BBA - Molecular Basis of Disease (v.1832, #2)
Editorial Board (i).
Insulin promotes iron uptake in human hepatic cell by regulating transferrin receptor-1 transcription mediated by hypoxia inducible factor-1 by Sudipta Biswas; Nisha Tapryal; Reshmi Mukherjee; Rajiv Kumar; Chinmay K. Mukhopadhyay (293-301).
Hepatic iron is known to regulate insulin signaling pathways and to influence insulin sensitivity in insulin resistance (IR) patients. However, the role of insulin on hepatic iron homeostasis remains unexplored. Here, we report that insulin promotes transferrin-bound iron uptake but shows no influence on non transferrin-bound iron uptake in human hepatic HepG2 cells. As a mechanism we detected increased transferrin receptor-1 (TfR1) expression both at protein and mRNA levels. Unaltered stability of protein and transcript of TfR1 suggested the regulation at transcriptional level that was confirmed by promoter activity. Involvement of transcription factor hypoxia inducible factor-1 (HIF-1) was shown by mutational analyses of the TfR1 promoter region and by electrophoretic mobility shift assay. When HepG2 cells were transfected with specific siRNA targeted to 3′UTR of HIF-1α, the regulatory subunit of HIF-1; insulin-induced TfR1 expression and iron uptake were inhibited. Transfection of cDNA expressing stable form of HIF-1α reversed the increased TfR1 expression and iron uptake. These results suggest a novel role of insulin in hepatic iron uptake by a HIF-1 dependent transcriptional regulation of TfR1.► Role of insulin on hepatic iron homeostasis is unknown. ► We report that insulin promotes transferrin-bound iron uptake in hepatic cell. ► Insulin activates hypoxia inducible factor-1 to transferrin receptor transcription. ► Our results show a novel link between energy and iron metabolism in hepatic cells. ► This also may implicate hepatic iron overload during insulin resistance.
Keywords: Insulin; Iron uptake; Transferrin receptor-1; Transcription; Hypoxia inducible factor-1; Hepatic iron overload;
Increasing βB1-crystallin sensitivity to proteolysis caused by the congenital cataract-microcornea syndrome mutation S129R by Sha Wang; Wei-Jie Zhao; Huihui Liu; Haipeng Gong; Yong-Bin Yan (302-311).
Congenital hereditary cataract, which is mainly caused by the deposition of crystallins in light-scattering particles, is one of the leading causes of newborn blindness in human beings. Recently, an autosomal dominant congenital cataract-microcornea syndrome in a Chinese family has been associated with the S129R mutation in βB1-crystallin. To investigate the underlying molecular mechanism, we examined the effect of the mutation on βB1-crystallin structure and thermal stability. Biophysical experiments indicated that the mutation impaired the oligomerization of βB1-crystallin and shifted the dimer–monomer equilibrium to monomer. Molecular dynamic simulations revealed that the mutation altered the hydrogen-bonding network and hydrophobic interactions in the subunit interface of the dimeric protein, which resulted in the opening of the tightly associated interacting sites to allow the infiltration of the solvent molecules into the interface. Despite the disruption of βB1-crystallin assembly, the thermal stability of βB1-crystallin was increased by the mutation accompanied by the reduction of thermal aggregation at high temperatures. Further analysis indicated that the mutation significantly increased the sensitivity of βB1-crystallin to trypsin hydrolysis. The digested fragments of the mutant were prone to aggregate and unable to protect βA3-crystallin against aggregation. These results indicated that the thermal stability-beneficial mutation S129R in βB1-crystallin provided an excellent model for discovering molecular mechanisms apart from solubility and stability. Our results also highlighted that the increased sensitivity of mutated crystallins towards proteases might play a crucial role in the pathogenesis of congenital hereditary cataract and associated syndrome.► We investigate the molecular mechanism underlying cataract-linked mutation S129R. ► S129R impairs the oligomerization, but increases the stability of βB1-crystallin. ► Molecular dynamic simulations revealed that S129R altered the intersubunit interactions. ► S129R increases βB1-crystallin sensitivity to trypsin hydrolysis. ► The trypsin-digested fragments are prone to aggregate.
Keywords: βB1-crystallin; Inherited mutation; Congenital cataract-microcornea syndrome; Molecular dynamic simulation; Protein aggregation; Limited proteolysis;
Role of the N-methyl-d-aspartate receptors complex in amyotrophic lateral sclerosis by Alida Spalloni; Michele Nutini; Patrizia Longone (312-322).
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease pathologically characterized by the massive loss of motor neurons in the spinal cord, brain stem and cerebral cortex. There is a consensus in the field that ALS is a multifactorial pathology and a number of possible mechanisms have been suggested. Among the proposed hypothesis, glutamate toxicity has been one of the most investigated. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated cell death and impairment of the glutamate-transport system have been suggested to play a central role in the glutamate-mediated motor neuron degeneration. In this context, the role played by the N-methyl-d-aspartate (NMDA) receptor has received considerable less attention notwithstanding its high Ca2 + permeability, expression in motor neurons and its importance in excitotoxicity. This review overviews the critical role of NMDA-mediated toxicity in ALS, with a particular emphasis on the endogenous modulators of the NMDAR.► Glutamate excitotoxicity is an important factor in ALS. ► NMDA receptors involvement in ALS has been overlooked. ► We discuss the role played by the NMDA receptors endogenous modulators in ALS.
Keywords: Amyotrophic lateral sclerosis; N-methyl-d-aspartate receptor; Excitotoxicity; Glutamate; Neurodegeneration;
BAMBI (BMP and activin membrane-bound inhibitor) protects the murine heart from pressure-overload biomechanical stress by restraining TGF-β signaling by Ana V. Villar; Raquel García; Miguel Llano; Manuel Cobo; David Merino; Aquilino Lantero; Mónica Tramullas; Juan M. Hurlé; María A. Hurlé; J. Francisco Nistal (323-335).
Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-β (TGF-βs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-β signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI−/−) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-β. BAMBI deletion led to a gain of myocardial TGF-β signaling through canonical (Smads) and non-canonical (TAK1–p38 and TAK1–JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI−/− mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-β targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI−/− mice involved TGF-βs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-β-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-β remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.► Cardiomyocytes and cardiac fibroblasts express BAMBI. ► BAMBI and TGF-β are up-regulated in the LV under pressure overload in humans and mice. ► BAMBI restrains TGF-β fibrogenic signaling through a negative feedback loop. ► BAMBI deletion exacerbates LV remodeling induced by TGF-β under pressure overload. ► BAMBI protects the pressure-overloaded myocardium against adverse remodeling.
Keywords: Myocardial remodeling; Aortic valve stenosis; Pressure overload; TGF-β; BAMBI; miR-21;
SR-A deficiency reduces myocardial ischemia/reperfusion injury; involvement of increased microRNA-125b expression in macrophages by Danyang Ren; Xiaohui Wang; Tuanzhu Ha; Li Liu; John Kalbfleisch; Xiang Gao; David Williams; Chuanfu Li (336-346).
The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A−/− and WT mice were subjected to ischemia (45 min) followed by reperfusion for up to 7 days. SR-A−/− mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A−/− heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A−/−) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A−/− macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A−/− macrophages. The levels of miR-125b in SR-A−/− macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A−/− macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.► SR-A deficiency attenuated ischemia/reperfusion-induced myocardial infarction. ► SR-A deficiency improved cardiac function after myocardial reperfusion up to 7 days. ► Increased expression of miR-125b was observed in SR-A−/− heart and macrophage. ► Overexpression of miR-125b in macrophages prevented hypoxia/reoxygenation-induced cell injury. ► Overexpression of miR-125b in macrophages suppressed p53-mediated apoptotic signaling.
Keywords: microRNA-125b; Macrophage SR-A; Hypoxia/reoxygenation; Myocardial I/R injury; Apoptosis;
Matrix Metalloproteinase 1 promotes tumor formation and lung metastasis in an intratibial injection osteosarcoma mouse model by Knut Husmann; Matthias J.E. Arlt; Roman Muff; Bettina Langsam; Josefine Bertz; Walter Born; Bruno Fuchs (347-354).
Proteolytic degradation of the extracellular matrix (ECM) is an important process during tumor invasion. Matrix Metalloproteinase 1 (MMP-1) is one of the proteases that degrade collagen type I, a major component of bone ECM. In the present study, the biological relevance of MMP-1 in osteosarcoma (OS) tumor growth and metastasis was investigated in vitro and in vivo. Human OS cells in primary culture expressed MMP-1 encoding mRNA at considerably higher levels than normal human bone cells. In addition, MMP-1 mRNA and protein expression in the highly metastatic human osteosarcoma 143-B cell line was remarkably higher than in the non-metastatic parental HOS cell line. Stable shRNA-mediated downregulation of MMP-1 in 143-B cells impaired adhesion to collagen I and anchorage-independent growth, reflected by a reduced ability to grow in soft agar. Upon intratibial injection into SCID mice, 143-B cells with shRNA-downregulated MMP-1 expression formed smaller primary tumors and significantly lower numbers of lung micro- and macrometastases than control cells. Conversely, HOS cells stably overexpressing MMP-1 showed an enhanced adhesion capability to collagen I and accelerated anchorage-independent growth compared to empty vector-transduced control cells. Furthermore, and most importantly, individual MMP-1 overexpression in HOS cells enabled the formation of osteolytic primary tumors and lung metastasis while the HOS control cells did not develop any tumors or metastases after intratibial injection. The findings of the present study reveal an important role of MMP-1 in OS primary tumor and metastasis formation to the lung, the major organ of OS metastasis.► MMP-1 expression in primary OS cells is enhanced. ► Modulation of MMP-1 expression influenced anchorage independent tumor cell growth. ► shRNA-mediated MMP-1 downregulation formed smaller primary tumors. ► shRNA-mediated MMP-1 downregulation lowered the number of lung metastases. ► MMP-1 overexpression enabled osteosarcoma tumor formation and lung metastasis.
Keywords: Osteosarcoma; Metastasis; MMP-1; In vivo; Intratibial mouse model;
High fat diets and pathology in the guinea pig. Atherosclerosis or liver damage? by Peng Ye; Irwin K. Cheah; Barry Halliwell (355-364).
Animal models have been widely used to investigate the relationship between diet and atherosclerosis and also to study disease etiology and possible interventions. Guinea pigs have been suggested to be a more “realistic” model for atherosclerosis due to their many similarities to humans. However, few published studies actually reported observations of characteristic atherosclerotic lesions and even fewer of advanced lesions. Studies, by our group, of guinea pigs fed on a high-fat diet revealed similar observations, with indications primarily of fatty streaks but little evidence of atherosclerotic plaques. This review discusses the feasibility of the guinea pig as a model for dietary-induced atherosclerosis. As it stands, current evidence raises doubt as to whether guinea pigs could serve as a realistic model for atherosclerosis. However, our own data and the literature suggest that they could be useful models for studying lipoprotein metabolism, non-alcoholic fatty liver disease, and dietary interventions which may help regulate these conditions.► Atherosclerosis and NAFLD are major causes of morbidity and mortality worldwide. ► Guinea pigs have been suggested to be a more “realistic” model for atherosclerosis. ► Very few studies in guinea pigs report observations of atherosclerotic lesions. ► Guinea pigs may be better models for studies into liver pathology. ► Future work should address variability in experimental conditions to allow comparison.
Keywords: Guinea pig; Atherosclerosis; Cholesterol; Dietary supplementation; Non-alcoholic fatty liver disease;
ARHGAP21 is a RhoGAP for RhoA and RhoC with a role in proliferation and migration of prostate adenocarcinoma cells by Mariana Lazarini; Fabiola Traina; João A. Machado-Neto; Karin S.A. Barcellos; Yuri B. Moreira; Marcelo M. Brandão; Sergio Verjovski-Almeida; Anne J. Ridley; Sara T. Olalla Saad (365-374).
Background: Several Rho GTPase-activating proteins (RhoGAPs) are implicated in tumor progression through their effects on Rho GTPase activity. ARHGAP21 is a RhoGAP with increased expression in head and neck squamous cell carcinoma and with a possible role in glioblastoma tumor progression, yet little is known about the function of ARHGAP21 in cancer cells. Here we studied the role of ARHGAP21 in two prostate adenocarcinoma cell lines, LNCaP and PC3, which respectively represent initial and advanced stages of prostate carcinogenesis. Results: ARHGAP21 is located in the nucleus and cytoplasm of both cell lines and its depletion resulted in decreased proliferation and increased migration of PC3 cells but not LNCaP cells. In PC3 cells, ARHGAP21 presented GAP activity for RhoA and RhoC and induced changes in cell morphology. Moreover, its silencing altered the expression of genes involved in cell proliferation and cytoskeleton organization, as well as the endothelin-1 canonical pathway. Conclusions: Our results reveal new functions and signaling pathways regulated by ARHGAP21, and indicate that it could contribute to prostate cancer progression.► The effects of ARHGAP21 silencing in prostate adenocarcinoma cells were evaluated. ► ARHGAP21 silencing decreased proliferation and increased migration of PC3 cells. ► Inhibition of ARHGAP21 modulates endothelin-1 canonical pathway. ► ARHGAP21 is a RhoGAP for RhoA and RhoC. ► ARHGAP21 might be involved in prostate cancer progression.
Keywords: ARHGAP21; RhoA; RhoC; Prostate adenocarcinoma; PC3; Endothelin-1;
The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site by Xuehui Liu; Chunyan Niu; Jintao Ren; Jiayu Zhang; Xiaodong Xie; Haining Zhu; Wei Feng; Weimin Gong (375-385).
Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.► The structure of the RNA recognition motif of FUS is determined. ► A unique positively charged “KK” loop is critical to nucleic acid binding. ► Substituting the lysine residues in the “KK” loop impaired the nucleic acid binding. ► RNA binding deficient mutation altered FUS subcellular localization.
Keywords: Fused in sarcoma; RNA recognition motif; Amyotrophic lateral sclerosis; Nucleic acid binding; NMR; Surface plasmon resonance;