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Nicoletta Zini

Publications and source records attributed to Nicoletta Zini.

8 recordsLinked to original sources
PubMed2006

Chondrogenic differentiation of murine and human mesenchymal stromal cells in a hyaluronic acid scaffold: differences in gene expression and cell morphology.

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGFbeta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type I combined with a significant increase in collagen type II (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGFbeta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

Animals↗
PubMed2006

Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts.

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-zeta might play some fundamental role during myogenic differentiation of C2C12 cells.

Amanitins↗
PubMed2006

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.

Acids↗
PubMed2005

Quantitative immunodetection of key elements of polyphosphoinositide signal transduction in osteoblasts from arthritic patients shows a direct correlation with cell proliferation.

Phosphoinositides play an essential role in diverse cellular functions such as cell proliferation, cytoskeletal regulation, intracellular vesicle trafficking, motility, cell metabolism and death. Alteration of these pathways is common to many diseases. In this study, we show that osteoblasts from patients affected by osteoarthritis (OA) and by rheumatoid arthritis (RA) present a decreased cell proliferation and a reduced expression of the key elements of polyphosphoinositide signal transduction such as phosphatidylinositol-3-kinase (PI 3K), phospholipase C gamma1 (PLCgamma1), and protein kinase C zeta (PKCzeta) compared to the post-traumatic (PT) patients. Our results suggest that a correlation may exist between the reduced osteoblast proliferation observed in OA and RA patients and the lowered expression of PI 3K, PLCgamma1, and PKCzeta enzymes. The reduced proliferation rate of osteoblasts in response to these signal transduction effectors could counteract the evolution of arthritic disease.

Aged↗
PubMed2005

Cellular and molecular events during chondrogenesis of human mesenchymal stromal cells grown in a three-dimensional hyaluronan based scaffold.

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff-11). Cellular differentiation was induced using two different concentrations of TGFbeta1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGFbeta MSCs did not survive while in the presence of TGFbeta the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGFbeta at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGFbeta (20 ng/ml) induce chondrogenesis of MSCs on Hyaff-11 scaffold better than 10 ng/ml of TGFbeta. This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.

Biocompatible Materials↗
PubMed2003

IL1-beta and TNF-alpha induce changes in the nuclear polyphosphoinositide signalling system in osteoblasts similar to that occurring in patients with rheumatoid arthritis: an immunochemical and immunocytochemical study.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are common joint diseases that can lead to destruction of cartilage and structural changes in the subchondral bone. In this study we show by western blot and quantitative immunocytochemistry that nuclear phospholipase C beta(1) (PLC beta(1)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)), two key elements of the polyphosphoinositide signal transduction system that regulate different cellular processes, increase in primary osteoblast cultures of RA patients when compared with post-traumatic after fall (PT) patients, whilst those of OA are not significantly affected. Moreover, we demonstrate that these alterations could be induced in PT osteoblasts by proinflammatory cytokines IL-1 beta and TNF-alpha. This suggests that proinflammatory cytokines, highly produced by RA infiltrating mononuclear cells, can modulate the nuclear polyphosphoinositide signalling pathway of the osteoblasts involved in bone remodelling.

Arthritis, Rheumatoid↗
PubMed2003

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration.

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.

Animals↗