Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1–10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and “on line” radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.     

Altered burst swimming in rainbow trout Oncorhynchus mykiss exposed to natural and synthetic oestrogens

Juvenile rainbow trout Oncorhynchus mykiss were exposed to two concentrations each of 17β‐oestradiol (E2; natural oestrogen hormone) or 17α‐ethinyl oestradiol (EE2; a potent synthetic oestrogen hormone) to evaluate their potential effects on burst‐swimming performance. In each of six successive burst‐swimming assays, burst‐swimming speed (U_burst) was lower in fish exposed to 0·5 and 1 µg l^?1 E2 and EE2 for four days compared with control fish. A practice swim (2 days prior to exposure initiation) in control fish elevated initial U_burst values, but this training effect was not evident in the 1 µg l^?1 EE2‐exposed fish. Several potential oestrogen‐mediated mechanisms for U_burst reductions were investigated, including effects on metabolic products, osmoregulation and blood oxygen‐carrying capacity. Prior to burst‐swimming trials, fish exposed to E2 and EE2 for 4 days had significantly reduced erythrocyte numbers and lower plasma glucose concentrations. After six repeated burst‐swimming trials, plasma glucose, lactate and creatinine concentrations were not significantly different among treatment groups; however, plasma Cl^? concentrations were significantly reduced in E2‐ and EE2‐treated fish. In summary, E2 and EE2 exposure altered oxygen‐carrying capacity ([erythrocytes]) and an osmoregulatory‐related variable ([Cl^?]), effects that may underlie reductions in burst‐swimming speed, which will have implications for fish performance in the wild.     

Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin‐1β

The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density. Few studies have investigated promoting corneal endothelial cell proliferation to increase the endothelial cell density. We hypothesize that pre‐transplantation treatment of proliferative tissue‐cultivated corneas may increase corneal endothelial cell density. We observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness. In this tissue culture system, we observed that lysophosphatidic acid increased the rabbit corneal endothelial cell density, number of BrdU‐positive cells and improve wound healing. We also observed an indirect effect of lysophosphatidic acid on corneal endothelial cell proliferation mediated by the stimulation of interleukin‐1β secretion from stromal cells. Human corneal tissues treated with lysophosphatidic acid or interleukin‐1β contained significantly more Ki‐67‐positive cells than untreated group. The lysophosphatidic acid‐ or interleukin‐1β‐treated cultured tissue remained hexagon‐shaped, with ZO‐1 expression and no evidence of the endothelial‐mesenchymal transition. Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Seasonal changes in gene expression of steroidogenic enzymes,androgen and estrogen receptors in frog testis

The anuran amphibian Pelophylax esculentus shows an annual cycle of sexual steroid production and spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we investigated the mRNA expression of key enzymes involved in the androgenic and oestrogenic biosynthesis pathways in the testis of frogs taken in the reproductive and postreproductive period. Furthermore, we also analysed androgen and oestrogen levels and their own receptor gene expressions. Our findings showed that during the reproductive period, 3β‐hydroxysteroid dehydrogenase, 17β‐hydroxysteroid dehydrogenase and 5α‐reductase mRNA levels were higher than those during the postreproductive period. High testosterone and 5α‐dihydrotestosterone titres as well as the expression levels of androgen receptors in the reproductive testis strongly confirmed that the androgenic pathway is necessary for spermatogenesis activation. Conversely, during the postreproductive period, the highest P450 aromatase, estrogen receptor α and β mRNA levels, paralleling with oestradiol titres, indicated that the oestrogenic pathway is essential for the interruption of the reproductive processes. Our findings demonstrated, for the first time in amphibians, that testicular endocrine cyclic activity could be modulated by the up‐regulation of key steroidogenic enzyme gene expressions. This in turn determines the activation of the androgenic pathway in reproductive phase and the oestrogenic one in postreproductive phase.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11β‐HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C‐terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K_m values for steroids and an unaltered or increased k_cat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild‐type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.     

Th17 can regulate silica‐induced lung inflammation through an IL‐1β‐dependent mechanism

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)‐1β is induced by silica and functions as the key pro‐inflammatory cytokine in this process. The Th17 response, which is induced by IL‐1β, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL‐1β and IL‐17 in silicosis, we used anakinra and an anti‐IL‐17 monoclonal antibody (mAb) to block the receptor of IL‐1β (IL‐RI) and IL‐17, respectively, in a mouse model of silicosis. We observed increased IL‐1β expression and an enhanced Th17 response after silica instillation. Treatment with an IL‐1 type I receptor (IL‐1RI) antagonist anakinra substantially decreased silica‐induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL‐17‐neutralized silicosis group. IL‐17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL‐22 and IL‐1β during the lung inflammation of silicosis. Silica may induce IL‐1β production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL‐1β/IL‐1RI‐dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL‐22 and IL‐1β. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.