BBA - Molecular Basis of Disease (v.1812, #7)

Osteoblasts from a mandibuloacral dysplasia patient induce human blood precursors to differentiate into active osteoclasts by Sofia Avnet; Rosanna Pallotta; Francesca Perut; Nicola Baldini; Maria Gabriela Pittis; Anita Saponari; Enrico Lucarelli; Barbara Dozza; Tiziana Greggi; Nadir M. Maraldi; Cristina Capanni; Elisabetta Mattioli; Marta Columbaro; Giovanna Lattanzi (711-718).
Mandibuloacral dysplasia type A (MADA) is a rare disease caused by mutations in the LMNA gene encoding A type lamins. Patients affected by mandibuloacral dysplasia type A suffer from partial lipodystrophy, skin abnormalities and accelerated aging. Typical of mandibuloacral dysplasia type A is also bone resorption at defined districts including terminal phalanges, mandible and clavicles. Little is known about the biological mechanism underlying osteolysis in mandibuloacral dysplasia type A. In the reported study, we analyzed an osteoblast primary culture derived from the cervical vertebrae of a mandibuloacral dysplasia type A patient bearing the homozygous R527H LMNA mutation. Mandibuloacral dysplasia type A osteoblasts showed nuclear abnormalities typical of laminopathic cells, but they proliferated in culture and underwent differentiation upon stimulation with dexamethasone and beta-glycerophosphate. Differentiated osteoblasts showed proper production of bone mineral matrix until passage 8 in culture, suggesting a good differentiation activity. In order to evaluate whether mandibuloacral dysplasia type A osteoblast-derived factors affected osteoclast differentiation or activity, we used a conditioned medium from mandibuloacral dysplasia type A or control cultures to treat normal human peripheral blood monocytes and investigated whether they were induced to differentiate into osteoclasts. A higher osteoclast differentiation and matrix digestion rate was obtained in the presence of mandibuloacral dysplasia type A osteoblast medium with respect to normal osteoblast medium. Further, TGFbeta 2 and osteoprotegerin expression were enhanced in mandibuloacral dysplasia type A osteoblasts while the RANKL/osteoprotegerin ratio was diminished. Importantly, inhibition of TGFbeta 2 by a neutralizing antibody abolished the effect of mandibuloacral dysplasia type A conditioned medium on osteoclast differentiation. These data argue in favor of an altered bone turnover in mandibuloacral dysplasia type A, caused by upregulation of bone-derived stimulatory cytokines, which activate non-canonical differentiation stimuli. In this context, TGFbeta 2 appears as a major player in the osteolytic process that affects mandibuloacral dysplasia type A patients.► Mandibuloacral dysplasia type A (MADA) osteoblasts show nuclear abnormalities. ► MADA osteoblasts undergo proper differentiation and produce bone mineral matrix. ► Osteoprotegerin and TGFbeta 2 expression are increased in MADA osteoblasts. ► MADA osteoblasts show altered RANKL/osteoprotegerin ratio. ► MADA osteoblast medium favors osteoclast differentiation and activity. ► A non-canonical TGFbeta 2-mediated pathway triggers osteoclast activation in MADA.
Keywords: Mandibuloacral dysplasia; Lamin A/C; Osteoblast; Osteoclast; Osteoprotegerin; TGFbeta 2;

Hyperglycemia-mediated oxidative stress plays a crucial role in the progression of diabetic nephropathy. Hence, the present study was hypothesized to explore the renoprotective nature of resveratrol by assessing markers of oxidative stress, proinflammatory cytokines and antioxidant competence in streptozotocin–nicotinamide-induced diabetic rats. Oral administration of resveratrol to diabetic rats showed a significant normalization on the levels of creatinine clearance, plasma adiponectin, C-peptide and renal superoxide anion, hydroxyl radical, nitric oxide, TNF-α, IL-1β, IL-6 and NF-κB p65 subunit and activities of renal aspartate transaminase, alanine transaminase and alkaline phosphatase in comparison with diabetic rats. The altered activities of renal aldose reductase, sorbitol dehydrogenase and glyoxalase-I and elevated level of serum advanced glycation end products in diabetic rats were also reverted back to near normalcy. Further, resveratrol treatment revealed a significant improvement in superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase activities and vitamins C and E, and reduced glutathione levels, with a significant decline in lipid peroxides, hydroperoxides and protein carbonyls levels in diabetic kidneys. Similarly, mRNA and protein analyses substantiated that resveratrol treatment notably normalizes the renal expression of Nrf2/Keap1and its downstream regulatory proteins in the diabetic group of rats. Histological and ultrastructural observations also evidenced that resveratrol effectively protects the kidneys from hyperglycemia-mediated oxidative damage. These findings demonstrated the renoprotective nature of resveratrol by attenuating markers of oxidative stress in renal tissues of diabetic rats.►Resveratrol protects diabetic kidney from hyperglycemia-mediated oxidative stress. ►This effect is facilitated by normalization of renal proinflammatory cytokines. ►Resveratrol regulates antioxidant enzyme activities through Nrf2–Keap signaling. ►Resveratrol provides a new strategy for diabetic nephropathy treatment.
Keywords: Advanced glycation end products; Diabetic kidney; Nrf2/Keap1 signaling; Oxidative stress; Proinflammatory cytokines; Resveratrol;

Mis-splicing of Tau exon 10 in myotonic dystrophy type 1 is reproduced by overexpression of CELF2 but not by MBNL1 silencing by C.M. Dhaenens; H. Tran; M.-L. Frandemiche; C. Carpentier; S. Schraen-Maschke; A. Sistiaga; M. Goicoechea; S. Eddarkaoui; E. Van Brussels; H. Obriot; A. Labudeck; M.H. Gevaert; F. Fernandez-Gomez; N. Charlet-Berguerand; V. Deramecourt; C.A. Maurage; L. Buée; A. Lopez de Munain; B. Sablonnière; M.L. Caillet-Boudin; N. Sergeant (732-742).
Tau is the proteinaceous component of intraneuronal aggregates common to neurodegenerative diseases called Tauopathies, including myotonic dystrophy type 1. In myotonic dystrophy type 1, the presence of microtubule-associated protein Tau aggregates is associated with a mis-splicing of Tau. A toxic gain-of-function at the ribonucleic acid level is a major etiological factor responsible for the mis-splicing of several transcripts in myotonic dystrophy type 1. These are probably the consequence of a loss of muscleblind-like 1 (MBNL1) function or gain of CUGBP1 and ETR3-like factor 1 (CELF1) splicing function. Whether these two dysfunctions occur together or separately and whether all mis-splicing events in myotonic dystrophy type 1 brain result from one or both of these dysfunctions remains unknown. Here, we analyzed the splicing of Tau exons 2 and 10 in the brain of myotonic dystrophy type 1 patients. Two myotonic dystrophy type 1 patients showed a mis-splicing of exon 10 whereas exon 2-inclusion was reduced in all myotonic dystrophy type 1 patients. In order to determine the potential factors responsible for exon 10 mis-splicing, we studied the effect of the splicing factors muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1), CUGBP1 and ETR3-like factor 2 (CELF2), and CUGBP1 and ETR3-like factor 4 (CELF4) or a dominant-negative CUGBP1 and ETR-3 like factor (CELF) factor on Tau exon 10 splicing by ectopic expression or siRNA. Interestingly, the inclusion of Tau exon 10 is reduced by CUGBP1 and ETR3-like factor 2 (CELF2) whereas it is insensitive to the loss-of-function of muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1) gain-of-function, or a dominant-negative of CUGBP1 and ETR-3 like factor (CELF) factor. Moreover, we observed an increased expression of CUGBP1 and ETR3-like factor 2 (CELF2) only in the brain of myotonic dystrophy type 1 patients with a mis-splicing of exon 10. Taken together, our results indicate the occurrence of a mis-splicing event in myotonic dystrophy type 1 that is induced neither by a loss of muscleblind-like 1 (MBNL1) function nor by a gain of CUGBP1 and ETR3-like factor 1 (CELF1) function but is rather associated to CUGBP1 and ETR3-like factor 2 (CELF2) gain-of-function.Display Omitted► Herein, we showed a mis-splicing of Tau exon 10 in myotonic dystrophy brain. ► This implies a deregulation of the splicing factor CELF2. ► Implication of MBNL1 or CELF1 is ruled out. ► Our work demonstrated a deregulation of CELF2 in myotonic dystrophy brain.
Keywords: Myotonic dystrophy; Triplet expansion disease; Microtubule-associated protein Tau; Splicing; CELF splicing factor family; MBNL1;

Cardiac sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) promotes Ca2+ uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr17 and Ser16 after 20–30 min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca2+ overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca2+-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30 min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser67. The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30 min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser67, and PLB dephosphorylation in ischemia. Reperfusion for 5 min following 30 min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser67, and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca2+ overload in early reperfusion.► Ischemia induces phospholamban (PLB) dephosphorylation via calcineurin activation. ► The activated calcineurin induces activation of PKC-α and protein phosphatase 1. ► Ischemia-induced PLB dephosphorylation contributes to calcium overload in reperfusion.
Keywords: Phospholamban; Calcineurin; Ischemia; Calcium; Protein kinase C;

Systemic administration of high-molecular weight hyaluronan stimulates wound healing in genetically diabetic mice by Mariarosaria Galeano; Francesca Polito; Alessandra Bitto; Natasha Irrera; Giuseppe M. Campo; Angela Avenoso; Margherita Calò; Patrizia Lo Cascio; Letteria Minutoli; Mauro Barone; Francesco Squadrito; Domenica Altavilla (752-759).
Hyaluronic acid (HA), an essential component of the extracellular matrix, is an efficient space filler that maintains hydration, serves as a substrate for assembly of proteoglycans and is involved in wound healing. Although numerous pieces of evidence demonstrate beneficial effects in promoting wound healing in diabetes, a systemic approach has never been tested. We used an incisional wound healing model in genetically diabetic mice to test the effects of systemic injection of HA. Diabetic (n  = 56) and normoglycemic (n  = 56) mice were subjected to incision and randomized (8 groups of 7 animals each) to receive HA at different doses, 7.5, 15 and 30 mg/kg/i.p., or vehicle (0.9% NaCl solution) for 12 days. At the end of the experiment animals were sacrificed and skin wounds were excised for histological, biochemical and molecular analysis. Histology revealed that the most effective dose to improve wound repair and angiogenesis in diabetic mice was 30 mg/kg. Furthermore HA injection (30 mg/kg) improved the altered healing pattern in diabetic animals, increased skin remodeling proteins TGF-β and transglutaminase-II and restored the altered expression of cyclin B1/Cdc2 complex. Evaluation of skin from diabetic animals injected with HA revealed also an increase in HA content, suggesting that systemic injection may be able to restore the reduced intracellular HA pool of diabetic mice. Finally HA markedly improved skin mechanical properties. These promising results, if confirmed in a clinical setting, may improve the care and management of diabetic patients.► In this study we evaluated systemic HA injection in diabetic animals. ► HA improved healing activating the cell cycle machinery. ► HA modulated matrix remodeling through TGF-β1 and transglutaminase-II expression.
Keywords: Hyaluronic acid; Diabetic mice; Cyclins; Transglutaminase-II;

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome by Carlos Campos; Rodrigo Guzmán; Encarnación López-Fernández; Ángela Casado (760-768).
Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H2O2) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n  = 78), some of them taking levothyroxine for hypothyroidism (n  = 24), and in their healthy age-matched controls (n  = 65). We found that levels of AGEs, diTyr, H2O2 and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H2O2 and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.► Urinary AGEs, diTyr, H2O2 and NOx could be used as oxidative stress biomarkers in DS. ► Urinary levels of 8-OHdG, 15-F2t-IsoP and TBARS are not altered in DS. ► Correlations with age were positive for diTyr and negative for Cr, TBARS, 15-F2t-IsoP and NOx in DS. ► Renal impairment could occur in DS and Cr adjustment may bias the results. ► Renal impairment due to hypothyroidism may bias the results in DS.
Keywords: Down syndrome; Oxidative stress; Nitrosative stress; Biomarker; Hypothyroidism; Urine;

Heat-shock protein 27 (Hsp27) as a target of methylglyoxal in gastrointestinal cancer by Tomoko Oya-Ito; Yuji Naito; Tomohisa Takagi; Osamu Handa; Hirofumi Matsui; Masaki Yamada; Keisuke Shima; Toshikazu Yoshikawa (769-781).
The molecular mechanisms underlying the posttranslational modification of proteins in gastrointestinal cancer are still unknown. Here, we investigated the role of methylglyoxal modifications in gastrointestinal tumors. Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins. By using a monoclonal antibody to methylglyoxal-modified proteins, we found that murine heat-shock protein 25 and human heat-shock protein 27 were the major adducted proteins in rat gastric carcinoma mucosal cell line and human colon cancer cell line, respectively. Furthermore, we found that heat-shock protein 27 was modified by methylglyoxal in ascending colon and rectum of patients with cancer. However, methylglyoxal-modified heat-shock protein 25/heat-shock protein 27 was not detected in non cancerous cell lines or in normal subject. Matrix-associated laser desorption/ionization mass spectrometry/mass spectrometry analysis of peptide fragments identified Arg-75, Arg-79, Arg-89, Arg-94, Arg-127, Arg-136, Arg-140, Arg-188, and Lys-123 as methylglyoxal modification sites in heat-shock protein 27 and in phosphorylated heat-shock protein 27. The transfer of methylglyoxal-modified heat-shock protein 27 into rat intestinal epithelial cell line RIE was even more effective in preventing apoptotic cell death than that of native control heat-shock protein 27. Furthermore, methylglyoxal modification of heat-shock protein 27 protected the cells against both the hydrogen peroxide- and cytochrome c-mediated caspase activation, and the hydrogen peroxide-induced production of intracellular reactive oxygen species. The levels of lactate converted from methylglyoxal were increased in carcinoma mucosal cell lines. Our results suggest that posttranslational modification of heat-shock protein 27 by methylglyoxal may have important implications for epithelial cell injury in gastrointestinal cancer.► Hsp25/Hsp27 is modified by methylglyoxal in carcinoma but not in healthy cell lines. ► Methylglyoxal modification of Hsp27 was necessary for its enhanced anti-apoptotic effects. ► Methylglyoxal modification of Hsp27 decreases intracellular reactive oxygen species. ► Lactate converted from methylglyoxal are elevated in carcinoma mucosal cell lines.
Keywords: Posttranslational modification; Proteomics; Heat-shock protein 27; Methylglyoxal; Apoptosis; Cancer;

GM1 gangliosidosis and Morquio B disease: An update on genetic alterations and clinical findings by Anna Caciotti; Scott C. Garman; Yadilette Rivera-Colón; Elena Procopio; Serena Catarzi; Lorenzo Ferri; Carmen Guido; Paola Martelli; Rossella Parini; Daniela Antuzzi; Roberta Battini; Michela Sibilio; Alessandro Simonati; Elena Fontana; Alessandro Salviati; Gulcin Akinci; Cristina Cereda; Carlo Dionisi-Vici; Francesca Deodato; Adele d'Amico; Alessandra d'Azzo; Enrico Bertini; Mirella Filocamo; Maurizio Scarpa; Maja di Rocco; Cynthia J. Tifft; Federica Ciani; Serena Gasperini; Elisabetta Pasquini; Renzo Guerrini; Maria Alice Donati; Amelia Morrone (782-790).
GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000–1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.► We produce an improved homology model of human GLB1. ► We note the importance of polymorphisms in determining phenotypes. ► We underline that severe cardiac valve involvement is typical of Morquio B disease. ► A continuum of phenotypes can be noted in all carefully examined patients.
Keywords: Beta-galactosidase; GM1 gangliosidosis; Morquio B; Mutation update; Homology modelling;

Hormone-like fibroblast growth factors and metabolic regulation by Yun Chau Long; Alexei Kharitonenkov (791-795).
The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called “hormone-like” subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.► This review covers the molecular properties of FGF19, FGF21 and FGF23 that confer their hormone-like functions. ► The novel and critical roles of FGF19, FGF21 and FGF23 in the regulation of diverse metabolic pathways are reviewed. ► This paper reviews the opportunities in targeting the hormone-like fibroblast growth factors for therapeutic applications.
Keywords: FGF19; FGF21; FGF23; Klotho; βKlotho; Metabolic regulation;

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc by Dae-Young Kim; Mi-Ju Kim; Hak-Bong Kim; Jae-Won Lee; Jae-Ho Bae; Dong-Wan Kim; Chi-Dug Kang; Sun-Hee Kim (796-805).
In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.► c-Myc is responsible for high susceptibility of MDR cancers to TRAIL. ► TRAIL increases cytotoxicity of anticancer drugs in MDR cells by degradation of P-gp. ► Up-regulation of DR5 contributes to sensitization of MDR cells to TRAIL. ► MDR is overcome by TRAIL in MDR cancer cells overexpressing c-Myc.
Keywords: TRAIL; Multidrug resistance; c-Myc; Cancer; P-gp;