SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na+ conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 mu M) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na+ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Prospective Evaluation of Clinical and Ultrasound Findings in Ankle Disease in Juvenile Idiopathic Arthritis: Importance of Ankle Ultrasound

Objective. To prospectively compare clinical examination of the ankle structures with ultrasound (US) findings. Methods. In 42 children with juvenile idiopathic arthritis (JIA; 25 girls, 17 boys, mean age 11.3 yrs, range 2.3–22.3 yrs), a total of 61 swollen/painful ankles were assessed clinically and ultrasonographically. Accurate clinical examination of the entire ankle joint was performed, focusing especially on 3 regions — tibiotalar joint and medial and lateral tendons. Clinical and US findings were both scored 0–3 (normal-severe). Results. US demonstrated no signs of tibiotalar joint effusion in 14 out of 43 ankles considered clinically involved. For the medial tendons, US showed tenosynovitis in 13 ankles out of 31 thought to be clinically normal; and for the lateral tendons, of the 19 deemed to be clinically involved, less than 50% had involvement on US. Very poor agreement was observed comparing the clinical and US scores for the 3 regions: tibiotalar joint, kappa = 0.3; medial tendons, kappa = 0.24; lateral tendons, kappa = 0.25. With regard to other ankle structures, only 39% of the subtalar (talocalcaneal) joints considered clinically involved were deemed abnormal on US. Finally, of the 10 ankles with talonavicular US effusion, only 2 were considered clinically involved. Conclusion. Using US findings as the “gold standard,” clinical examination of the ankle in children with JIA was found to be inadequate in identifying the structures involved. US assessment prior to any glucocorticoid injection should be considered to improve the outcome. A prospective study comparing the outcome following clinical- versus US-guided ankle joint injection should be undertaken, to confirm our findings.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy. © FASEB.

An in vitro investigation of endocrine disrupting effects of tricothecenes deoxynivalenol (DON), T-2, HT-2 toxins

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Preterm cerebellar growth impairment after postnatal exposure to glucocorticoids

As survival rates of preterm newborns improve as a result of better medical management, these children increasingly show impaired cognition. These adverse cognitive outcomes are associated with decreases in the volume of the cerebellum. Because animals exhibit reduced preterm cerebellar growth after perinatal exposure to glucocorticoids, we sought to determine whether glucocorticoid exposure and other modifiable factors increased the risk for these adverse outcomes in human neonates. We studied 172 preterm neonatal infants from two medical centers, the University of British Columbia and the University of California, San Francisco, by performing serial magnetic resonance imaging examinations near birth and again near term-equivalent age. After we adjusted for associated clinical factors, antenatal betamethasone was not associated with changes in cerebellar volume. Postnatal exposure to clinically routine doses of hydrocortisone or dexamethasone was associated with impaired cerebellar, but not cerebral, growth. Alterations in treatment after preterm birth, particularly glucocorticoid exposure, may help to decrease risk for adverse neurological outcome after preterm birth.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

Endocrine disruptor activity in bottled mineral and flavoured water

A panel of reporter gene assays (RGAs) coupled with a single solid phase extraction (SPE) step was developed and used to screen bottled mineral water for the presence of four classes of endocrine disruptors (EDs), oestrogens, androgens, progestagens and glucocorticoids.

Fourteen brands of bottled mineral water in triplicate (42 samples) were analysed. Overall, hormonal activity was found in 78% of the samples. Oestrogenic, androgenic, progestagenic and glucocorticoid activity was found in 38%, 38%, 36% and 55% of the samples, respectively at an average concentration of 10 ng/l 17 beta-estradiol equivalent (EEQ), 26 ng/l testosterone equivalent (TEQ), 123 ng/l progesterone equivalent (PEQ) and 13.5 ng/l hydrocortisone equivalent (HEQ).

The level of oestrogenic, androgenic and progestagenic activity observed is not considered a matter of concern for the consumers' health. It is unknown whether the glucocorticoid levels observed are safe. The ED source, long term exposure and mixture effects remain to be investigated. (C) 2012 Elsevier Ltd. All rights reserved.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

Interventions to prevent steroid-induced osteoporosis and osteoporotic fractures in Duchenne muscular dystrophy (Protocol)

This is the protocol for a review and there is no abstract. The objectives are as follows:

Primary objective: To assess the effects of interventions to delay or treat osteoporosis in DMD patients treated with glucocorticoid steroids.

Secondary objectives: To assess the effects of interventions to delay or treat osteoporosis in DMD on the frequency of vertebral fragility fractures and long bone fractures in DMD patients treated with glucocorticoid steroids.

Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Differential regulations of AQP4 and Kir4.1 by triamcinolone acetonide and dexamethasone in the healthy and inflamed retina.

PURPOSE: Glucocorticoids are used to treat macular edema, although the mechanisms underlying this effect remain largely unknown. The authors have evaluated in the normal and endotoxin-induced uveitis (EIU) rats, the effects of dexamethasone (dex) and triamcinolone acetonide (TA) on potassium channel Kir4.1 and aquaporin-4 (AQP4), the two main retinal Müller glial (RMG) channels controlling retinal fluid movement. METHODS: Clinical as well as relatively low doses of dex and TA were injected in the vitreous of normal rats to evaluate their influence on Kir4.1 and AQP4 expression 24 hours later. The dose-dependent effects of the two glucocorticoids were investigated using rat neuroretinal organotypic cultures. EIU was induced by footpad lipopolysaccharide injection, without or with 100 nM intraocular dex or TA. Glucocorticoid receptor and channel expression levels were measured by quantitative PCR, Western blot, and immunohistochemistry. RESULTS: The authors found that dex and TA exert distinct and specific channel regulations at 24 hours after intravitreous injection. Dex selectively upregulated Kir4.1 (not AQP4) in healthy and inflamed retinas, whereas TA induced AQP4 (not Kir4.1) downregulation in normal retina and upregulation in EIU. The lower concentration (100 nM) efficiently regulated the channels. Moreover, in EIU, an inflammatory condition, the glucocorticoid receptor was downregulated in the retina, which was prevented by intravitreous injections of the low concentration of dex or TA. CONCLUSIONS: The results show that dex and TA are far from being equivalent to modulate RMG channels. Furthermore, the authors suggest that low doses of glucocorticoids may have antiedematous effects on the retina with reduced toxicity.

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

The role of teriparatide in sequential and combination therapy of osteoporosis.

Osteoporosis is complicated by the occurrence of fragility fractures. Over past years, various treatment options have become available, mostly potent antiresorptive agents such as bisphosphonates and denosumab. However, antiresorptive therapy cannot fully and rapidly restore bone mass and structure that has been lost because of increased remodelling. Alternatively recombinant human parathyroid hormone (rhPTH) analogues do increase the formation of new bone material. The bone formation stimulated by intermittent PTH analogues not only increases bone mineral density (BMD) and bone mass but also improves the microarchitecture of the skeleton, thereby reducing incidence of vertebral and nonvertebral fractures. Teriparatide, a recombinant human PTH fragment available in Switzerland, is reimbursed as second-line treatment in postmenopausal women and men with increased fracture risk, specifically in patients with incident fractures under antiresorptive therapy or patients with glucocorticoid-induced osteoporosis and intolerance to antiresorptives. This position paper focuses on practical aspects in the management of patients on teriparatide treatment. Potential first-line indications for osteoanabolic treatment as well as the benefits and limitations of sequential and combination therapy with antiresorptive drugs are discussed.