Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation.

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation.

The Horizon of a Therapy for Rare Genetic Diseases: A "Druggable" Future for Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic condition characterized by progressive extra-skeletal ossification leading to cumulative and severe disability. FOP has an extremely variable and episodic course and can be induced by trauma, infections, iatrogenic harms, immunization or can occur in an unpredictable way, without any recognizable trigger. The causative gene is ACVR1, encoding the Alk-2 type I receptor for bone morphogenetic proteins (BMPs). The signaling is initiated by BMP binding to a receptor complex consisting of type I and II molecules and can proceed into the cell through two main pathways, a canonical, SMAD-dependent signaling and a p38-mediated cascade. Most FOP patients carry the recurrent R206H substitution in the receptor Glycine-Serine rich (GS) domain, whereas a few other mutations are responsible for a limited number of cases. Mutations cause a dysregulation of the downstream BMP-dependent pathway and make mutated ACVR1 responsive to a non-canonical ligand, Activin A. There is no etiologic treatment for FOP. However, many efforts are currently ongoing to find specific therapies targeting the receptor activity and the downstream aberrant pathway at different levels or targeting cellular components and/or processes that are important in modifying the local environment leading to bone neo-formation.

A case of Fibrodysplasia Ossificans Progressiva associated with a novel variant of the ACVR1 gene.

BACKGROUND: Fibrodysplasia Ossificans Progressiva (FOP) is a rare autosomal dominant disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of soft tissues leading to cumulative disability. The genetic cause of FOP are mutations in the ACVR1 gene that encodes a type I receptor of Bone Morphogenetic Proteins. The most recurrent mutation in FOP patients is R206H affecting the Glycine-Serine rich domain and causing the hyper-activation of the receptor and the responsivity to the non-canonical ligand, Activin A. In the present study, we described a 3-years old child with early and highly suggestive clinical features of FOP who was found negative for the recurrent p.R206H substitution. METHODS: Molecular screening of the whole ACVR1 coding sequence and functional characterization in transfection-based assays. RESULTS AND CONCLUSIONS: We identified a novel, de novo variant in the fifth ACVR1 coding exon (NM_001111067.4:c.772A>T; NP_001104537.1:p.(R258W)). This substitution, never reported in association with FOP, affects a conserved arginine residue in the kinase domain of the protein. In silico analysis predicted the pathogenicity of this substitution, demonstrated by in vitro assays showing that the p.R258W ACVR1 mutated receptor acquires the ability to transduce the aberrant Activin A-mediated signaling, as observed for the gene variants associated with FOP.

Genetic and Acquired Heterotopic Ossification: A Translational Tale of Mice and Men.

Heterotopic ossification is defined as an aberrant formation of bone in extraskeletal soft tissue, for which both genetic and acquired conditions are known. This pathologic process may occur in many different sites such as the skin, subcutaneous tissue, skeletal muscle and fibrous tissue adjacent to joints, ligaments, walls of blood vessels, mesentery and other. The clinical spectrum of this disorder is wide: lesions may range from small foci of ossification to massive deposits of bone throughout the body, typical of the progressive genetically determined conditions such as fibrodysplasia ossificans progressiva, to mention one of the most severe and disabling forms. The ectopic bone formation may be regarded as a failed tissue repair process in response to a variety of triggers and evolving towards bone formation through a multistage differentiation program, with several steps common to different clinical presentations and distinctive features. In this review, we aim at providing a comprehensive view of the genetic and acquired heterotopic ossification disorders by detailing the clinical and molecular features underlying the different human conditions in comparison with the corresponding, currently available mouse models.

P63 modulates the expression of the WDFY2 gene which is implicated in cancer regulation and limb development.

TP63 is a member of the TP53 gene family, sharing a common gene structure that produces two groups of mRNAs' encoding proteins with different N-terminal regions (ΔN and TA isoforms); both transcripts are also subjected to alternative splicing mechanisms at C-terminus, generating a variety of isoforms. p63 is a master regulator of epidermal development and homoeostasis as well as an important player in tumorigenesis and cancer progression with both oncogenic and tumour suppressive roles. A number of studies have aimed at the identification of p63 target genes, allowing the dissection of the molecular pathways orchestrated by the different isoforms. In the present study we investigated in more detail the p63 responsiveness of the WDFY2 (WD repeat and FYVE domain containing 2) gene, encoding for an endosomal protein identified as a binding partner of the PI-3K/AKT signalling pathway. We showed that overexpression of different p63 isoforms was able to induce WDFY2 expression in TP53-null cells. The p63-dependent transcriptional activation was associated with specific response elements (REs) that have been identified by a bioinformatics tool and validated by yeast- and mammal-based assays. Interestingly, to confirm that WDFY2 belongs to the p63 network of cancer regulation, we analysed the impact of WDFY2 alterations, by showing its frequent deletion in different types of tumours and suggesting its expression level as a prognostic biomarker. Lastly, we identified a chromosomal translocation involving the WDFY2 locus in a patient affected by a rare congenital limb anomaly, indicating WDFY2 as a possible susceptibility gene placed downstream p63 in the network of limb development.

Hints on transcriptional control of essential players in heterotopic ossification of Fibrodysplasia Ossificans Progressiva.

Signaling of the Bone Morphogenetic Protein (BMP) pathway is influenced by the level of expression of its components, in particular receptors, intracellular molecules and target genes which largely depends on gene transcription. One peculiar aspect of Fibrodysplasia Ossificans Progressiva (FOP) relates to the cell types in which the genetic mutation exerts its effects, then not only those involved in the heterotopic ossification processes but also others that participate in the inflammatory phases preceding and triggering heterotopic ossification. Such effects are in part detectable as variation in gene expression, which is also variably manifesting in term of time of appearance in different phases of the inflammatory or ossification processes.

Peripheral Blood Mononuclear Cell Immunophenotyping in Fibrodysplasia Ossificans Progressiva Patients: Evidence for Monocyte DNAM1 Up-regulation.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by sporadic heterozygous mutations in ACVR1 gene which progressively leads to severe heterotopic ossification. FOP is characterized by episodic flare-ups triggered by different factors such as viral infections, tissue injuries, vaccinations, or occurring without a recognizable cause. The sporadic course of the disease, the documented presence of an important inflammatory reaction in early lesions and the partial response to corticosteroids support the idea that the immune system, and in particular the innate component, may play a role in FOP pathogenesis. However, an extensive expression profile of the peripheral blood mononuclear cells (PBMC) of FOP patients has never been done. METHODS: In this study, we carried out a wide PBMC immunophenotyping on a cohort of FOP patients and matching controls by multiparametric analysis of the expression of a panel of 37 markers associated with migration, adhesion, inhibition, activation, and cell death of circulating immune cells. RESULTS: We observed a statistically significant increase of the expression of DNAM1 receptor in patients' monocytes as compared to controls, and little but significant differences in the expression profile of CXCR1 (CD181), CD62L, CXCR4 (CD184), and HLA-DR molecules. CONCLUSIONS: DNAM1 had been previously shown to play a pivotal role in monocyte migration through the endothelial barrier and the increased expression detected in patients' monocytes might suggest a role of this surface receptor during the early phases of FOP flare-ups in which the activation of the immune response is believed to represent a crucial event. © 2017 International Clinical Cytometry Society.

High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

The ACVR1 gene encodes a type I receptor of bone morphogenetic proteins (BMPs). Activating mutations in ACVR1 are responsible for fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by congenital toe malformation and progressive heterotopic endochondral ossification leading to severe and cumulative disability. Until now, no therapy has been available to prevent soft-tissue swelling (flare-ups) that trigger the ossification process. With the aim of finding a new therapeutic strategy for FOP, we developed a high-throughput screening (HTS) assay to identify inhibitors of ACVR1 gene expression among drugs already approved for the therapy of other diseases. The screening, based on an ACVR1 promoter assay, was followed by an in vitro and in vivo test to validate and characterize candidate molecules. Among compounds that modulate the ACVR1 promoter activity, we selected the one showing the highest inhibitory effect, dipyridamole, a drug that is currently used as a platelet anti-aggregant. The inhibitory effect was detectable on ACVR1 gene expression, on the whole Smad-dependent BMP signaling pathway, and on chondrogenic and osteogenic differentiation processes by in vitro cellular assays. Moreover, dipyridamole reduced the process of heterotopic bone formation in vivo Our drug repositioning strategy has led to the identification of dipyridamole as a possible therapeutic tool for the treatment of FOP. Furthermore, our study has also defined a pipeline of assays that will be useful for the evaluation of other pharmacological inhibitors of heterotopic ossification.

Identification and characterization of regulatory elements in the promoter of ACVR1, the gene mutated in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: The ACVR1 gene encodes a type I receptor for bone morphogenetic proteins (BMPs). Mutations in the ACVR1 gene are associated with Fibrodysplasia Ossificans Progressiva (FOP), a rare and extremely disabling disorder characterized by congenital malformation of the great toes and progressive heterotopic endochondral ossification in muscles and other non-skeletal tissues. Several aspects of FOP pathophysiology are still poorly understood, including mechanisms regulating ACVR1 expression. This work aimed to identify regulatory elements that control ACVR1 gene transcription. METHODS AND RESULTS: We first characterized the structure and composition of human ACVR1 gene transcripts by identifying the transcription start site, and then characterized a 2.9 kb upstream region. This region showed strong activating activity when tested by reporter gene assays in transfected cells. We identified specific elements within the 2.9 kb region that are important for transcription factor binding using deletion constructs, co-transfection experiments with plasmids expressing selected transcription factors, site-directed mutagenesis of consensus binding-site sequences, and by protein/DNA binding assays. We also characterized a GC-rich minimal promoter region containing binding sites for the Sp1 transcription factor. CONCLUSIONS: Our results showed that several transcription factors such as Egr-1, Egr-2, ZBTB7A/LRF, and Hey1, regulate the ACVR1 promoter by binding to the -762/-308 region, which is essential to confer maximal transcriptional activity. The Sp1 transcription factor acts at the most proximal promoter segment upstream of the transcription start site. We observed significant differences in different cell types suggesting tissue specificity of transcriptional regulation. These findings provide novel insights into the molecular mechanisms that regulate expression of the ACVR1 gene and that could be targets of new strategies for future therapeutic treatments.

The role of the 3'UTR region in the regulation of the ACVR1/Alk-2 gene expression.

BACKGROUND: The ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3'UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression. RESULTS: The ACVR1/Alk-2 3'UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3'UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3'UTR sequence thereby modulating its expression. CONCLUSION: In this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3'UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3'UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches.