Catia Bellucci

P253R fibroblast growth factor receptor‐2 mutation induces RUNX2 transcript variants and calvarial osteoblast differentiation

Unregulated fibroblast growth factor 2 (FGF2) signaling caused by mutations in the fibroblast growth factor receptor (FGFR2) leads to human craniosynostosis such as the Apert syndrome. In an in vitro control model of calvarial osteoblasts from Apert patients carrying the FGFR2 P253R mutation, we studied the changes in cellular phenotype and evaluated the effects of FGF2. Compared with wild‐type controls, osteocalcin mRNA was down‐regulated in Apert osteoblasts, Runt‐related transcription factor‐2 (RUNX2) mRNA was differentially spliced, and FGF2 secretion was greater. Total protein synthesis, fibronectin and type I collagen secretion were up‐regulated, while protease and glycosidase activities and matrix metalloproteinase‐13 (MMP‐13) transcription were decreased, suggesting an altered ECM turnover. Adding FGF2 increased protease and glycosidase activities and down‐regulated fibronectin and type I collagen secretion in Apert osteoblasts. High affinity FGF2 receptors were up‐regulated in Apert osteoblasts and analysis of signal transduction showed elevated levels of Grb2 tyrosine phosphorylation and the Grb2‐p85 beta association, which FGF2 stimulation strongly reduced. All together these findings suggest increased constitutive receptor activity in Apert mutant osteoblasts and an autocrine loop involving the FGF2 pathway in modulation of Apert osteoblast behavior.

Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation.

BackgroundWe focused on the phenotype of non-mineralizing MG63 and mineralizing TE85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated.Materials and MethodsOsteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay.ResultsOsteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE85 cell proliferation and reduced TE85 procollagen and osteocalcin production.ConclusionsThe different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

Apert's syndrome: differential in vitro production of matrix macromolecules and its regulation by interleukins

During embryonic development, variations in the composition of the extracellular matrix (ECM) macromolecules influence bone tissue differentiation. We present novel findings on the in vitro phenotypic expression of periosteal fibroblasts obtained from patients affected by Apert’s syndrome, a rare craniofacial malformation, and the effects that interleukins (ILs) induce on the phenotype. Apert fibroblasts synthesized greater quantities of glycosaminoglycans (GAGs) and intracellular type I collagen, and also produced more type III collagen and fibronectin. The amount of hyaluronic acid (HA) secreted by Apert fibroblasts was much higher than that secreted by normal fibroblasts, but, as the absolute values of heparan sulphate (HS), chondroitin sulphate (CS) and dermatan sulphate (DS) also rose in Apert media, the HA–sulphated GAG ratio was similar in the media obtained from both populations. Both ILs triggered elevations of HA in normal cells, although relative percentage secretion remained unaltered, but significantly reduced HA secretion by Apert cells. IL‐1 significantly increased CS in normal and Apert media, whereas IL‐6 enhanced HS and DS in media of both populations. HA–sulphated GAG ratio decreased in Apert media after IL treatment. Both ILs boosted fibronectin production by Apert fibroblasts, whereas IL‐1 increased type III but not type I collagen. Taken together, these data demonstrate that the synthesis and secretion of ECM macromolecules are markedly altered in Apert fibroblasts. The fact that treatment with ILs further modifies the Apert phenotype suggests that ILs may be implicated in the pathophysiology of the malformations during skull morphogenesis.

A regulatory role of fibroblast growth factor in the expression of decorin, biglycan, betaglycan and syndecan in osteoblasts from patients with Crouzon's syndrome

Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzons syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzons syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.

TGFβ Isoforms and Decorin Gene Expression are Modified in Fibroblasts Obtained from Non-syndromic Cleft Lip and Palate Subjects

Interaction between extracellular matrix (ECM) and cytokines is thought to be crucial for palatal development. The localization of transforming growth factors (TGFa and TGFβ isoforms) in craniofacial tissues suggests that they carry out multiple functions during development. In the present report, we studied TGFa, TGFβ1, and TGFβ3 expressions and their effects on ECM macromolecule production of normal and cleft palatal fibroblasts in vitro, to investigate the mechanisms by which the phenotypic modulation of fibroblasts occurs during the cleft palate process. The results indicated that, while TGFa mRNA was not evidenced in CLP or normal fibroblasts, a reduced TGFβ1 hybridization signal was detected in CLP fibroblasts. In addition, these secreted more active TGFβ3 than TGFβ 1, as evaluated in a biological assay. The CLP phenotype, which differed from the normal one because of its higher PG decorin expression and greater production of GAG and collagen, was further modified by the addition of growth factors. In fact, in CLP fibroblasts, TGFa and TGFβ1 down-regulated PG decorin transcript, TGFβ1 increased collagen and GAG in both cellular and extracellular compartments, and TGFβ3 promoted secretory processes of cells. In conclusion, the data represent the first report in a human model in vitro that TGFβ1 and β 3 are differently expressed and are correlated to the CLP phenotype. Thus, strength is given to the hypothesis that TGFβ3 isoforms are the potential inducers of phenotypic expression in palatal fibroblasts during development and that an autocrine growth factor production mechanism may be responsible for the phenotypic modifications.

Interleukin pattern of Apert fibroblasts in vitro.

The phenotype of cultured fibroblasts from patients affected by Aperts syndrome, a rare connective disorder, differs from that of normal cells in its extracellular matrix macromolecule composition (glycosaminoglycans, collagens and fibronectin) and is further modulated by treatment with interleukins (ILs). As the mechanisms responsible for the changes are unknown, we used our recently described model system for Apert periosteal fibroblasts to ascertain whether the pattern of ILs they secrete into the medium is comparable to that of normal fibroblasts. The results obtained by enzyme-linked immunosorbent assay (ELISA) show that the levels of interleukin-1 (IL-1) and interleukin-6 (IL-6) were lower in Apert than in normal media, whereas levels of IL-1 receptor antagonist (IL-1ra), the natural inhibitor of IL-1, were markedly higher. IL-1 specific bio-activity on thymocyte proliferation was also decreased in Apert supernatants. As we provided also evidence that active transforming growth factor beta (TGFbeta1), an IL-1 antagonist, was not secreted in greater amount in Apert media with respect to normals, the enhancement of IL-1ra appeared critical in down-regulating IL-1. Northern blot analysis of cytokine mRNA revealed no detectable IL-1 or IL-6 gene expression in normal fibroblasts, but high amounts of IL-6 mRNA transcripts in Apert cells. As the increased IL-6 gene expression did not translate into a parallel increase of secreted IL-6, the control of IL-6 secretion may be mainly post-transcriptional. Furthermore, the result that a treatment of the cultures with IL-1ra was able to induce a decrease of IL-6 secretion, suggests that the observed decreased secretion of IL-6 may be due to the autocrine action of overproduction of IL-1ra. The observed imbalance in the production of ILs which we show for the first time suggests ILs may be the natural autocrine regulators of ECM production in Apert fibroblasts. We hypothesize that in vitro differences previously reported in fibroblast phenotypes and several clinical features of Aperts syndrome may correlate with different cytokine patterns.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism†

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine‐treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age‐matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real‐time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF‐beta, retinoic acid (RA), and GABA‐ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin α2, and MMP13 genes were concordantly modulated, while integrin β5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition. J. Cell. Physiol. 222: 748–756, 2010.

Reversal of experimental Laron Syndrome by xenotransplantation of microencapsulated porcine Sertoli cells.

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans.

Effects of interleukin-1β on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants

Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1 beta is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1 beta acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1 beta treatment by morphological, histochemical and biochemical methods. Interleukin-1 beta stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1 beta-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1 beta modifies the ECM turnover without toxic effect on chondrocytes.

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy

Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre‐clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti‐inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre‐transplant storage stability of specific pathogen‐free pSCs (SPF‐pSCs) and evaluated the in vivo long‐term viability and safety of grafts.