) cells by modulating activity and/or expression of HDACs. We found that ßOHB significantly enhanced cisplatin-induced apoptosis and cleavage of caspase-3 and -8 in HCC cells. Further, ßOHB significantly decreased the expression of HDCA 3/5/6 and survivin in liver hepatocellular (HepG2) cells. In HDAC3/6 gene silencing, survivin expression was significantly decreased, and cisplatin-induced cleavage of caspase-3 was significantly enhanced compared with control in HepG2 cells. In conclusion, ßOHB enhanced cisplatin-induced apoptosis via HDAC3/6 inhibition/survivin axis in HepG2 cells, which suggests that ßOHB could be a new adjuvant agent for cisplatin chemotherapy.

Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Transcriptional Regulation of Ovarian Steroidogenic Genes: Recent Findings Obtained from Stem Cell-Derived Steroidogenic Cells.

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein ß to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein ß (C/EBPß) with cAMP-stimulation. C/EBPß expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPß via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

Novel regulatory systems for acetylcholine release in rat striatum and anti-Alzheimer's disease drugs.

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 µM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

ß-Hydroxybutyrate, a ketone body, reduces the cytotoxic effect of cisplatin via activation of HDAC5 in human renal cortical epithelial cells.

AIMS: ß-Hydroxybutyrate (ßOHB) is a metabolic intermediate that constitutes about 70% of ketone bodies produced in liver from oxidation of fatty acids released from adipose tissue. A recent study showed that ßOHB inhibits HDAC1, 3 and 4 (classes I and IIa) in human embryonic kidney 293 (HEK293) cells. Therefore, ßOHB could regulate epigenetics via modulating HDACs. However, little is known about the protective effect of ßOHB on renal cells through epigenetics. The aim of this study is to investigate whether ßOHB reduces cisplatin-induced nephrotoxicity in human renal cortical epithelial (HRCE) cells by modulating HDACs. MAIN METHODS: In this study, we used human renal cortical epithelial (HRCE) cells. The anti-apoptotic effect of ßOHB was evaluated using flow cytometry analysis. The expression of apoptosis-related proteins and HDACs was evaluated by western immunoblot. KEY FINDINGS: The results showed that ßOHB significantly reduced cisplatin-induced apoptosis in HRCE cells. Furthermore, ßOHB significantly reduced cisplatin-induced cleavage of caspase-3, acetylation of histone H3, and phosphorylation of AMP-activated kinase. This anti-apoptotic effect of ßOHB was markedly attenuated by an inhibitor of HDAC4/5, and ßOHB-mediated suppression of cleavage of caspase3 was significantly blocked by siRNA-induced gene silencing of HDAC5. SIGNIFICANCE: ßOHB attenuates cisplatin-induced apoptosis by activation of HDAC5 in HRCE cells, suggesting that ßOHB may be a new therapeutic agent for cisplatin nephropathy.