ABSTRACT
We aimed to study the mechanisms involved in bone-related iron impairment by using the osteoblast-like MG-63 cell line. Our results indicate that iron impact the S1P/S1PR signalizing axis and suggest that iron can affect the S1P process and favor the occurrence of osteoporosis during chronic iron overload. INTRODUCTION: Systemic iron excess favors the development of osteoporosis, especially during genetic hemochromatosis. The cellular mechanisms involved are still unclear despite numerous data supporting a direct effect of iron on bone biology. Therefore, the aim of this study was to characterize mechanisms involved in the iron-related osteoblast impairment. METHODS: We studied, by using the MG-63 cell lines, the effect of iron excess on SPNS2 gene expression which was previously identified by us as potentially iron-regulated. Cell-type specificity was investigated with hepatoma HepG2 and enterocyte-like Caco-2 cell lines as well as in iron-overloaded mouse liver. The SPNS2-associated function was also investigated in MG-63 cells by fluxomic strategy which led us to determinate the S1P efflux in iron excess condition. RESULTS: We showed in MG-63 cells that iron exposure strongly increased the mRNA level of the SPNS2 gene. This was not observed in HepG2, in Caco-2 cells, and in mouse livers. Fluxomic study performed concomitantly on MG-63 cells revealed an unexpected decrease in the cellular capacity to export S1P. Iron excess did not modulate SPHK1, SPHK2, SGPL1, or SGPP1 gene expression, but decreased COL1A1 and S1PR1 mRNA levels, suggesting a functional implication of low extracellular S1P concentration on the S1P/S1PR signalizing axis. CONCLUSIONS: Our results indicate that iron impacts the S1P/S1PR signalizing axis in the MG-63 cell line and suggest that iron can affect the bone-associated S1P pathway and favor the occurrence of osteoporosis during chronic iron overload.
Subject(s)
Anion Transport Proteins/biosynthesis , Iron Overload/metabolism , Lysophospholipids/metabolism , Osteoblasts/metabolism , Sphingosine/analogs & derivatives , Up-Regulation/physiology , Animals , Anion Transport Proteins/genetics , Caco-2 Cells , Cells, Cultured , Collagen Type I/biosynthesis , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , Disease Models, Animal , Gene Silencing , Hemochromatosis/metabolism , Hep G2 Cells , Humans , Iron/metabolism , Iron/pharmacology , Liver/metabolism , Male , Mice, Knockout , Osteoblasts/drug effects , RNA, Messenger/genetics , Sphingosine/metabolismABSTRACT
UNLABELLED: In order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis. INTRODUCTION: Iron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested. METHODS: We investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes. RESULTS: FAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure. CONCLUSION: We showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.
Subject(s)
Iron Overload/metabolism , Osteoblasts/metabolism , Cation Transport Proteins/biosynthesis , Cation Transport Proteins/genetics , Cells, Cultured , Citric Acid , Ferric Compounds/pharmacology , Ferrous Compounds/pharmacology , Gene Expression Regulation/drug effects , Hemochromatosis Protein , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Humans , Iron Overload/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Osteoblasts/drug effects , Oxidative Stress/drug effects , Phenotype , Quaternary Ammonium Compounds/pharmacologyABSTRACT
The field of hereditary iron overload has known, in the recent period, deep changes mainly related to major advances in molecular biology. It encompasses now a series of genetic entities. The mechanistic understanding of iron overload development and iron toxicity has greatly improved. The diagnostic approach has become essentially noninvasive with a major role for biological tests. From the therapeutic viewpoint, the phlebotomy treatment is now enriched by the possibility of resorting to oral chelation and by innovative perspectives directly linked to our improvement in the molecular understanding of these diseases.
Subject(s)
Iron Overload/genetics , Antimicrobial Cationic Peptides/deficiency , Antimicrobial Cationic Peptides/genetics , Cation Transport Proteins/deficiency , Cation Transport Proteins/genetics , Ceruloplasmin/deficiency , Ceruloplasmin/genetics , Chelation Therapy , Forecasting , Genetic Counseling , Hemochromatosis/classification , Hemochromatosis/diagnosis , Hemochromatosis/drug therapy , Hemochromatosis/genetics , Hemochromatosis/therapy , Hemochromatosis Protein , Hemosiderosis/genetics , Hemosiderosis/metabolism , Hepcidins , Histocompatibility Antigens Class I/genetics , Humans , Iron/metabolism , Iron Metabolism Disorders/genetics , Iron Overload/diagnosis , Iron Overload/drug therapy , Iron Overload/physiopathology , Iron Overload/therapy , Liver/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Molecular Diagnostic Techniques , Neurodegenerative Diseases/genetics , PhlebotomyABSTRACT
Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor.
Subject(s)
Gene Silencing , Homeodomain Proteins/metabolism , Interferon Type I/genetics , Promoter Regions, Genetic/genetics , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , DNA/genetics , DNA/metabolism , DNA Probes , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, Reporter , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Humans , Mice , Newcastle disease virus/physiology , Paired Box Transcription Factors , Protein Binding , Protein Structure, Tertiary , RNA, Antisense/genetics , RNA, Antisense/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Response Elements/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Transfection , Two-Hybrid System TechniquesABSTRACT
Interferon-A (IFN-A) differential gene expression is modulated by a complex interplay between cis-acting DNA elements and the corresponding specific trans-regulating factors. Substitutions in the proximal virus-responsive element of the interferon-A (IFN-A) promoters contribute to their differential gene expression. The 5' distal silencing region in the weakly virus-inducible murine IFN-A11 gene has been previously delimited. DNase I footprinting experiments and transient gene expression assays demonstrate identical silencing activity in equivalent regions of the genes for IFN-A11 and IFN-A4 promoters. A minimal 20-mer distal negative regulatory element (DNRE) in both promoters is necessary and sufficient for the silencing and a region in the highly inducible IFN-A4 promoter located between the silencer and the virus-responsive element overrides the silencer activity. Mutations in the central region of the DNRE, causing derepression, also altered the formation of one of the two major DNA-protein complexes. One of these contains a protein related to or identical to the high mobility group I(Y) proteins, while the other complex contains a major protein present in uninduced and virus-induced cells with a molecular mass of 38 kDa, which may be related to the silencer activity. Similar DNREs are present in other virus-uninducible IFN-A promoters, and these data suggest that a common silencer may mediate the transcriptional repression in different genes of this family.
