Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Genetically modified mouse models to investigate thyroid development, function and growth.

The thyroid gland produces thyroid hormones (TH), which are essential regulators for growth, development and metabolism. The thyroid is mainly controlled by the thyroid-stimulating hormone (TSH) that binds to its receptor (TSHR) on thyrocytes and mediates its action via different G protein-mediated signaling pathways. TSH primarily activates the Gs-pathway, and at higher concentrations also the Gq/11-pathway, leading to an increase of intracellular cAMP and Ca2+, respectively. To date, the physiological importance of other G protein-mediated signaling pathways in thyrocytes is unclear. Congenital hypothyroidism (CH) is defined as the lack of TH at birth. In familial cases, high-throughput sequencing methods have facilitated the identification of novel mutations. Nevertheless, the precise etiology of CH yet remains unraveled in a proportion of cases. Genetically modified mouse models can reveal new pathophysiological mechanisms of thyroid diseases. Here, we will present an overview of genetic mouse models for thyroid diseases, which have provided crucial insights into thyroid gland development, function, and growth with a special focus on TSHR and microRNA signaling.

Thyrocyte-specific Dicer1 deficiency alters thyroid follicular organization and prevents goiter development.

MicroRNAs (miRNAs) are important regulators of posttranscriptional gene expression and involved in embryonic development, regulation of cell differentiation, and growth. Dicer1 is a key enzyme in the maturation process of functional miRNAs. However, miRNA-mediated regulation of normal thyroid function and growth is largely unknown. To understand the role of miRNAs in the thyroid, we generated constitutive and tamoxifen-inducible, thyrocyte-specific Dicer1 knockout mice. The mice with perinatal Dicer1 deletion (cTgDcrKO) showed impaired follicular organization, increased fibrosis, and accumulation of adipocytes in the thyroid. Similar histological changes were observed in tamoxifen-induced adult Dicer1-deficient mice (iTgDcrKO). The thyroid phenotype in both knockout (KO) lines was associated with significantly down-regulated mRNA expression of thyroid transcription factor 1 (Ttf-1/Nkx2-1), thyroid peroxidase, and thyroglobulin (Tg) and up-regulated expression of genes involved in Tgf-ß signaling. Furthermore, in cTgDcrKO mice, which developed mild hypothyroidism, the protein expression of Nkx2-1, thyroglobulin, Paired box 8, and TSH receptor were clearly down-regulated compared with controls. Despite similar down-regulation of Dicer1 in cTgDcrKO and iTgDcrKO compared with controls, Dicer1 deletion in adult mice thyrocytes did not lead to acute hypothyroidism. No significant differences in thyroid weights between cTgDcrKO, iTgDcrKO, and controls were observed. However, a goitrogenic diet induced a 4-fold increase in thyroid weight in control animals, whereas it had no effect on iTgDcrKO thyroids. In summary, Dicer1 deficiency in thyrocytes is associated with intrathyroid fibrosis, adipogenesis, and enhanced expression of Tgf-ß signaling genes. Furthermore, our data indicate that Dicer1 is required for thyroid follicular organization, thyrocyte differentiation, and goiter development.

Sphingosine kinase as an oncogene: autocrine sphingosine 1-phosphate modulates ML-1 thyroid carcinoma cell migration by a mechanism dependent on protein kinase C-alpha and ERK1/2.

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Health-related quality of life in adult survivors after paediatric allo-SCT.

Fifty-three adults who had received SCT as children responded to questionnaires on health-related quality of life (HRQoL) (Swedish HRQoL survey (SWED-QUAL)), sense of coherence (SOC), anxiety and depression (HAD) and a health and symptom inventory. Late effects were classified following the Common Terminology Criteria for Adverse Events (CTCAE) v. 3.0. HRQoL was below norm in 9 of 13 SWED-QUAL domains. Poorest domains (P<0.001) were satisfaction with physical health, general health, partner relations and sexual function, whereas emotional wellbeing and satisfaction with family life were on par with the norm. Older age, time elapsed post-SCT and fewer self-reported symptoms correlated with better HRQoL. Unfavourable late effect scores had no or limited impact, whereas age at SCT or TBI did not adversely affect HRQoL. Most subjects were well subjectively and objectively, whereas 24% had more complicated late effects. The median Karnofsky score was 90, 13% of subjects having scores below 80. In total, 53% reported pain, whereas 42.5% had memory and concentration problems. Anxiety and/or depression, reported by 35%, were associated with lower HRQoL and SOC ratings. Overall, 55% reported infertility and expressed difficulty with this. To conclude, childhood SCT did not negatively affect overall health for most of these adult long-term survivors, although poorer HRQoL with psychological and cognitive problems are common.

Quality of life and health in children following allogeneic SCT.

A total of 52 children, age 9 or over and at least 3 years (median=8) beyond SCT for leukaemia (n=32) or nonmalignant diseases, participated in a single-centre study of health and quality of life (QoL). QoL and self-esteem were assessed with SCHQ-CF87, a generic multidimensional self-report instrument, and with 'I think I am'. As a group, the children had good QoL, but were below norm in the bodily pain (P<0.05), general health and self-esteem dimensions (P<0.01). Lansky or Karnofsky function levels were at a median of 90. Sense of coherence (SOC-13) was normal and correlated with SCHQ-CF87. Most children were subjectively and objectively in good health according to a self-assessment symptom inventory or by a medical record-based scoring of late effects, although pain was commonly reported. A total of 25% of the patients were rated as having moderate to severe late effects, without considering cataracts or infertility. Neither age at SCT, gender, malignant vs nonmalignant disease, nor stature influenced QoL significantly. Children with moderate to severe chronic graft-versus-host disease or cognitive deficits had lower QoL in some dimensions. No correlation was, however, found between the physician-rated total late effects score and overall QoL. Contrarily, QoL was clearly related to the degree of self-rated symptoms.

Control of ureogenesis.

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made. Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium. The relationship between ornithine-cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near-physiological conditions the ornithine-cycle enzymes are far from being saturated with their subsidiaries. Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine. Activation of carbamoyl-phosphate synthetase (ammonia) by addition of N-carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine-cycle flux to the steady-state ammonia concentration was shifted to lower concentrations of ammonia. The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl-phosphate synthetase. It is concluded that in vivo the function of activity changes in carbamoyl-phosphate synthetase, via the well-documented alterations in the intramitochondrial concentration of N-acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl-phosphate synthetase.

Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene.

Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene. The Michaelis constants for NH3, MgATP and HCO-3 were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a Ka of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free Mg2+ ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mg2+ is due to an increased affinity of the enzyme for N-acetylglutamate. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process: at N-acetylglutamate concentrations below 0.5 mM maximal binding was not completed within 30 min. The rate of binding increased with increasing N-acetylglutamate concentrations. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte.

Fluorimetric determination of carbamoyl phosphate.

A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present.

Activity of carbamoyl-phosphate synthetase (ammonia) in isolated rat-liver mitochondria: cycling of carbamoyl phosphate in the absence of ornithine.

1. When NH3 was added to isolated rat-liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate. 2. Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mitochondria had been preincubated with NH3 and unlabelled bicarbonate, revealed that flux through carbamoyl-phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated. 3. It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non-enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl-phosphate synthetase.

Transport of N-acetylglutamate in rat-liver mitochondria.

The permeability properties of the rat-liver mitochondrial membrane for N-acetylglutamate, the activator of carbamoyl-phosphate synthetase (ammonia), were studied. 1. Transport of N-acetylglutamate into the mitochondria was only observed in partially or fully de-energized mitochondria and when the extramitochondrial concentration was unphysiologically high (in the mM range). However, even under these conditions the intramitochondrial concentration of N-acetylglutamate was much lower than that outside. 2. Mitochondrial N-acetylglutamate efflux only occurs when the mitochondria are in an energized state. At 25 degrees C, at an intramitochondrial N-acetylglutamate concentration of 0.7-1.0 nmol/mg protein, efflux proceeds at a rate of about 0.05 nmol X min-1 X mg protein-1. This is 10-fold lower than the maximal rate of N-acetylglutamate synthesis in the mitochondria. 3. Homologous exchange between intramitochondrial N-[14C]acetylglutamate and extramitochondrial unlabelled N-acetylglutamate could not be demonstrated. 4. It is concluded that transport of N-acetylglutamate in vivo is effectively unidirectional, out of the mitochondria. This behaviour is in accordance with the physiological requirement for efflux of N-acetylglutamate from the mitochondria in order to be degraded in the cytosol.