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Jessika BERTACCHINI
Professore Associato
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa
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Pubblicazioni
2023
- Experimental measurements and CFD modelling of hydroxyapatite scaffolds in perfusion bioreactors for bone regeneration
[Articolo su rivista]
D’Adamo, Alessandro; Salerno, Elisabetta; Corda, Giuseppe; Ongaro, Claudio; Zardin, Barbara; Ruffini, Andrea; Orlandi, Giulia; Bertacchini, Jessika; Angeli, Diego
abstract
In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors.
2023
- Flow-dependent shear stress affects the biological properties of pericyte-like cells isolated from human dental pulp
[Articolo su rivista]
Bertani, Giulia; Di Tinco, Rosanna; Bertoni, Laura; Orlandi, Giulia; Pisciotta, Alessandra; Rosa, Roberto; Rigamonti, Luca; Signore, Michele; Bertacchini, Jessika; Sena, Paola; De Biasi, Sara; Villa, Erica; Carnevale, Gianluca
abstract
Background: Human dental pulp stem cells represent a mesenchymal stem cell niche localized in the perivascular area of dental pulp and are characterized by low immunogenicity and immunomodulatory/anti-inflammatory properties. Pericytes, mural cells surrounding the endothelium of small vessels, regulate numerous functions including vessel growth, stabilization and permeability. It is well established that pericytes have a tight cross talk with endothelial cells in neoangiogenesis and vessel stabilization, which are regulated by different factors, i.e., microenvironment and flow-dependent shear stress. The aim of this study was to evaluate the effects of a pulsatile unidirectional flow in the presence or not of an inflammatory microenvironment on the biological properties of pericyte-like cells isolated from human dental pulp (hDPSCs). Methods: Human DPSCs were cultured under both static and dynamic conditions with or without pre-activated peripheral blood mononuclear cells (PBMCs). Pulsatile unidirectional flow shear stress was generated by using a specific peristaltic pump. The angiogenic potential and inflammatory properties of hDPSCs were evaluated through reverse phase protein microarrays (RPPA), confocal immunofluorescence and western blot analyses. Results: Our data showed that hDPSCs expressed the typical endothelial markers, which were up-regulated after endothelial induction, and were able to form tube-like structures. RPPA analyses revealed that these properties were modulated when a pulsatile unidirectional flow shear stress was applied to hDPSCs. Stem cells also revealed a downregulation of the immune-modulatory molecule PD-L1, in parallel with an up-regulation of the pro-inflammatory molecule NF-kB. Immune-modulatory properties of hDPSCs were also reduced after culture under flow-dependent shear stress and exposure to an inflammatory microenvironment. This evidence was strengthened by the detection of up-regulated levels of expression of pro-inflammatory cytokines in PBMCs. Conclusions: In conclusion, the application of a pulsatile unidirectional flow shear stress induced a modulation of immunomodulatory/inflammatory properties of dental pulp pericyte-like cells.
2023
- Human dental pulp stem cells (hDPSCs) promote the lipofibroblast transition in the early stage of a fibro-inflammatory process
[Articolo su rivista]
Pisciotta, Alessandra; DI TINCO, Rosanna; Bertani, Giulia; Orlandi, Giulia; Bertoni, Laura; Pignatti, Elisa; Orciani, Monia; Sena, Paola; Bertacchini, Jessika; Salvarani, Carlo; Carnevale, Gianluca
abstract
Introduction: In autoimmune diseases, particularly in systemic sclerosis and chronic periaortitis, a strict correlation between chronic inflammation and fibrosis exists. Since the currently used drugs prove mostly effective in suppressing inflammation, a better comprehension of the molecular mechanisms exerted by cell types implicated in fibro-inflammation is needed to develop novel therapeutic strategies. Mesenchymal stromal/stem cells (MSCs) are being matter of deep investigation to unveil their role in the evolution of fibrogenetic process. Several findings pointed out the controversial implication of MSCs in these events, with reports lining at a beneficial effect exerted by external MSCs and others highlighting a direct contribution of resident MSCs in fibrosis progression. Human dental pulp stem cells (hDPSCs) have demonstrated to hold promise as potential therapeutic tools due to their immunomodulatory properties, which strongly support their contribution to tissue regeneration.
Methods: Our present study evaluated hDPSCs response to a fibro-inflammatory microenvironment, mimicked in vitro by a transwell co-culture system with human dermal fibroblasts, at early and late culture passages, in presence of TGF-β1, a master promoter of fibrogenesis.
Results and Discussion: We observed that hDPSCs, exposed to acute fibro-inflammatory stimuli, promote a myofibroblast-to-lipofibroblast transition, likely based on BMP2 dependent pathways. Conversely, when a chronic fibro-inflammatory microenvironment is generated, hDPSCs reduce their anti-fibrotic effect and acquire a pro-fibrotic phenotype. These data provide the basis for further investigations on the response of hDPSCs to varying fibro-inflammatory conditions.
2022
- An Alternative Solution for Microfluidic Chip Fabrication
[Relazione in Atti di Convegno]
Ongaro, C.; Betti, A.; Zardin, B.; Siciliani, V.; Orazi, L.; Bertacchini, J.; Borghi, M.
abstract
2022
- Characterization of Dental Pulp Stem Cells Response to Bone Substitutes Biomaterials in Dentistry
[Articolo su rivista]
Di Tinco, R.; Consolo, U.; Pisciotta, A.; Orlandi, G.; Bertani, G.; Nasi, M.; Bertacchini, J.; Carnevale, G.
abstract
Bone substitute biomaterials (BSBs) represent a promising alternative to bone autografts, due to their biocompatibility, osteoconduction, slow resorption rates, and the ability to define and maintain volume for bone gain in dentistry. Many biomaterials are tailored to provide structural and biological support for bone regeneration, and allow the migration of bone-forming cells into the bone defect. Neural crest-derived stem cells isolated from human dental pulp (hDPSCs) represent a suitable stem cell source to study the biological effects of BSBs on osteoprogenitor cells involved in the physiological bone regenerative processes. This study aimed to evaluate how three different BSBs affect the stem cell properties, osteogenic differentiation, and inflammatory properties of hDPSCs. Our data highlight that BSBs do not alter cell proliferation and stemness markers expression, nor induce any inflammatory responses. Bone metabolism data show that hDPSCs exposed to the three BSBs distinctively secrete the factors supporting osteoblast activity and osteoclast activity. Our data indicate that (i) hDPSCs are a suitable stem cell source to study the effects of BSBs, and that (ii) the formulation of BSBs may condition the biological properties of stem cells, suggesting their versatile suitability to different dentistry applications.
2022
- Liquid flow in scaffold derived from natural source: experimental observations and biological outcome
[Articolo su rivista]
Salerno, Elisabetta; Orlandi, Giulia; Ongaro, Claudio; D'Adamo, Alessandro; Ruffini, Andrea; Carnevale, Gianluca; Zardin, Barbara; Bertacchini, Jessika; Angeli, Diego
abstract
This study investigates the biological effects on a 3D scaffold based on hydroxyapatite cultured with MC3T3 osteoblasts in response to flow-induced shear stress (FSS). The scaffold adopted here (B-HA) derives from the biomorphic transformation of natural wood and its peculiar channel geometry mimics the porous structure of the bone. From the point of view of fluid dynamics, B-HA can be considered a network of micro-channels, intrinsically offering the advantages of a microfluidic system. This work, for the first time, offers a description of the fluid dynamic properties of the B-HA scaffold, which are strongly connected to its morphology. These features are necessary to determine the FSS ranges to be applied during in vitro studies to get physiologically relevant conditions. The selected ranges of FSS promoted the elongation of the attached cells along the flow direction and early osteogenic cell differentiation. These data confirmed the ability of B-HA to promote the differentiation process along osteogenic lineage. Hence, such a bioactive and naturally derived scaffold can be considered as a promising tool for bone regeneration applications.
2021
- Autoimmunity profiles as prognostic indicators in patients with colorectal cancer versus those with cancer at other sites: A prospective study
[Articolo su rivista]
Sena, P.; Mancini, S.; Bertacchini, J.; Carnevale, G.; Pedroni, M.; Roncucci, L.
abstract
Colorectal cancer represents a paradigmatic model of inflammatory carcinogenesis accom-panied by the production of several kinds of tumor-associated autoantibodies (TAABs). The specific aim of this study is to define the clinical impact of the presence of non-specific circulating TAABs in a cohort of cancer patients and to establish whether significant differences were present between colorectal cancer and cancers at other sites. For this aim a prospective study was developed and a five-year survival analysis performed. Indirect immunofluorescence on rat tissues for non-organ specific autoantibodies (NOSAs: liver-kidney-stomach), on rat colon substrates (colon-related autoantibodies, CAAs) and on HEp-2 cell lines was performed. NOSA positivity was more frequent in patients with colorectal cancer than in those with cancer at other sites. Survival analysis demonstrated a significantly worse prognosis in cancer patients positive for TAABs. CAA positivity is a predictor of survival, independently from the presence of comorbidities, and HEp-2 reactivity was a strong predictor of survival in a stepwise Cox-regression model, including stage at diagnosis. Overall overproduction of TAABs is associated with advanced oncological disease, the presence of metastasis, and poorer prognosis of cancer patients.
2021
- Evaluation of antimicrobial effect of air-polishing treatments and their influence on human dental pulp stem cells seeded on titanium disks
[Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Bertacchini, J.; Colombari, B.; Conserva, E.; Blasi, E.; Consolo, U.; Carnevale, G.
abstract
Dental implants are one of the most frequently used treatment options for tooth replacement, and titanium is the metal of choice due to its demonstrated superiority in resisting corrosion, lack of allergic reactions and mechanical strength. Surface roughness of titanium implants favors the osseointegration process; nevertheless, its topography may provide a suitable substrate for bacterial biofilm deposition, causing peri-implantitis and leading to implant failure. Subgingival prophylaxis treatments with cleansing powders aimed to remove the bacterial accumulation are under investigation. Two different air-polishing powders-glycine and tagatose-were assayed for their cleaning and antimicrobial potential against a Pseudomonas biofilm and for their effects on human dental pulp stem cells (hDPSCs), seeded on sandblasted titanium disks. Immunofluorescence analyses were carried out to evaluate cell adhesion, proliferation, stemness and osteogenic differentiation. The results demonstrate that both the powders have a great in vitro cleaning potential in the early period and do not show any negative effects during hDPSCs osteogenic differentiation process, suggesting their suitability for enhancing the biocompatibility of titanium implants. Our data suggest that the evaluated cleansing systems reduce microbial contamination and allow us to propose tagatose as an adequate alternative to the gold standard glycine for the air-polishing prophylaxis treatment.
2021
- Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk
[Articolo su rivista]
Magarò, Maria Sara; Bertacchini, Jessika; Florio, Francesca; Zavatti, Manuela; Potì, Francesco; Cavani, Francesco; Amore, Emanuela; De Santis, Ilaria; Bevilacqua, Alessandro; Reggiani Bonetti, Luca; Torricelli, Pietro; Maurel, Delphine B.; Biressi, Stefano; Palumbo, Carla
abstract
Bone and muscle have been recognized as endocrine organs since they produce and
secrete “hormone-like factors” that can mutually influence each other and other tissues, giving rise
to a “bone–muscle crosstalk”. In our study, we made use of myogenic (C2C12 cells) and osteogenic
(2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation
process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell
differentiation and we identified sclerostin as one of the myokines produced by muscle cells.
Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered
a negative regulator of bone growth due to its role as an antagonist of the Wnt/β-catenin pathway.
Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how
it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin
synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting,
real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of
sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics.
This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed
that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan.
Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age)
via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects
of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the
muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests
that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and
potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our
data point out a role for muscle as a new source of sclerostin.
2021
- Mechanisms involved in the promoting activity of fibroblasts in HTLV-1-mediated lymphomagenesis: Insights into the plasticity of lymphomatous cells
[Articolo su rivista]
Rigotto, G.; Montini, B.; Mattiolo, A.; Lazzari, N.; Piano, M. A.; Remondini, D.; Marmiroli, S.; Bertacchini, J.; Chieco-bianchi, L.; Calabro, M. L.
abstract
Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.
2021
- Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells
[Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Pignatti, E.; Maccaferri, M.; Bertacchini, J.; Sena, P.; Vallarola, A.; Tupler, R.; Croci, S.; Bonacini, M.; Salvarani, C.; Carnevale, G.
abstract
Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.
2021
- Surface properties modulate protein corona formation and determine cellular uptake and cytotoxicity of silver nanoparticles
[Articolo su rivista]
Barbalinardo, M.; Bertacchini, J.; Bergamini, L.; Magaro, M. S.; Ortolani, L.; Sanson, A.; Palumbo, C.; Cavallini, M.; Gentili, D.
abstract
Nanoparticles (NPs) have been studied for biomedical applications, ranging from prevention, diagnosis and treatment of diseases. However, the lack of the basic understanding of how NPs interact with the biological environment has severely limited their delivery efficiency to the target tissue and clinical translation. Here, we show the effective regulation of the surface properties of NPs, by controlling the surface ligand density, and their effect on serum protein adsorption, cellular uptake and cytotoxicity. The surface properties of NPs are tuned through the controlled replacement of native ligands, which favor protein adsorption, with ligands capable of increasing protein adsorption resistance. The extent and composition of the protein layer adsorbed on NPs are strongly correlated to the degree of ligands replaced on their surface and, while BSA is the most abundant protein detected, ApoE is the one whose amount is most affected by surface properties. On increasing the protein resistance, cellular uptake and cytotoxicity in mouse embryonic fibroblasts of NPs are drastically reduced, but the surface coating has no effect on the process by which NPs mainly induce cell death. Overall, this study reveals that the tuning of the surface properties of NPs allows us to regulate their biological outcomes by controlling their ability to adsorb serum proteins. This journal is
2021
- The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine
[Articolo su rivista]
Paganelli, F.; Chiarini, F.; Palmieri, A.; Martinelli, M.; Sena, P.; Bertacchini, J.; Roncucci, L.; Cappellini, A.; Martelli, A. M.; Bonucci, M.; Fiorentini, C.; Ferri, I. H.
abstract
The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with im-munomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer.
2020
- Scleral ossicles: Angiogenic scaffolds, a novel biomaterial for regenerative medicine applications
[Articolo su rivista]
Checchi, M.; Bertacchini, J.; Cavani, F.; Magaro, M. S.; Reggiani Bonetti, L.; Pugliese, G. R.; Tamma, R.; Ribatti, D.; Maurel, D. B.; Palumbo, C.
abstract
Given the current prolonged life expectancy, various pathologies affect increasingly the aging subjects. Regarding the musculoskeletal apparatus, bone fragility induces more susceptibility to fractures, often not accompanied by good ability of self-repairing, in particular when critical-size defects (CSD) occur. Currently orthopedic surgery makes use of allografting and autografting which, however, have limitations due to the scarce amount of tissue that can be taken from the donor, the possibility of disease transmission and donor site morbidity. The need to develop new solutions has pushed the field of tissue engineering (TE) research to study new scaffolds to be functionalized in order to obtain constructs capable of promoting tissue regeneration and achieve stable bone recovery over time. This investigation focuses on the most important aspect related to bone tissue regeneration: the angiogenic properties of the scaffold to be used. As an innovative solution, scleral ossicles (SOs), previously characterized as natural, biocompatible and spontaneously decellularized scaffolds used for bone repair, were tested for angiogenic potential and biocompatibility. To reach this purpose, in ovo Chorioallantoic Membrane Assay (CAM) was firstly used to test the angiogenic potential; secondly, in vivo subcutaneous implantation of SOs (in a rat model) was performed in order to assess the biocompatibility and the inflammatory response. Finally, thanks to the analysis of mass spectrometry (LCMSQE), the putative proteins responsible for the SO angiogenic properties were identified. Thus, a novel natural biomaterial is proposed, which is (i) able to induce an angiogenic response in vivo by subcutaneous implantation in a non-immunodeficient animal model, (ii) which does not induce any inflammatory response, and (iii) is useful for regenerative medicine application for the healing of bone CSD.
2020
- WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis
[Articolo su rivista]
Smargiassi, A.; Bertacchini, J.; Checchi, M.; Poti, F.; Tenedini, E.; Montosi, G.; Magaro, M. S.; Amore, E.; Cavani, F.; Ferretti, M.; Grisendi, G.; Maurel, D. B.; Palumbo, C.
abstract
The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
2019
- Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit
[Articolo su rivista]
Bertacchini, Jessika; Mediani, Laura; Beretti, Francesca; Guida, Marianna; Ghalali, Aram; Brugnoli, Federica; Bertagnolo, Valeria; Petricoin, Emanuel; Poti, Francesco; Arioli, Jessica; Anselmi, Laura; Bari, Alessia; Mccubrey, James; Martelli, Alberto M.; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra
abstract
Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.
2019
- Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche
[Articolo su rivista]
Sola, Antonella; Bertacchini, Jessika; D'Avella, Daniele; Anselmi, Laura; Maraldi, Tullia; Marmiroli, Sandra; Messori, Massimo
abstract
The need for new approaches to investigate ex vivo the causes and effects of tumor and to achieve improved cancer treatments and medical therapies is particularly urgent for malignant pathologies such as lymphomas and leukemias, whose tissue initiator cells interact with the stroma creating a three-dimensional (3D) protective environment that conventional mono- and bi-dimensional (2D) models are not able to simulate realistically. The solvent-casting particulate leaching (SCPL) technique, that is already a standard method to produce polymer-based scaffolds for bone tissue repair, is proposed here to fabricate innovative 3D porous structures to mimic the bone marrow niche in vitro. Two different polymers, namely a rigid polymethyl methacrylate (PMMA) and a flexible polyurethane (PU), were evaluated to the purpose, whereas NaCl, in the form of common salt table, resulted to be an efficient porogen. The adoption of an appropriate polymer-to-salt ratio, experimentally defined as 1:4 for both PMMA and PU, gave place to a rich and interconnected porosity, ranging between 82.1 vol% and 91.3 vol%, and the choice of admixing fine-grained or coarse-grained salt powders allowed to control the final pore size. The mechanical properties under compression load were affected both by the polymer matrix and by the scaffold's architecture, with values of the elastic modulus indicatively varying between 29 kPa and 1283 kPa. Preliminary tests performed with human stromal HS-5 cells co-cultured with leukemic cells allowed us to conclude that stromal cells grown associated to the supports keep their well-known protective and pro-survival effect on cancer cells, indicating that these devices can be very useful to mimic the bone marrow microenvironment and therefore to assess the efficacy of novel therapies in pre-clinical studies.
2019
- Gnrh antagonists produce differential modulation of the signaling pathways mediated by gnrh receptors
[Articolo su rivista]
Sperduti, S.; Limoncella, S.; Lazzaretti, C.; Paradiso, E.; Riccetti, L.; Turchi, S.; Ferrigno, I.; Bertacchini, J.; Palumbo, C.; Poti, F.; Longobardi, S.; Millar, R. P.; Simoni, M.; Newton, C. L.; Casarini, L.
abstract
Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix,.
2019
- Interaction among Calcium Diet Content, PTH (1-34) Treatment and Balance of Bone Homeostasis in Rat Model: The Trabecular Bone as Keystone
[Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Roli, Laura; Checchi, Marta; Magarò, Maria Sara; Bertacchini, Jessika; Palumbo, Carla
abstract
The present study is the second step (concerning normal diet restoration) of the our previous study (concerning the calcium-free diet) to determine whether normal diet restoration, with/without concomitant PTH (1-34) administration, can influence amounts and deposition sites of the total bone mass. Histomorphometric evaluations and immunohistochemical analysis for Sclerostin expression were conducted on the vertebral bodies and femurs in the rat model. The final goals are (i) to define timing and manners of bone mass changes when calcium is restored to the diet, (ii) to analyze the different involvement of the two bony architectures having different metabolism (i.e., trabecular versus cortical bone), and (iii) to verify the eventual role of PTH (1-34) administration. Results evidenced the greater involvement of the trabecular bone with respect to the cortical bone, in response to different levels of calcium content in the diet, and the effect of PTH, mostly in the recovery of trabecular bony architecture. The main findings emerged from the present study are (i) the importance of the interplay between mineral homeostasis and skeletal homeostasis in modulating and guiding bone's response to dietary/metabolic alterations and (ii) the evidence that the more involved bony architecture is the trabecular bone, the most susceptible to the dynamical balance of the two homeostases.
2019
- Trabecular Bone as Keystone for the Interplay Among Calcium Diet Content, PTH(1-34) Treatment and Balance of Bone Homeostases in Rat Model
[Capitolo/Saggio]
Ferretti, Marzia; Cavani, Francesco; Checchi, Marta; Magaro', MARIA SARA; Amore, Emanuela; Bertacchini, Jessika; Palumbo, Carla
abstract
The present study aims to determine whether normal-diet restoration, with/without concomitant PTH(1-34) administration, can influence amounts and
deposition sites of the total bone mass. Histomorphometric evaluations and immunohistochemical analysis for Sclerostin expression were conducted on the vertebral bodies and femurs in the rat model.
Final goals 1) to define timing and manners of bone mass changes when calcium is restored to the diet; 2) to analyze the different involvement of the two bony architectures having different metabolism (i.e. trabecular versus cortical bone); 3) to verify the eventual role of PTH(1-34) administration. Results evidenced the greater involvement of the trabecular bone with respect to the cortical bone, in response to differing levels of calcium content in the diet, and the effect of PTH, mostly in the recovery of trabecular bony architecture.
The main findings emerged are: i) the importance of the interplay between mineral homeostasis and skeletal homeostasis in modulating and guiding bone’s response to dietary/metabolic alterations and ii) the evidence that the more involved bony architecture is trabecular bones, the most susceptible to the dynamical balance of mineral and skeletal homeostasis.
2018
- Aberrant DNA Methylation in Chronic Myeloid Leukemia: Cell Fate Control, Prognosis, and Therapeutic Response
[Articolo su rivista]
Behzad, Masumeh Maleki; Shahrabi, Saeid; Jaseb, Kaveh; Bertacchini, Jessika; Ketabchi, Neda; Saki, Najmaldin
abstract
Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy characterized by the expression of the BCR-ABL1 fusion gene with different chimeric transcripts. Despite the crucial impact of constitutively active tyrosine kinase in CML pathogenesis, aberrant DNA methylation of certain genes plays an important role in disease progression and the development of drug resistance. This article reviews recent findings relevant to the effect of DNA methylation pattern of regulatory genes on various cellular activities such as cell proliferation and survival, as well as cell-signaling molecules in CML. These data might contribute to defining the role of aberrant DNA methylation in disease initiation and progression. However, further studies are needed on the validation of specific aberrant methylation markers regarding the prognosis and prediction of response among the CML patients.
2018
- Angiogenic and inflammatory potential of Scleral Ossicles, novel natural biomaterials for bone regeneration
[Abstract in Rivista]
Checchi, Marta; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Sola, Antonella; Bisi, Francesca; Messori, Massimo; Ribatti, Domenico; Maurel, Delphine; Palumbo, Carla
abstract
The aim of this work is the analysis of the angiogenic and inflammatory potential of the Scleral Ossicles (SOs), already analysed by the structural viewpoint, and the development of a functionalized-SOs-construct. The final goal is to improve the healing of critical-size bone fractures.
2018
- Dual inhibition of PI3K/mTOR signaling in chemoresistant AMLprimary cells.
[Articolo su rivista]
Bertacchini, Jessika; Frasson, Chiara; Chiarini, Francesca; D'Avella, Daniele; Accordi, Benedetta; Anselmi, Laura; Barozzi, Patrizia; Foghieri, Fabio; Luppi, Mario; Martelli, Alberto M.; Basso, Giuseppe; Najmaldin, Saki; Khosravi, Abbas; Rahim, Fakher; Marmiroli, Sandra
abstract
A main cause of treatment failure for AML patients is resistance to chemotherapy. Survival of AML cells may depend on mechanisms that elude conventional drugs action and/or on the presence of leukemia initiating cells at diagnosis, and their persistence after therapy. MDR1 gene is an ATP-dependent drug efflux pump known to be a risk factor for the emergence of resistance, when combined to unstable cytogenetic profile of AML patients. In the present study, we analyzed the sensitivity to conventional chemotherapeutic drugs of 26 samples of primary blasts collected from AML patients at diagnosis. Detection of cell viability and apoptosis allowed to identify two group of samples, one resistant and one sensitive to in vitro treatment. The cells were then analyzed for the presence and the activity of P-glycoprotein. A comparative analysis showed that resistant samples exhibited a high level of MDR1 mRNA as well as of P-glycoprotein content and activity. Moreover, they also displayed high PI3K signaling. Therefore, we checked whether the association with signaling inhibitors might resensitize resistant samples to chemo-drugs. The combination showed a very potent cytotoxic effect, possibly through down modulation of MDR1, which was maintained also when primary blasts were co-cultured with human stromal cells. Remarkably, dual PI3K/mTOR inactivation was cytotoxic also to leukemia initiating cells. All together, our findings indicate that signaling activation profiling associated to gene expression can be very useful to stratify patients and improve therapy.
2018
- Interaction between mineral and skeletal homeostasis in rats fed different calcium content diets with/without PTH (1-34)
[Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Bertacchini, Jessika; Checchi, Marta; Magaro', MARIA SARA; Palumbo, Carla
abstract
Aim of the study is to analyze how mineral and skeletal homeostases influence both the bone loss due to calcium-diet deprivation and the successive bone mass recovery after calcium-diet restoration, with/without concomitant PTH(1-34) administration.
2018
- Muscle-to-bone crosstalk: the Wnt/-catenin pathway is a candidate mechanism mediating the signalling between C2C12 muscle cells and 2T3 osteoblasts
[Abstract in Rivista]
Magaro', MARIA SARA; Bertacchini, Jessika; Poti', Francesco; Checchi, Marta; Benincasa, Marta; Sena, Paola; Palumbo, Carla
abstract
The study aims to determine whether myokines can potentially regulate osteogenesis,
2018
- Osteocytes Specific GSK3 Inhibition Affects In Vitro Osteogenic Differentiation.
[Articolo su rivista]
Bertacchini, J; Magaro', MARIA SARA; Potì, F; Palumbo, C
abstract
Osteocytes, the most important regulators of bone processes, are producers of molecules (usually proteins) that act as signals in order to communicate with nearby cells. These factors control cell division (proliferation), differentiation, and survival. Substantial evidence showed different signaling pathways activated by osteocytes and involved in osteoblast differentiation, in particular in the last decade, when the Wingless-related integration site (WNT) pathway assumed a critical large importance. WNT activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism, making GSK3 a potential therapeutic target for bone diseases. In our study, we hypothesized an important role of the osteocyte MLO-Y4 conditioned medium in controlling the differentiation process of osteoblast cell line 2T3. We found an effect of diminished differentiation capability of 2T3 upon conditioning with medium from murine long bone osteocyte-Y4 cells (MLO-Y4) pre-treated with GSK3 inhibitor CHIR2201. The novel observations of this study provide knowledge about the inhibition of GSK3 in MLO-Y4 cells. This strategy could be used as a plausible target in osteocytes in order to regulate bone resorption mediated by a loss of osteoblasts activity through a paracrine loop.
2018
- Proposal of a Novel Natural Biomaterial, the Scleral Ossicle, for the Development of Vascularized Bone Tissue In Vitro
[Articolo su rivista]
Checchi, Marta; Bertacchini, Jessika; Grisendi, Giulia; Smargiassi, Alberto; Sola, Antonella; Messori, Massimo; Palumbo, Carla
abstract
Recovering of significant skeletal defects could be partially abortive due to the perturbations that affect the regenerative process when defects reach a critical size, thus resulting in a non-healed bone. The current standard treatments include allografting, autografting, and other bone implant techniques. However, although they are commonly used in orthopedic surgery, these treatments have some limitations concerning their costs and their side effects such as potential infections or malunions. On this account, the need for suitable constructs to fill the gap in wide fractures is still urgent. As an innovative solution, scleral ossicles (SOs) can be put forward as natural scaffolds for bone repair. SOs are peculiar bony plates forming a ring at the scleral-corneal border of the eyeball of lower vertebrates. In the preliminary phases of the study, these ossicles were structurally and functionally characterized. The morphological characterization was performed by SEM analysis, MicroCT analysis and optical profilometry. Then, UV sterilization was carried out to obtain a clean support, without neither contaminations nor modifications of the bone architecture. Subsequently, the SO biocompatibility was tested in culture with different cell lines, focusing the attention to the differentiation capability of endothelial and osteoblastic cells on the SO surface. The results obtained by the above mentioned analysis strongly suggest that SOs can be used as bio-scaffolds for functionalization processes, useful in regenerative medicine.
2018
- Wisp2 overexpression induced by short Teriparatide treatment affects IDG-SW3 osteogenic differentiation.
[Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Magaro', MARIA SARA; Poti', Francesco; Tenedini, Elena; Montosi, Giuliana; Magaro', MARIA SARA; Vinet, Jonathan; Maurel, Delphine; Palumbo, Carla
abstract
The study supports the importance of osteocytes in controlling the action of the other bone cells and suggests that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
2017
- Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function
[Articolo su rivista]
Morelli F., F.; Verbeek D., S.; Bertacchini, Jessika; Vinet, Jonathan; Mediani, Laura; Marmiroli, Sandra; Cenacchi, G.; Nasi, Milena; DE BIASI, Sara; Brunsting J., F.; Lammerding, J.; Pegoraro, E.; Angelini, C.; Tupler, Rossella; Alberti, S.; Carra, Serena
abstract
Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy.
2017
- An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8
[Articolo su rivista]
Morelli, FEDERICA FRANCESCA; Mediani, Laura; Heldens, Lonneke; Bertacchini, Jessika; Bigi, Ilaria; Carrà, Arianna Dorotea; Vinet, Jonathan; Carra, Serena
abstract
The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction.
2017
- Biocompatibility Analyses of Al₂O₃-Treated Titanium Plates Tested with Osteocyte and Fibroblast Cell Lines
[Articolo su rivista]
Smargiassi, Alberto; Bertacchini, Jessika; Checchi, Marta; Cavani, Francesco; Ferretti, Marzia; Palumbo, Carla
abstract
Osseointegration of a titanium implant is still an issue in dental/orthopedic implants durable over time. The good integration of these implants is mainly due to their surface and topography. We obtained an innovative titanium surface by shooting different-in-size particles of Al₂O₃ against the titanium scaffolds which seems to be ideal for bone integration. To corroborate that, we used two different cell lines: MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) and tested the titanium scaffolds untreated and treated (i.e., Al₂O₃ shot-peened titanium surfaces). Distribution, density, and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM). DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin, and vitronectin, under CM; cell distribution was analyzed after gold-palladium sputtering of samples by SEM. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: (i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and (ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of stable dental function over time.
2017
- Cross-talk between the CK2 and AKT signaling pathways in cancer
[Articolo su rivista]
Ruzzene, Maria; Bertacchini, Jessika; Toker, Alex; Marmiroli, Sandra
abstract
CK2 and AKT display a high degree of cross-regulation of their respective functions, both directly, through physical interaction and phosphorylation, and indirectly, through an intense cross-talk of key downstream effectors, ultimately leading to sustained AKT activation.Being CK2 and AKT attractive targets for therapeutic intervention, here we would like to emphasize how AKT and CK2 might influence cell fate through their complex isoform-specific and contextual-dependent cross-talk, to the extent that such functional interplay should be considered when devising therapies that target one or both these key signaling kinases.
2017
- Expression and functional proteomic analyses of osteocytes from Xenopus laevis tested under mechanical stress conditions: preliminary observations on an appropriate new animal model
[Articolo su rivista]
Bertacchini, Jessika; Benincasa, Marta; Checchi, Marta; Cavani, Francesco; Smargiassi, Alberto; Ferretti, Marzia; Palumbo, Carla
abstract
Hitherto, the role of the osteocyte as transducer of mechanical stimuli into biological signals is far from settled. In this study, we used an appropriate model represented by the cortex of Xenopus laevis long bone diaphysis lacking (unlike the mammalian one) of vascular structures and containing only osteocytes inside the bone matrix. These structural features allow any change of protein profile that might be observed upon different experimental conditions, such as bone adaptation to stress/mechanical loading, to be ascribed specifically to osteocytes. The study was conducted by combining ultrastructural observations and two-dimensional electrophoresis for proteomic analysis. The osteocyte population was extracted from long bones of lower limbs of amphibian skeletons after different protocols (free and forced swimming). The experiments were performed on 210 frogs subdivided into five trials, each including free swimming frogs (controls) and frogs submitted to forced swimming (stressed). The stressed groups were obliged to swim (on movable spheres covering the bottom of a pool on a vibrating plate) continuously for 8 h, and killed 24 h later along with the control groups. Long bones free of soft tissues (periosteum, endosteum and bone marrow), as well as muscles of posterior limbs, were processed and analyzed for proteins differentially expressed or phosphorylated between the two sample groups. The comparative analysis showed that protein phosphorylation profiles differ between control and stressed groups. In particular, we found in long bones of stressed samples that both Erk1/2 and Akt are hyperphosphorylated; moreover, the different phosphorylation of putative Akt substrates (recognized by specific Akt phosphosubstrates-antibody) in stressed vs. control samples clearly demonstrated that Akt signaling is boosted by forced swimming (leading to an increase of mechanical stress) of amphibian long bones. In parallel, we found in posterior limb muscles that the expression of heat shock protein HSP27 and HSP70 stress markers increased upon the forced swimming condition. Because the cortexes of frog long bones are characterized by the presence of only osteocytes, all our results establish the suitability of the X. laevis animal model to study the bone response to stress conditions mediated by this cell type and pave the way for further analysis of the signaling pathways involved in these signal transduction mechanisms.
2017
- Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization
[Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Tenedini, Elena; Montosi, Giuliana; Vinet, Jonathan; Ferretti, Marzia; Palumbo, Carla
abstract
PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.
2017
- Scleral ossicles as natural biomaterials on which vascular-like network is promoted from Mouse Aortic Endothelial cells (MAECs): preliminary results
[Abstract in Rivista]
Checchi, Marta; Grisendi, Giulia; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Benincasa, Marta; Sena, Paola; Cavani, Francesco; Palumbo, Carla
abstract
When a severe fracture is difficult to self-recovered, it is defined as “critical-size” bone
defect. Till now, many efforts have been made by the tissue engineering (TE) to generate
scaffolds suitable for recovering of this type of fracture, but the main obstacle remains
the lack of an appropriate vascularization of the scaffolds. In the field of the regenerative
medicine, the TE has developed many different biomaterials, with various features and
peculiar functions, to be used in combination with cells and growth factors, in the generation
of specialized constructs. Our proposal of natural scaffolds useful to obtain complex
constructs concerns peculiar bony chips extracted from the eye bulb of adult chickens:
the scleral ossicles (SOs). This proposed model is interesting because once SOs reach the
definitive size in the adult animal, they are devoted only to mechanical stereotyped stress
for their lifetime so that the activation of the bone remodelling should be avoided and, to
do this, the osteocytes undergo massive apoptosis, making the ossicles like decellularized
bones [1]. The novelty of our proposal is that the scaffolds do not require surface treatment
(like further matrix deposition on the SO surface) since they are characterized, like
all bones, by the well-known organic components such as type I-collagen fibres, proteoglycans
and glycoproteins. The latter, for example, play the role of adhesion proteins and
therefore can mediate the adhesion of the endothelial cells that should develop the vascular
network. Our final goal is to obtain an in vitro 3D-vascularized natural constructs,
from scaffolds easily available in nature to use in vivo for the healing of “critical-size”
bone defeats. Previously [2] we identified the best preparation methods to obtain suitable
SO surface for cell culture. Recently, we have performed a series of in vitro experiments to
test the biocompatibility properties of the support; then, cell adhesion tests, viability and
proliferation assay were carried out. Further, we tried to induce a vascular-like network
organization of Mouse Aortic Endothelial Cells (MAECs) directly on the SOs surface, stimulating
the cells with a known angiogenic factor, the Vascular Endothelial Growth Factor
(VEGF), getting encouraging preliminary results.
2017
- Significance of Inactivated Genes in Leukemia: Pathogenesis and Prognosis
[Articolo su rivista]
Heidari, N.; Abroun, S.; Bertacchini, J.; Vosoughi, T.; Rahim, F.; Saki, N.
abstract
Epigenetic and genetic alterations are two mechanisms participating in leukemia, which can inactivate genes involved in leukemia pathogenesis or progression. The purpose of this review was to introduce various inactivated genes and evaluate their possible role in leukemia pathogenesis and prognosis. By searching the mesh words "Gene, Silencing AND Leukemia" in PubMed website, relevant English articles dealt with human subjects as of 2000 were included in this study. Gene inactivation in leukemia is largely mediated by promoter's hypermethylation of gene involving in cellular functions such as cell cycle, apoptosis, and gene transcription. Inactivated genes, such as ASPP1, TP53, IKZF1 and P15, may correlate with poor prognosis in acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), respectively. Gene inactivation may play a considerable role in leukemia pathogenesis and prognosis, which can be considered as complementary diagnostic tests to differentiate different leukemia types, determine leukemia prognosis, and also detect response to therapy. In general, this review showed some genes inactivated only in leukemia (with differences between B-ALL, T-ALL, CLL, AML and CML). These differences could be of interest as an additional tool to better categorize leukemia types. Furthermore; based on inactivated genes, a diverse classification of Leukemias could represent a powerful method to address a targeted therapy of the patients, in order to minimize side effects of conventional therapies and to enhance new drug strategies.
2017
- Understanding the endocrine crosstalk between bone and muscle: molecular investigation of the impact of myokines on osteogenesis using C2C12 myoblast and 2T3 osteoblast cell lines
[Abstract in Rivista]
Magaro', MARIA SARA; Bertacchini, Jessika; Checchi, Marta; Palumbo, Carla
abstract
Bones and skeletal muscles interact mechanically to allow locomotion in vertebrate and even invertebrate organisms. Until the last decade of research, the interactions between them had been gathered under the umbrella of the “mechanical coupling” theory, where muscles are the load suppliers and bones provide the attachment sites [1]. However, bone and skeletal muscle have recently been identified as endocrine organs, that secrete cytokines and chemokines, through which they interact to promote locomotion. This molecular and biochemical interplay has been named “bone-muscle crosstalk”. The bi-directional flow of signals between bone and muscle has been investigated experimentally by differentiating bone or skeletal muscle progenitor cells in a medium conditioned by myotubes or osteocytes respectively [2][3]. These studies have demonstrated that osteocyte (osteokines) and myotube (myokines) secreted factors have an inhibitory influence on myogenesis and osteogenesis respectively, since they reduce the majority of the mRNA levels of genes associated with differentiation. We propose to study the effects of myokines on osteogenesis by differentiating 2T3 osteoblastic cells in a medium conditioned by either early (3-5 days) or late (7-10 days) myo-tubes. We will then analyze mRNA and protein levels of marker genes of differentia-tion, to establish the effect of early and late patterns of myokines. Besides, we will characterize the differentiation process from a functional point of view by studying alkaline phosphatase activity and the deposition of mineralized matrix. As expected results, early and late myotube-conditioned media should affect differently the osteoblast lineage in the course of differentiation. If this is the case, we will proceed with a metabolomic profiling of the conditioned medium, to identify the cytokines most abundantly expressed. This first set of results will pave the way for further experiments of myoblast and osteoblast co-culture aimed at a real-time tracking of the bi-directional signaling betweeen these tissues and its impact on all stages of differentiation. The results of this study will deepen our understanding of how the muscle secretome protects osteocytes and preserve their function and vice versa how bone factors maintain muscle function. Such knowledge will help identify potential new therapies for bone and muscle diseases, especially when they co-exist, as is the case of the twin syndrome of osteoporosis and sarcopenia.
2016
- Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling
[Articolo su rivista]
Mediani, Laura; Gibellini, Federica; Bertacchini, Jessika; Frasson, Chiara; Bosco, Raffaella; Accordi, Benedetta; Basso, Giuseppe; Bonora, Massimo; Calabrò, Maria Luisa; Mattiolo, Adriana; Sgarbi, Gianluca; Baracca, Alessandra; Pinton, Paolo; Riva, Giovanni; Rampazzo, Enrico; Petrizza, Luca; Prodi, Luca; Milani, Daniela; Luppi, Mario; Potenza, Leonardo; De Pol, Anto; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra
abstract
PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.
2016
- Synergistic cytotoxic effects of bortezomib and CK2 inhibitor CX-4945 in acute lymphoblastic leukemia: Turning offthe prosurvival ER chaperone BIP/Grp78 and turning on the proapoptotic NF-κB
[Articolo su rivista]
Buontempo, Francesca; Orsini, Ester; Lonetti, Annalisa; Cappellini, Alessandra; Chiarini, Francesca; Evangelisti, Camilla; Evangelisti, Cecilia; Melchionda, Fraia; Pession, Andrea; Bertaina, Alice; Locatelli, Franco; Bertacchini, Jessika; Neri, Luca Maria; Mccubrey, James A.; Martelli, Alberto Maria
abstract
The proteasome inhibitor bortezomib is a new targeted treatment option for refractory or relapsed acute lymphoblastic leukemia (ALL) patients. However, a limited efficacy of bortezomib alone has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. Recently, it has been documented that UPR disruption could be considered a selective anti-leukemia therapy. CX- 4945, a potent casein kinase (CK) 2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. In this study, we analyzed in T- and B-ALL preclinical settings, the molecular mechanisms of synergistic apoptotic effects observed after bortezomib/CX-4945 combined treatment. We demonstrated that, adding CX-4945 after bortezomib treatment, prevented leukemic cells from engaging a functional UPR in order to buffer the bortezomibmediated proteotoxic stress in ER lumen. We documented that the combined treatment decreased pro-survival ER chaperon BIP/Grp78 expression, via reduction of chaperoning activity of Hsp90. Bortezomib/CX-4945 treatment inhibited NF-κB signaling in T-ALL cell lines and primary cells from T-ALL patients, but, intriguingly, in B-ALL cells the drug combination activated NF-κB p65 pro-apoptotic functions. In fact in B-cells, the combined treatment induced p65-HDAC1 association with consequent repression of the anti-apoptotic target genes, Bcl-xL and XIAP. Exposure to NEMO (IKKγ)-binding domain inhibitor peptide reduced the cytotoxic effects of bortezomib/CX-4945 treatment. Overall, our findings demonstrated that CK2 inhibition could be useful in combination with bortezomib as a novel therapeutic strategy in both T- and B-ALL.
2015
- Inhibition of Ras-mediated signaling pathways in CML stem cells
[Articolo su rivista]
Bertacchini, Jessika; Ketabchi, Neda; Mediani, Laura; Capitani, Silvano; Marmiroli, Sandra; Saki, Najmaldin
abstract
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of the BCR-ABL1 oncoprotein in cells with a hematopoietic stem cell (HSC) origin. BCR-ABL1 tyrosine kinase activity leads to constitutive activation of Ras, which in turn acts as a branch point to initiate multiple downstream signaling pathways governing proliferation, self-renewal, differentiation and apoptosis. As aberrant regulation of these cellular processes causes transformation and disease progression particularly in advanced stages of CML, investigation of these signaling pathways may uncover new therapeutic targets for the selective eradication of CML stem cells. Transcription factors play a crucial role in unbalancing the Ras signaling network and have recently been investigated as potential modulators in this regard. In this review, we first briefly summarize the Ras-associated molecular pathways that are involved in the regulation of CML stem cell properties. Next we discuss the relevance of Ras-associated transcription factors as nuclear targets in combination treatment strategies for CML.
2015
- Targeting PI3K/AKT/mTOR network for treatment of leukemia
[Articolo su rivista]
Bertacchini, Jessika; Heidari, Nazanin; Mediani, Laura; Capitani, Silvano; Shahjahani, Mohammad; Ahmadzadeh, Ahmad; Saki, Najmaldin
abstract
Objective: Increased activity of PI3K/AKT/mTOR pathway has been observed in a huge number of malignancies. This pathway can function as a prosurvival factor in leukemia stem cells and early committed leukemic precursors and its inhibition is regarded as a therapeutic approach. Accordingly, the aim of this review is to evaluate the PI3K/Akt/mTOR inhibitors used in leukemia models. Discussion: Inhibition of the PI3K/AKT/mTOR pathway has been reported to have beneficial therapeutic effects in leukemias, both in vitro in leukemia cell lines and in vivo in animal models. Overall, the use of dual PI3K/mTOR inhibitor, dual Akt/RTK inhibitor, Akt inhibitor, selective inhibitor of PI3K, mTOR inhibitor and dual PI3K/PDK1 inhibitor in CML, AML, APL, CLL, B-ALL and T-ALL has a better therapeutic effect than conventional treatments. Conclusions: Targeting the PI3K/Akt/mTOR pathway may have pro-apoptotic and antiproliferative effects on hematological malignancies. Furthermore, modulation of miRNA can be used as a novel therapeutic approach to regulate the PI3K/Akt/mTOR pathway. However, both aspects require further clinical studies.
2015
- The role of HDACs as leukemia therapy targets using HDI
[Articolo su rivista]
Ahmadzadeh, A.; Khodadi, E.; Shahjahani, M.; Bertacchini, J.; Vosoughi, T.; Saki, N.
abstract
Histone deacetylases (HDACs) are the enzymes causing deacetylation of histone and non-histone substrates. Histone deacetylase inhibitors (HDIs) are a family of drugs eliminating the effect of HDACs in malignant cells via inhibition of HDACs. Due to extensive effects upon gene expression through interference with fusion genes and transcription factors, HDACs cause proliferation and migration of malignant cells, inhibiting apoptosis in these cells via tumor suppressor genes. Over expression evaluation of HDACs in leukemias may be a new approach for diagnosis of leukemia, which can present new targets for leukemia therapy. HDIs inhibit HDACs, increase acetylation in histones, cause up- or down regulation in some genes and result in differentiation, cell cycle arrest and apoptosis induction in malignant cells via cytotoxic effects. Progress in identification of new HDIs capable of tracking several targets in the cell can result in novel achievements in treatment and increase survival in patients. In this review, we examine the role of HDACs as therapeutic targets in various types of leukemia as well as the role of HDIs in inhibition of HDACs for treatment of these malignancies.
2014
- Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis
[Articolo su rivista]
Bertacchini, Jessika; Guida, M; Accordi, B; Mediani, Laura; Martelli, A. M; Barozzi, Patrizia; Petricoin, E; Liotta, L; Milani, G; Giordan, M; Luppi, Mario; Forghieri, Fabio; DE POL, Anto; Cocco, L; Basso, G; Marmiroli, Sandra
abstract
Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.
2014
- Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics
[Articolo su rivista]
Cenni, Vittoria; Capanni, Cristina; Mattioli, Elisabetta; Columbaro, Marta; Wehnert, Manfred; Ortolani, Michela; Fini, Milena; Novelli, Giuseppe; Bertacchini, Jessika; Maraldi, Nadir M; Marmiroli, Sandra; D'Apice, Maria Rosaria; Prencipe, Sabino; Squarzoni, Stefano; Lattanzi, Giovanna
abstract
Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.
2013
- The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression
[Articolo su rivista]
Bertacchini, Jessika; Beretti, Francesca; Vittoria, Cenni; Guida, Marianna; Federica, Gibellini; Mediani, Laura; Oriano, Marin; Nadir M., Maraldi; DE POL, Anto; Giovanna, Lattanzi; Lucio, Cocco; Marmiroli, Sandra
abstract
The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to name those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and their precursor prelamin A (collectively called A-type lamins) are type V intermediate filaments proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions, and are therefore bona fide substrates of Akt. We describe here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt regulates also Lmna transcription. All together, our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamins physiology to therapeutic approaches for lamin A-linked disorders.
2011
- Reverse-phase protein microarrays (RPPA) as a diagnosticand therapeutic guide in multidrug resistant leukemia
[Articolo su rivista]
Maraldi, Tullia; Bertacchini, Jessika; Benincasa, Marta; M., Guida; DE POL, Anto; L., Liotta LA; E., Petricoin; L., Cocco; Marmiroli, Sandra
abstract
Abstract. Reverse-phase microarray assays using phosphospecificantibodies (RPPA) can directly measure levels ofphosphorylated protein isoforms. In the current study, lysatesfrom parental and multidrug resistant (MDR) CEM leukemiacells were spotted onto reverse-phase protein microarraysand probed with a panel of phospho-antibodies to ERK, PCKand Akt pathways. In particular, the Akt pathway is consideredto play significant roles in leukemia and Akt inhibitor therapyhas been proposed as a potential tool in the treatment of thisdisease. The RPPA data prompted us to investigate deeperthis pathway. Here, we found that whereas total Akt1 proteinlevel is higher in parental CEM cells, the activated isoformcontent, p-Akt1, increases in doxorubicin-selected CEM cells(MDR-CEM). This was backed up by Western blot analysis,confirming that Akt1 activity/phosphorylation may be upregulatedin MDR-CEM cells. Further exploration of inhibitorytherapy in this system was evaluated. The TNF-relatedapoptosis-inducing ligand, TRAIL, has been shown toselectively kill tumor cells. Herein, we describe that in MDRCEMcells TRAIL responsiveness correlates with a reducedexpression of endogenous Akt1, suggesting that the MDRphenotype associated to P-gp sensitizes cells to TRAIL therapy.
2009
- A-type lamins and signaling: the PI 3-kinase/Akt pathway moves forward
[Articolo su rivista]
Marmiroli, Sandra; Bertacchini, Jessika; Beretti, Francesca; V., Cenni; M., Guida; DE POL, Anto; N. M., Maraldi; G., Lattanzi
abstract
Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship.
2009
- Human MATER localization in specific cell domains of oocytes and follicular cells
[Articolo su rivista]
Sena, Paola; Riccio, Massimo; Marzona, Laura; A., Nicoli; T., Marsella; Marmiroli, Sandra; Bertacchini, Jessika; Fano, Rita Adriana; LA SALA, Giovanni Battista; DE POL, Anto
abstract
MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.
2009
- MATER protein as substrate of PKCepsilon in human cumulus cells.
[Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Sena, Paola; Marzona, Laura; A., Nicoli; Marca, A. L.; Marmiroli, Sandra; Bertacchini, Jessika; LA SALA, Giovanni Battista; DE POL, Anto
abstract
High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.
2009
- Molecular targeting of the PKC-β inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression
[Articolo su rivista]
Verdelli, D; Nobili, L; Todoerti, K; Intini, D; Cosenza, Maria; Civallero, Monica; Bertacchini, Jessika; Deliliers, Gl; Sacchi, Stefano; Lombardi, L; Neri, A.
abstract
The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin-treated KMS-26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B-cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin-treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM.
2008
- Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12 Cells
[Articolo su rivista]
Vittoria, Cenni; Bertacchini, Jessika; Beretti, Francesca; Giovanna, Lattanzi; Alberto, Bavelloni; Riccio, Massimo; Maria, Ruzzene; Oriano, Marin; Giorgio, Arrigoni; Veena, Parnaik; Manfred, Wehnert; Nadir M., Maraldi; DE POL, Anto; Lucio, Cocco; Marmiroli, Sandra
abstract
Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.
2008
- The oral protein-kinase C beta inhibitor enzastaurin (LY317615) suppresses signalling through the AKT pathway, inhibits proliferation and induces apoptosis in multiple myeloma cell lines
[Articolo su rivista]
Antonino, Neri; Marmiroli, Sandra; Pierfrancesco, Tassone; Luigia, Lombardi; Lucia, Nobili; Donata, Verdelli; Monica, Civallero; Maria, Cosenza; Bertacchini, Jessika; Federico, Massimo; DE POL, Anto; Giorgio Lambertenghi, Deliliers; Sacchi, Stefano
abstract
Deregulation of the protein kinase C (PKC) signalling pathway has been implicated in tumor progression. Here we investigated the PKC inhibitor enzastaurin for its activity against multiple myeloma (MM) cells. Enzastaurin suppresses cell proliferation in a large panel of human myeloma cell lines (HMCLs), with IC50 values ranging from 1.3 to 12.5 mu M and induces apoptosis, which is prevented by the ZVAD-fmk broad caspase inhibitor. These results are consistent with decreased phosphorylation of AKT and GSK3-beta, a downstream target of the AKT pathway and a pharmacodynamic marker for enzastaurin. Furthermore, enzastaurin cytotoxicity is retained when HMCLs were cocultured with multipotent mesenchymal stromal cells. Enzastaurin has additive or synergistic cytotoxic effects with bortezomib or thalidomide. Considering the strong anti-myeloma activity of enzastaurin in vitro and in animal models and its safe toxicity profile, phase II studies in MM patients of enzastaurin alone or in combination with other drugs are warranted.
2007
- Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level
[Articolo su rivista]
DI MAIRA, G; Brustolon, F; Bertacchini, Jessika; Tosoni, K; Marmiroli, Sandra; Pinna, La; Ruzzene, M.
abstract
Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R.-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2 alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2 alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.