Is There a Biological Basis for Treatment of Fibrodysplasia Ossificans Progressiva with Rosiglitazone? Potential Benefits and Undesired Effects

Thiazolidinediones (TZDs), among which Rosiglitazone, are known agonists of the peroxisome-proliferator-activated receptor γ (PPARγ) commonly used for treatment of hyperglycemia. A recently published article describing a case report on a patient affected by Fibrodysplasia Ossificans Progressiva (FOP) treated with Rosiglitazone has prompted interest for careful analysis of the rational basis of such treatment. This article reviews the effects of PPARγ agonists in relationship with various pathogenic steps that occur during the course of FOP by reviewing the particularly rich literature on the effects of Rosiglitazone, to underscore their relevance to FOP and to consider possible adverse effects

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

Glial cell line-derived neurotrophic factor-stimulated phosphatidylinositol 3-kinase and Akt activities exert opposing effects on the ERK pathway: importance for the rescue of neuroectodermic cells.

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in rescuing neural crest cells from apoptosis during their migration in the foregut. This survival factor binds to the heterodimer GDNF family receptor alpha1/Ret, inducing the Ret tyrosine kinase activity. ret loss-of-function mutations result in Hirschsprung's disease, a frequent developmental defect of the enteric nervous system. Although critical to enteric nervous system development, the intracellular signaling cascades activated by GDNF and their importance in neuroectodermic cell survival still remain elusive. Using the neuroectodermic SK-N-MC cell line, we found that the Ret tyrosine kinase activity is essential for GDNF to induce phosphatidylinositol 3-kinase (PI3K)/Akt and ERK pathways as well as cell rescue. We demonstrate that activation of PI3K is mandatory for GDNF-induced cell survival. In addition, evidence is provided for a critical up-regulation of the ERK pathway by PI3K at the level of Raf-1. Conversely, Akt inhibits the ERK pathway. Thus, both PI3K and Akt act in concert to finely regulate the level of ERK. We found that Akt activation is indispensable for counteracting the apoptotic signal on mitochondria, whereas ERK is partially involved in precluding procaspase-3 cleavage. Altogether, these findings underscore the importance of the Ret/PI3K/Akt pathway in GDNF-induced neuroectodermic cell survival

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\u2019 encoding proteins with different N-terminal regions ( 06N 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

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

open12noopenCappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello E.; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R.Cappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R

Severe Heterotopic Ossification in the Skeletal Muscle and Endothelial Cells Recruitment to Chondrogenesis Are Enhanced by Monocyte/Macrophage Depletion

Altered macrophage infiltration upon tissue damage results in inadequate healing due to inappropriate remodeling and stem cell recruitment and differentiation. We investigated in vivo whether cells of endothelial origin phenotypically change upon heterotopic ossification induction and whether infiltration of innate immunity cells influences their commitment and alters the ectopic bone formation. Liposome-encapsulated clodronate was used to assess macrophage impact on endothelial cells in the skeletal muscle upon acute damage in the ECs specific lineage-tracing Cdh5CreER(T2):R26REYFP/dtTomato transgenic mice. Macrophage depletion in the injured skeletal muscle partially shifts the fate of ECs toward endochondral differentiation. Upon ectopic stimulation of BMP signaling, monocyte depletion leads to an enhanced contribution of ECs chondrogenesis and to ectopic bone formation, with increased bone volume and density, that is reversed by ACVR1/SMAD pathway inhibitor dipyridamole. This suggests that macrophages contribute to preserve endothelial fate and to limit the bone lesion in a BMP/injury-induced mouse model of heterotopic ossification. Therefore, alterations of the macrophage-endothelial axis may represent a novel target for molecular intervention in heterotopic ossification

Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at Cterminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and Nterminal truncated P53 family isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites

Fibrodysplasia Ossificans Progressiva: what have we achieved and where are we now? follow-up to the 2015 Lorentz Workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics

Is There a Biological Basis for Treatment of Fibrodysplasia Ossificans Progressiva with Rosiglitazone? Potential Benefits and Undesired Effects

Thiazolidinediones (TZDs), among which Rosiglitazone, are known agonists of the peroxisome-proliferator-activated receptor γ (PPARγ) commonly used for treatment of hyperglycemia. A recently published article describing a case report on a patient affected by Fibrodysplasia Ossificans Progressiva (FOP) treated with Rosiglitazone has prompted interest for careful analysis of the rational basis of such treatment. This article reviews the effects of PPARγ agonists in relationship with various pathogenic steps that occur during the course of FOP by reviewing the particularly rich literature on the effects of Rosiglitazone, to underscore their relevance to FOP and to consider possible adverse effects. Fibrodysplasia Ossificans Progressiva Fibrodysplasia Ossificans Progressiva (FOP) (MIM 135100) is a rare and extremely disabling disorder, characterized by congenital malformation of the great toes and progressive heterotopic ossification Heterotopic ossification generally starts in the infancy and continues in an episodic and progressive way. Typically, the disorder presents with alternate quiescent periods, that sometimes last also years, and acute phases (flareups) through which ectopic bone formation at the level of axial and appendicular skeleton occurs and causes progressive and severe joint ankyloses and formation of an ectopic "second skeleton" The molecular defect causing the disorder is a gain of function mutation in the ACVR1/ALK2 gene, that encodes a Type I receptor for bone morphogenetic proteins (BMPs) The quite mild congenital malformation in the great toes can be interpreted in the context of the complex interplay of developmental pathways, including the BMP pathway, controlling development of limb elements. The most critical and still poorly understood point in the pathogenic mechanism is the type of stimuli that trigger flareup episodes and subsequent ossification. Flareups seem to be induced by trauma, medical or surgical or dental interventions, intramuscular injections, and infections. However, in some cases flareups start with no 2 PPAR Research apparent stimulus. The disorder is highly variable in term of onset, frequency, and severity of ectopic ossification episodes Pathological findings described after analysis of bioptic specimens from misdiagnosed patients have highlighted two important points: the phases of tissue composition at the sites of flareup and the type of bone produced in ectopic lesions Bone formation is preceded by the appearance of bumps that very often cause misdiagnosis by erroneous attribution of the initial lesion to cancer or other disorders like aggressive juvenile fibromatosis or lymphoedema FOP ectopic bone appears as a normal endochondrally formed lamellar bone with normal bone marrow elements. Radiological and scintigraphic characterization of ectopic bone in 47 FOP patients showed characteristics of resistance, stress response, and remodeling comparable to normal bone in unaffected people Thus, an inflammatory stimulus that in some way reaches a mutated receptor and stimulates a hyper-responsive signaling pathway are essential elements in the pathogenic process. Many efforts and expectations are directed to find approaches to interfere with the most critical steps of the pathogenic mechanism that could beneficially affect patients' quality of life. Very recently an article that describes successful results of treatment of an FOP patient with Rosiglitazone has been published The aim of this article was to review a particularly rich literature on the effects of Rosiglitazone and try to underscore their relevance to FOP. Rosiglitazone Rosiglitazone (ROSI) belongs to the class of Thiazolidinediones (TZDs) and has been approved as drug for treatment of hyperglycemia Rosiglitazone is mainly used as an orally active hypoglycemic agent for the treatment of noninsulin-dependent diabetes mellitus or Type 2 Diabetes (T2D). Mechanism of Action: ROSI as an Antidiabetic Drug. One of the characteristics of T2D is impaired response to normal circulating insulin levels at target tissues (muscle, liver, adipose tissue). Pancreatic beta cells partly compensate this resistance by increasing their own mass and/or efficiency of insulin secretion, however, when this compensatory ability is insufficient, hyperglycemia occurs. ROSI acts by the highly selective activation of the Peroxisome-ProliferatorActivated Receptor gamma (PPAR-γ), a ligand dependent transcription factor that belongs to the family of nuclear hormone receptors Because of its actions at different levels, adverse effects such as appearance or worsening of cardiovascular events, overweight, edema, and increased risk of fractures are observed. Peroxisome-Proliferator-Activated Receptor Gamma (PPAR-γ) PPAR-γ, the transcription factor for which ROSI is a potent and selective agonist, is involved in several different physiological and pathological processes such as differentiation, inflammation, aging, obesity, infertility, and cancer (i) PPAR-α is expressed in tissues with high catalytic activity of fatty acids as liver, skeleton, cardiac muscle, renal cortex. (ii) PPAR-δ has a wide expression pattern in particular in tissues where it exerts control of fatty acid oxidation as skin, brain, adipose tissue, muscle. (iii) PPAR-γ, the most extensively studied, is highly expressed in adipose tissue where it acts as the central molecule for adipocytic differentation, in pancreatic beta cells, vascular endothelial cells, and different cells involved in immune response as monocytes, macrophages, dendritic cells. PPAR-γ isoforms, differing for alternative promoters, and mRNA processing, are reported The different PPAR molecules share a four domain structural oganization Another mechanism of transcriptional repression is due to ligand-dependent sumoylation at the ligand-interaction domain of PPAR-γ. Sumoylation causes formation of a complex including PPAR-γ, a nuclear receptor corepressor (NcoR) and histone deacetylase-3 (HDAC3). This complex can be localized at PPREs in promoters of proinflammatory genes related to the NFκB that are therefore repressed PPAR-γ as an Anti-Inflammatory Molecule. PPAR-γ is normally expressed in antigen presenting cells in the immune system, as monocytes, macrophages, and dendritic cells Generation of PPAR-γ conditional knock out mice in macrophages further underscored its importance in control of inflammation and oxidative metabolism Macrophages represent a first line defense against pathogens and, in response to microenvironment stimuli, may become activated and differentiate by different ways. The classic activation is induced by Interferon-γ and promotes a proinflammatory response to elimininate pathogens Other evidences underscore the anti-inflammatory role of PPAR-γ. Animal models of inflammatory and autoimmune disorders as asthma, rheumatoid arthritis, multiple sclerosis, and type 2 diabetes associated nephropathy Together with the above evidences, a common PPAR-γ (Pro12Ala) nonsynonymous polymorphism that causes substitution of the conserved Pro12 residue in the ligandindependent DNA binding domain and other less common variants have been evaluated in association studies and also investigated by functional studies Besides mediating the antidiabetic and antiinflammatory effects of TZD agonists, PPAR-γ was also reported as a target of 5-aminosalicylic acid (5-ASA) antiinflammatory effects in an animal model of inflammatory bowel disease PPAR-γ: Adipogenesis versus Osteogenesis. Osteoblasts and adipocytes share common progenitors, mesenchymal stem cells. PPAR-γ expression is early induced by different key factors in the adipogenesis pathway and is considered the master transcriptional regulator of adipogenesis Generation of PPAR-γ knock out mouse models were critical for defining the effects of changes in PPAR-γ levels in vivo. Null homozygous mice showed lethality during embryo development at 10.5-11.5 dpc because of placental dysfunction Another mouse model, the PPAR-γ hyp/hyp , in which a hypomorphic mutation at the PPAR-γ locus causes absence of PPAR-γ expression in white adipose tissue (WAT), has highlighted the contribution of PPAR-γ to bone homeostasis PPAR-γ and Osteoclastogenesis. Considering the observed reduced bone mass after treatment with TZD PPAR-γ agonists, both in animal models and in clinical studies in diabetic human patients, the question whether these drugs, besides affecting osteoblastogenesis, also affect osteoclastogenesis also arises. Osteoclasts are multinucleated cells derived from hematopoietic precursors, contrary to osteoblasts that derive from mesenchymal stem cells. As mentioned above, PPAR-γ hyp/hyp mice showed increased osteoclast activity documented by increased expression of genes typical of the osteoclastic lineage, tartrate-resistant acid phosphatase (Trap) and cathepsin K (CathK) On the other hand, mice generated with conditional knock out of PPAR-γ in hematopoietic and endothelial cells showed evident splenomegaly with megakaryocyte accumulation, extramedullary hematopoiesis, pale bones, increased bone volume with reduced marrow cavity In summary, although with some contradiction in published data (increased osteoclast activity in Rosiglitazone and Bone: Effect of Treatment in Animal Models. The effect of ROSI on bone metabolism has been studied in vivo in animals to verify possible adverse effects. The examination of the in vivo effect of ROSI administration on the skeletons of adult (6 months old) male nondiabetic C57BL/6 mice Another article described the effect of treatment of adult (5 months old) male and female mice with rosiglitazone, providing other informations on the mechanism of bone loss PPAR Research 5 Morphologic changes in bone and cell differentiative potential were accompanied by changes in markers specific of bone development, as Runx2, Osterix, Col1A1, and osteocalcin, in favor of markers specific of adipogenesis. Rosiglitazone: Effect of Treatment in Humans. ROSI is approved and currently utilized as antidiabetic drug in patients affected by type 2 diabetes. Data on efficacy and adverse events are available in large studies in which, besides evaluating glycemic control, the most frequent adverse events are reported: risk of cardiovascular events, weight gain, fluid retention and bone fracture. We focus especially on the risk of bone fractures since other adverse events are more connected with diabetes complications while the effect on bone metabolism has a direct relevance to FOP disease. A large comparative study in which participated 500 centers in North America, Canada, and Europe, known as A Diabetes Outcome Progression Trial (ADOPT), involved an initial group of 4360 patients The first published results of ADOPT [51] reported comparable efficacy for the three drugs in term of control of glycemia, with greater durability of glycemic control in ROSI treated patients. Expected adverse events were described: in particular for ROSI, weight gain, increased LDL cholesterol, increased frequency of edema, reduction of hematocrit and C reactive protein level. The authors reported as unexpected a higher rate of fractures in females and not in males (humerus, hand, foot), in the group receiving rosiglitazone compared to metformin and glyburide In a more recent study, the effect of Pioglitazone and ROSI was evaluated in a group of 1819 male and female over 40 years old patients who underwent bone fracture and TZD drug exposure A confirmation of risk of bone fracture in patients treated with TZD drugs is reported in a recently published article describing results of a cross-sectional study Another article by the group of authors who participated in the ADOPT study, described results of measures of bone biomarkers. Although not providing conclusive explanation for the clinical findings, it would suggest an effect of TZD drugs primarily on increased bone resorptio