IFITM5 Research Articles

DMP1 and IFITM5 Regulate Osteogenic Differentiation of MC3T3-E1 on PEO-Treated Ti-6Al-4V-Ca2+/Pi surface.

Despite numerous studies on various surface modifications on titanium and its alloys, it remains unclear what kind of titanium-based surface modifications are capable of controlling cell activity. This study aimed to understand the mechanism at the cellular and molecular levels and investigate the in vitro response of osteoblastic MC3T3-E1 cultured on the Ti-6Al-4V surface modified by plasma electrolytic oxidation (PEO) treatment. A Ti-6Al-4V surface was prepared by PEO at 180, 280, and 380 V for 3 or 10 min in an electrolyte containing Ca2+/Pi ions. Our results showed that PEO-treated Ti-6Al-4V-Ca2+/Pi surfaces enhanced the cell attachment and differentiation of MC3T3-E1 compared to the untreated Ti-6Al-4V control but did not affect cytotoxicity as shown by cell proliferation and cell death. Interestingly, on the Ti-6Al-4V-Ca2+/Pi surface treated by PEO at 280 V for 3 or 10 min, MC3T3-E1 showed a higher initial adhesion and mineralization. In addition, the alkaline phosphatase (ALP) activity significantly increased in MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi (280 V for 3 or 10 min). In RNA-seq analysis, the expression of dentin matrix protein 1 (DMP1), sortilin 1 (Sort1), signal-induced proliferation-associated 1 like 2 (SIPA1L2), and interferon-induced transmembrane protein 5 (IFITM5) was induced during the osteogenic differentiation of MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi. DMP1 and IFITM5 silencing decreased the expression of bone differentiation-related mRNAs and proteins and ALP activity in MC3T3-E1. These results suggest that the PEO-treated Ti-6Al-4V-Ca2+/Pi surface induces osteoblast differentiation by regulating the expression of DMP1 and IFITM5. Therefore, surface microstructure modification through PEO coatings with Ca2+/Pi ions could be used as a valuable method to improve biocompatibility properties of titanium alloys.

Abnormal signal pathways and tumor heterogeneity in osteosarcoma

BackgroundOsteosarcoma (OS) is the most frequent and aggressive primary malignant sarcoma among adolescents and chemotherapy has not substantially progressed for decades. New insights into OS development and therapeutic strategies are urgently needed.MethodsWe analyzed integrated single-cell transcriptomes, bulk RNA-seq, and microarray data from Gene Expression Omnibus (GEO) datasets. We also used Weighted Gene Co-expression Network Analysis (WGCNA), Gene set enrichment analysis (GSEA), and Gene set variation analysis (GSVA), along with Simple ClinVar and Enrichr web servers.ResultsThe findings of integrated single-cell analysis showed that OS arises from imperfect osteogenesis during development. Novel abnormalities comprised deficient TGFβ and P53 signal pathways, and cell cycle pathway activation, and a potentially new driver mutation in the interferon induced transmembrane protein 5 (IFITM5) that might function as a pathogenic factor in OS. Osteosarcoma is characterized by oncocyte heterogeneity, especially in immunogenic and adipocyte-like subtypes that respectively promote and hamper OS treatment. Etoposide is a promising chemotherapeutic that provides palliation by affecting the subtype of OS and correcting the abnormal pathways.ConclusionVarious abnormal signal pathways play indispensable roles in OS development. We explored the heterogeneity and underlying mechanisms of OS and generated findings that will assist with OS assessment and selecting optimal therapies.

Effects of the overexpression of IFITM5 and IFITM5 c.-14C>T mutation on human osteosarcoma cells.

The present study aimed to investigate the effects of overexpression of interferon-induced transmembrane protein 5 (IFITM5) and IFITM5 c.-14C>T mutation on osteogenic differentiation, and the proliferation, migration and invasion of SaOS2 cells. SaOS2 cells were transfected with plasmids containing wild type IFITM5 (W) or IFITM5 containing the c.-14C>T mutation (MU). The mRNA and protein expression levels of IFITM5 in SaOS2 cells were respectively detected by reverse transcription quantitative polymerase chain reaction and western blotting. The proliferative, migratory and invasive ability of SaOS2 cells was also examined. In addition, the expression levels of osteogenic differentiation markers alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (Runx2) were detected. Mineralized nodules were detected by Alizarin Red S staining and were quantified by measuring absorbance. The mRNA and protein expression levels of IFITM5 were high in cells transfected with IFITM5 and IFITM5 c.-14C>T mutation, and were higher in cells transfected with IFITM5 c.-14C>T mutation. There was no difference in proliferation between the control group (C) and the W and MU groups. However, overexpression of IFITM5 and IFITM5 c.-14C>T mutation increased apoptotic rate, decreased invasive capacity, increased the expression of ALP, OCN and Runx2, and increased the number of mineralized nodules following osteogenic induction. In addition, compared with C and W groups, cells transfected with IFITM5 c.-14C>T mutation exhibited decreased migratory ability. In conclusion, overexpression of IFITM5 and IFITM5 c.-14C>T mutation promotes tumor cell apoptosis, inhibits tumor invasion and promotes osteogenic differentiation. These findings may provide a theoretical basis for the development of a novel treatment method that targets IFITM5, and provides a platform for the potential treatment of human osteosarcoma.

IFITM5 mutations and osteogenesis imperfecta.

Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5' untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.

Journal of Bone and Mineral Research: Volume 30, Number 3, March 2015

Abstract Cover: Transgenic mice overexpressing the mutant Ifi tm5. Skeletal preparations of mutant transgenics (Mut Tg) at E18.5 show reduced alizarin red staining and fractures which are not evident in wild type transgenic (WT Tg) or non-transgenic (NT) littermates. (see Lietman et al. beginning on page 489)

Effects of targeted modulation of miR-762 on expression of the IFITM5 gene in Saos-2 cells.

Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that plays an important role in the mineralization of the matrix in mature osteoblasts. However, understanding of the regulatory mechanism of IFITM5 expression is limited. Emerging evidence indicates that microRNAs (miRNAs) act as pivotal regulators in various biological processes including osteoblast proliferation and differentiation. This study aimed to investigate the impact of miRNAs on IFITM5 expression. Bioinformatic analyses predicted that miR-762 would be a potential regulator of IFITM5. A Dual-Luciferase Reporter Assay System indicated that miR-762 could bond with the 3'untranslated region (3'UTR) of IFITM5 via wild-type or mutant recombinant vectors and Western blotting verified that miR-762 negatively regulated IFITM5 expression. Collectively, these data indicate that miR-762 is a novel regulator of IFITM5 and that it suppresses the expression of IFITM5 in Saos-2 cells.

Genotype–phenotype study in type V osteogenesis imperfecta

Type V osteogenesis imperfecta (OI) presents with moderate-to-severe skeletal deformity and is characterized by hyperplastic callus formation at fracture sites and calcification of the interosseous membranes of the forearm and lower leg. The facial dysmorphism is not well characterized and has not been described in previous reports. Inheritance is autosomal dominant, although the genetic aetiology remained unknown until very recently. The aims of this study were to establish the genetic aetiology in patients with type V OI and further characterize patients with this condition, and to ascertain whether they have a similar clinical phenotype and facial dysmorphism. Three families (one mother-daughter pair and two singletons) were identified with the above features and further investigations (molecular genetic analysis and skin biopsy including electron microscopy, histology and collagen species analysis) were performed. Accurate phenotyping of patients with type V OI was performed. PCR amplification was performed using the Sheffield Diagnostic Genetics Service pyrosequencing assay for the interferon-induced transmembrane protein-5 (IFITM5) gene. All the patients had been confirmed to have a heterozygous variant, c.[-14C>T];[=], in the 5'-UTR of the IFITM5 gene, which is located in the transcribed region of this gene. This recurrent mutation, in IFITM5, also known as bone-restricted interferon-induced transmembrane protein-like protein or BRIL, encodes a protein with a function in bone formation and plays an important role in osteoblast formation. All four patients in this study appear to have similar clinical features and facial dysmorphism, including a short, up-turned nose, a small mouth, a prominent chin and greyish-blue sclerae. Skin biopsy in one patient showed clumping of elastic fibres and normal biochemical analysis of collagen. We have been able to characterize patients with type V OI further and confirm the genetic aetiology in this distinct form of OI. Accurate phenotyping (including describing the common facial features) before investigations is important to enable the useful interpretation of genetic results and/or target-specific gene testing.

Phenotypic Variability of Osteogenesis Imperfecta Type V Caused by an IFITM 5 Mutation

In a large cohort of osteogenesis imperfecta type V (OI type V) patients (17 individuals from 12 families), we identified the same mutation in the 5′ untranslated region (5′UTR) of the interferon-induced transmembrane protein 5 (IFITM5) gene by whole exome and Sanger sequencing (IFITM5 c.–14C > T) and provide a detailed description of their phenotype. This mutation leads to the creation of a novel start codon adding five residues to IFITM5 and was recently reported in several other OI type V families. The variability of the phenotype was quite large even within families. Whereas some patients presented with the typical calcification of the forearm interosseous membrane, radial head dislocation and hyperplastic callus (HPC) formation following fractures, others had only some of the typical OI type V findings. Thirteen had calcification of interosseous membranes, 14 had radial head dislocations, 10 had HPC, 9 had long bone bowing, 11 could ambulate without assistance, and 1 had mild unilateral mixed hearing loss. The bone mineral density varied greatly, even within families. Our study thus highlights the phenotypic variability of OI type V caused by the IFITM5 mutation.

Natural antisense transcripts enhance bone formation by increasing sense IFITM5 transcription

Interferon induced transmembrane protein 5 (IFITM5) has been recognized as an osteoblast differentiation factor. Its regulation, however, is still unclear. In this report, four novel naturally occurring antisense transcripts of rat IFITM2 and IFITM5 transcribed from the opposite strand of the IFITM gene locus, were isolated and characterized. They are alternatively transcribed from rat chromosome 1 and expressed at relatively high levels during early differentiation of primary isolates of rat osteoblast cells. There are two common fragments in all of the isoform cDNA sequences that are complimentary to both IFITM2 and IFITM5 respectively. There is an additional unique region in one isoform, immediately downstream of the putative IFITM5 complimentary region, which is also complimentary to IFITM cDNA sequence. Reading frame analysis showed that these antisense transcripts are non protein coding mRNAs. We investigated the expression of these antisense transcripts and their effects on IFITM expression as well as osteoblast differentiation. All isoforms were positively correlated with IFITM5 expression and antisense specific siRNAs inhibited osteoblast differentiation significantly. In contrast, these antisense transcripts had no effect on the expression of IFITM2. We speculate that IFITM5 may be regulated by antisense transcripts.

A Single Recurrent Mutation in the 5′-UTR of IFITM5 Causes Osteogenesis Imperfecta Type V

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.

A Mutation in the 5′-UTR of IFITM5 Creates an In-Frame Start Codon and Causes Autosomal-Dominant Osteogenesis Imperfecta Type V with Hyperplastic Callus

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder associated with bone fragility and susceptibility to fractures after minimal trauma. OI type V has an autosomal-dominant pattern of inheritance and is not caused by mutations in the type I collagen genes COL1A1 and COL1A2. The most remarkable and pathognomonic feature, observed in ~65% of affected individuals, is a predisposition to develop hyperplastic callus after fractures or surgical interventions. To identify the molecular cause of OI type V, we performed whole-exome sequencing in a female with OI type V and her unaffected parents and searched for de novo mutations. We found a heterozygous de novo mutation in the 5'-untranslated region of IFITM5 (the gene encoding Interferon induced transmembrane protein 5), 14 bp upstream of the annotated translation initiation codon (c.-14C>T). Subsequently, we identified an identical heterozygous de novo mutation in a second individual with OI type V by Sanger sequencing, thereby confirming that this is the causal mutation for the phenotype. IFITM5 is a protein that is highly enriched in osteoblasts and has a putative function in bone formation and osteoblast maturation. The mutation c.-14C>T introduces an upstream start codon that is in frame with the reference open-reading frame of IFITM5 and is embedded into a stronger Kozak consensus sequence for translation initiation than the annotated start codon. In vitro, eukaryotic cells were able to recognize this start codon, and they used it instead of the reference translation initiation signal. This suggests that five amino acids (Met-Ala-Leu-Glu-Pro) are added to the N terminus and alter IFITM5 function in individuals with the mutation.