New frontiers of inositide-specific phospholipase C in nuclear signalling

Strong evidence has been obtained during the last 16 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. A number of advances has resulted in the discovery that nuclear phosphoinositides and their metabolizing enzymes are deeply involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Even though nuclear inositol lipids generate second messengers such as diacyglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence functions such as pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and up-dated findings about inositol lipid metabolism in the nucleus

Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2α distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies

Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step

Histological and clinical survey of polylactic-polyglicolic acid and dextrane copolymer in maxillary sinus lift:a pilot in vivo study

Of various proposed alternatives to autogenous bone, a synthetic, degradable copolymer of PLA-GLA and dextrane seems to be a promising biomaterial for maxillary sinus lift. Consecutive partially edentulous patients showing severe monolateral posterior maxillary atrophy were treated via sinus lift using PLA-GLA-dextrane copolymer as the sole filler. Delayed implant positioning was performed and cores of regenerated tissues and native bone controls were retrieved and evaluated by light and electron microscopy, histomorphometry, microhardness and qualitative X-ray analysis. Seven sinuses in 7 patients were augmented with PLA-GLA-dextrane copolymer. Six to nine months after the copolymer ‘graft’, 17 bone cores were retrieved: all histological sections contained newly synthesized, mineralized material and new bone in various stages of development. Histomorphometry revealed average Trabecular Bone Volume (TBV) values ranging from 51% (6 months) to 77% (9 months). Backscattered scanning electron microscopy (BSE) in experimental and control samples confirmed histology findings. Microhardness values suggested newly formed bone at nine months was not as hard as native bone. Ca and P content was similar in 9-month regenerated and native bone. Seventeen implants were inserted in the second stage of surgery: resulting Implant Success (SR) and Cumulative Success (CSR) up to 3 years were 100% following Albrektsson’s criteria. Sinus lift augmentation using PLA-GLA-dextrane copolymer as the sole filler resulted in uneventful surgeries. New bone formation was evident histologically and its maturation was still in progress after 9 months. Successful, staged implant positioning was achieved in regenerated tissue

Nuclear damages and oxidative stress: new perspectives for laminopathies

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients

3-D reconstruction of nuclear lamina shape

The localization of lamins A and C has been revealed by a FITC-conjugated monoclonal antibody in Swiss 3T3 fibroblasts by using confocal microscopy. 3-D reconstruction reveals that in these flattened cells, the nuclear lamina delimits coin-shaped nuclei. The relationship between the lamina and the chromatin has been studied by double labelling with the anti-lamin antibody and propidium iodide

Nuclear lipid-dependent signal transduction in human osteosarcoma cells.

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1