BBA - Molecular Basis of Disease (v.1802, #2)

Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus by Juan Carlos Molero; Scott Lee; Ilit Leizerman; Ayelet Chajut; Adrian Cooper; Ken Walder (235-239).
Objective: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic Psammomys obesus using novel electron microscopy technologies. Background: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle. Methods: Assessment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy X-ray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemius muscle. Results: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls. Conclusions: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.
Keywords: Intramyocellular lipid; Rosiglitazone; Thiazolidinedione; Psammomys obesus;

Sensitization to alloxan-induced diabetes and pancreatic cell apoptosis in acatalasemic mice by Yoko Kikumoto; Hitoshi Sugiyama; Tatsuyuki Inoue; Hiroshi Morinaga; Keiichi Takiue; Masashi Kitagawa; Naomi Fukuoka; Mizuho Saeki; Yohei Maeshima; Da-Hong Wang; Keiki Ogino; Noriyoshi Masuoka; Hirofumi Makino (240-246).
Human acatalasemia may be a risk factor for the development of diabetes mellitus. However, the mechanism by which diabetes is induced is still poorly understood. The impact of catalase deficiency on the onset of diabetes has been studied in homozygous acatalasemic mutant mice or control wild-type mice by intraperitoneal injection of diabetogenic alloxan. The incidence of diabetes was higher in acatalasemic mice treated with a high dose (180 mg/kg body weight) of alloxan. A higher dose of alloxan accelerated severe atrophy of pancreatic islets and induced pancreatic β cell apoptosis in acatalasemic mice in comparison to wild-type mice. Catalase activity remained low in the acatalasemic pancreas without the significant compensatory up-regulation of glutathione peroxidase or superoxide dismutase. Furthermore, daily intraperitoneal injection of angiotensin II type 1 (AT1) receptor antagonist telmisartan (0.1 mg/kg body weight) prevented the development of alloxan-induced hyperglycemia in acatalasemic mice. This study suggests that catalase plays a crucial role in the defense against oxidative-stress-mediated pancreatic β cell death in an alloxan-induced diabetes mouse model. Treatment with telmisartan may prevent the onset of alloxan-induced diabetes even under acatalasemic conditions.
Keywords: Acatalasemia; Diabetes mellitus; Oxidative stress; Catalase; Apoptosis; Angiotensin II; Receptor antagonist;

Functional studies of new GLA gene mutations leading to conformational fabry disease by C. Filoni; A. Caciotti; L. Carraresi; C. Cavicchi; R. Parini; D. Antuzzi; A. Zampetti; S. Feriozzi; P. Poisetti; S.C. Garman; R. Guerrini; E. Zammarchi; M.A. Donati; A. Morrone (247-252).
Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of α-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants in FD patients which lead to α-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations, c.155G > A (p.C52Y), c.548G > C (p.G183A), c.647A > G (p.Y216C) in as many individuals (two male; one female) and performed in vitro expression studies and Western blot analysis in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When mutant proteins overexpressed in COS-1 cells and in patients' lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients, but it is not always possible to issue the enzyme's active form in all involved organs. Our study endorses the hypothesis that an active site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.
Keywords: α-Galactosidase; 1-Deoxygalactonojirimicin; Chaperone; Fabry disease;

Mutations alter secretion of fukutin-related protein by Pei J. Lu; Allen Zillmer; XiaoHua Wu; Hanns Lochmuller; Judy Vachris; Derek Blake; Yiumo Michael Chan; Qi L. Lu (253-258).
Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker–Warburg syndrome, muscle–eye–brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases.
Keywords: FKRP mutation; LGMD2I; Secretion; Glycosylation; Read-through;

Vacuolization and alterations of lysosomal membrane proteins in cochlear marginal cells contribute to hearing loss in neuraminidase 1-deficient mice by Xudong Wu; Katherine A. Steigelman; Erik Bonten; Huimin Hu; Wenxuan He; Tianying Ren; Jian Zuo; Alessandra d’Azzo (259-268).
The neuraminidase-1 (Neu1) knockout mouse model is a phenocopy of the lysosomal storage disease (LSD) sialidosis, characterized by multisystemic and neuropathic symptoms, including hearing loss. We have characterized the auditory defects in Neu1 / mice and found that hearing loss involves both conductive and sensorineural components. Auditory brainstem response (ABR) thresholds were significantly elevated in Neu1 / mice at P21 (48   –55 dB), and hearing loss appeared progressive (53   –66 dB at P60). At these ages Neu1 / mice accumulated cerumen in the external ear canal and had a thickened mucosa and inflammation in the middle ear. In cochleae of adult wild-type mice, Neu1 was expressed in several cell types in the stria vascularis, the organ of Corti, and spiral ganglion. Progressive morphological abnormalities such as extensive vacuolization were detected in the Neu1 / cochleae as early as P9. These early morphologic changes in Neu1 / cochleae were associated with oversialylation of several lysosomal associated membrane proteins (Lamps) in the stria vascularis. A marked increase in the expression and apical localization of Lamp-1 in marginal cells of the stria vascularis predicts exacerbation of lysosomal exocytosis into the endolymph. Consequently, the endolymphatic potential in Neu1 / mice was reduced by approximately 20 mV at ages P31–P44, which would cause dysfunction of transduction in sensory hair cells. This study suggests a molecular mechanism that contributes to hearing loss in sialidosis and identifies potential therapeutic targets.
Keywords: Cochlea; Sialidosis; Hearing loss; Lysosomes; Endolymphatic potential;

Quantitative in vitro and in vivo characterization of the human P32T mutant ITPase by Greg Herting; Katie Barber; Maria R. Zappala; Richard P. Cunningham; Nicholas E. Burgis (269-274).
Human ITPase, encoded by the ITPA gene, and its orthologs (RdgB in Escherichia coli and HAM1 in Saccharomyces cerevisiae) exclude noncanonical nucleoside triphosphates (NTPs) from NTP pools. Deoxyinosine triphosphate (dITP) and 2′-deoxy-N-6-hydroxylaminopurine triphosphate are both hydrolyzed by ITPase to yield the corresponding deoxynucleoside monophosphate and pyrophosphate. In addition, metabolites of thiopurine drugs such as azathioprine have been shown to be substrates for ITPase. The ITPA 94C>A [P32T] variant is one of two polymorphisms associated with decreased ITPase activity. Furthermore, the ITPA 94C>A [P32T] variant is associated with an increased risk of adverse drug reactions for patients treated with azathioprine. The nature of the observed phenotypes for ITPA 94C>A [P32T] variant individuals is currently unclear. Our biochemical assays indicate the P32T ITPase has 55% activity with dITP compared to wild-type ITPase. Complementation experiments at 37 °C show that N-6-hydroxylaminopurine sensitivity of E. coli rdgB mutants is reduced with a plasmid bearing the ITPA 94C>A [P32T] gene approximately 50% less than with a plasmid bearing the wild-type ITPA gene. The reduction in sensitivity is less at 42 °C. Experiments with synthetic lethal E. coli recA(ts) rdgB mutants show that the ITPA 94C>A [P32T] gene also complements the recA(ts) rdgB growth deficiency at 42 °C approximately 40% lower than wild-type ITPA gene. Western blot analysis indicates that the expression level of P32T ITPase is reduced in these cells relative to wild type. Our data support the idea that P32T ITPase is a functional protein, albeit with a reduced rate of noncanonical NTP pyrophosphohydrolase activity and reduced protein stability.
Keywords: ITPA; ITPase; RdgB; Noncanonical purines; dITP; N-6-hydroxylaminopurine;

A suggested role for mitochondria in Noonan syndrome by Icksoo Lee; Alena Pecinova; Petr Pecina; Benjamin G. Neel; Toshiyuki Araki; Raju Kucherlapati; Amy E. Roberts; Maik Hüttemann (275-283).
Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.
Keywords: Cardio-facio-cutaneous syndrome; Cytochrome c oxidase; Noonan syndrome; Mitochondria; Oxidative phosphorylation; PTPN11; Reactive oxygen species; SHP2;

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation by Sanjay K. Banerjee; Kenneth R. McGaffin; Xueyin N. Huang; Ferhaan Ahmad (284-291).
Human mutations in PRKAG2, the gene encoding the γ2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TGT400N), we previously reported initial cardiac hypertrophy (ages 2–8 weeks) followed by dilation and failure (ages 12–20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TGT400N mice showed significantly increased cardiac mass/body mass ratios up to ∼ 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were ∼ 2- and ∼ 5-fold higher, respectively, in TGT400N relative to wildtype (WT) mice at age 2 weeks. NF-κB activity and nuclear translocation of the p50 subunit were increased ∼ 2- to 3-fold in TGT400N hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated ∼ 2-fold as early as age 2 weeks. To ascertain whether these changes in TGT400N mice were a consequence of increased AMPK activity, we crossbred TGT400N with TGα2DN mice, which express a dominant negative, kinase dead mutant of the AMPK α2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-κB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-κB and Akt signaling pathway, which mediates cardiac hypertrophy.
Keywords: AMPK; Hypertrophy; AKT; NF-κB; Transgenic Mouse; Heart;

An intronic polymorphism of IRF4 gene influences gene transcription in vitro and shows a risk association with childhood acute lymphoblastic leukemia in males by Thuy N. Do; Esma Ucisik-Akkaya; Charronne F. Davis; Brittany A. Morrison; M. Tevfik Dorak (292-300).
The interferon regulatory factor (IRF) family of DNA-binding proteins regulates expression of interferon-inducible genes with roles in the immune response and carcinogenesis. IRF4 is involved in the differentiation of B and T cells and is overexpressed in B-cell malignancies as a result of c-REL (NF-κB) hyperactivation. IRF4 polymorphisms are associated with susceptibility to chronic lymphoid leukemia (CLL) and non-Hodgkin lymphoma (NHL). We examined 13 IRF4 SNPs in 114 cases of childhood acute lymphoblastic leukemia (ALL) and 388 newborn controls from Wales (U.K.) using TaqMan assays. IRF4 intron 4 SNP rs12203592 showed a male-specific risk association (OR = 4.4, 95% CI = 1.5 to 12.6, P  = 0.007). Functional consequences of the C > T substitution at this SNP were assessed by cell-based reporter assays using three different cell lines. We found a repressive effect of the rs12203592 wildtype allele C on IRF4 promoter activity (P  < 0.001) but no repression by the variant allele in any cell line tested. Thus, homozygosity for the rs12203592 variant allele would result in increased IRF4 expression. This increase would be compounded by high levels of NF-κB activity in males due to the absence of estrogen. IRF4 differs from other IRFs in its anti-interferon activity which interferes with immune surveillance. We propose that a detailed study of IRF4 can provide information on the mechanism of the sex effect and the role of immune surveillance in childhood ALL development.
Keywords: Genetic Predisposition to Disease; Lymphoblastic Leukemia, Acute, Childhood; Interferon Regulatory Factor 4; Polymorphism, Single Nucleotide; Luciferase Reporter Assay; Sex Effect;

δEF1 promotes breast cancer cell proliferation through down-regulating p21 expression by Fen Hu; Chuan Wang; Jun Du; Wei Sun; Jidong Yan; Dong Mi; Jie Zhang; Yuhuan Qiao; Tianhui Zhu; Shuang Yang (301-312).
Although the zinc finger-homeodomain transcription factor δEF1 is implied as a regulatory factor at the crossroad between proliferation and differentiation in carcinogenesis, its potential effect in the regulation of cell cycle progression has not been well elucidated. In our present study, we provide novel finding that, in breast cancer, the ectopic expression of δEF1 in MDA-MB-231 cells significantly promoted cell proliferation by increasing the cell number in S phase of the cell cycle. In contrast, δEF1 knockdown by RNA interference exhibited an opposite effect, highlighting a potent role of δEF1 to promote G1-S transition of breast cancer cells. Moreover, we demonstrated that δEF1 down-regulated p21 and concurrently up-regulated the expressions of CDK2 and CDK4 during this process. Further, δEF1 inhibited p21 transcription by recruiting to the E2 box element on the p21 promoter. Depletion of endogenous δEF1 in MDA-MB-231 cells was sufficient to allow an inherent release of p21 expression, thus resulting in the cell cycle arrest. In addition, the stimulatory effect of δEF1 on cell proliferation through p21 regulation was supported by an inverse correlation of δEF1 and p21 expressions observed in both breast cancer cell lines and clinical tumor specimens. Taken together, these observations suggest a dual effect of δEF1 in promoting breast cancer cell proliferation, by differentially regulating the cell cycle regulatory proteins.
Keywords: δEF1; p21; CDK; Cell cycle; Breast cancer proliferation;