It is known that nuclear lipids play a role in proliferation, differentiation, and apoptotic process. Cellular nuclei contain high levels of phosphatidylcholine and sphingomyelin, which are partially linked with cholesterol and proteins to form lipid-protein complexes. These lipids are also associated with transcription factors and newly synthesized RNA but, up to date, their organization is still unknown. The aim of the present work was to study if these specific lipid-protein interactions could be nuclear membrane microdomains and to evaluate their possible role. The results obtained demonstrate for the first time the existence of nuclear microdomains characterized by a specific lipid composition similar to that of intranuclear lipid-protein complexes previously described. Nuclear microdomain lipid composition changes during cell proliferation when the content of newly synthesized RNA increases. Because previous data show a correlation between nuclear lipids and transcription process, the role of nuclear microdomains in cellular functions is discussed. INTRODUCTIONExtensive research on biological membranes has led to the modification of the Singer-Nicolson fluid-mosaic model and has indicated that certain classes of lipids and proteins are not randomly distributed over the membrane but form distinct microdomains (Lichtenberg et al., 2005) that can exist in equilibrium (Brown, 2006). The most studied class of microdomain are the cholesterol (CHO) and sphingomyelin (SM)-enriched lipid rafts (Edidin, 2003). However, the distinct concepts of lipid rafts, detergent-resistant membranes and liquid-ordered lipid phases are often confused in the current literature (Lichtenberg et al., 2005). Rafts are described as transient detergent-resistant membrane microdomains enriched in sphingolipids and CHO, whereas "detergent-resistant membranes" are formed after detergent treatment and do not correspond to any membrane structure. In contrast, "liquid-ordered lipid phase domains" are the result of interactions between high levels of CHO and phospholipid fatty acyl chains (Lichtenberg et al., 2005). On the other hand, several lines of investigation have supported the idea that detergent-resistant membranes are not detergent-induced artifacts, but do exist as domains in cellular membranes (Brown and London, 1997). As visualization of rafts in living cells is difficult, their existence and function rely on indirect methods such as detergent extraction (Munro, 2003). However, Schuck et al. (2003) have found that various detergents differ considerably in their ability to selectively solubilize membrane proteins and enrich the content of sphingolipids and CHO. Insolubility in detergents like Triton X-100 was observed in lipid bilayers, which exist in physical states where lipid packing is tight (London and Brown, 2000). However, Braccia et al. (2003) have demonstrated that isolation of lipid rafts from microvillar membrane with "conventional" Triton X-100 at low temperature and with Brij 98 at 37°C have essentially similar profiles ...

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCß1 having a role in the onset of DNA synthesis and PC-PLCc1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an en...

Although the role of 1alpha,25-dihydroxyvitamin D3 in calcium homeostasis of bone tissue is clear, evidence of the involvement of vitamin D3 in the central nervous system functions is increasing. In fact, vitamin D3 regulates vitamin D receptor and nerve growth factor expression, modulates brain development, and reverses experimental autoimmune encephalomyelitis. Only few studies, however, address vitamin D3 effect on embryonic hippocampal cell differentiation. In this investigation, the HN9.10e cell line was used as experimental model; these cells, that are a somatic fusion product of hippocampal cells from embryonic day-18 C57BL/6 mice and N18TG2 neuroblastoma cells, show morphological and cytoskeletal features similar to their neuronal precursors. By this model, we have studied the time course of vitamin D3 localization in the nucleus and its effect on proteins involved in proliferation and/or differentiation. We found that the translocation of vitamin D3 from cytoplasm to the nucleus is transient, as the maximal nuclear concentration is reached after 10 h of incubation with (3)H-vitamin D3 and decreases to control values by 12 h. The appearance of differentiation markers such as Bcl2, NGF, STAT3, and the decrease of proliferation markers such as cyclin-1 and PCNA are late events. Moreover, physiological concentrations of vitamin D3 delay cell proliferation and induce cell differentiation of embryonic cells characterized by modification of soma lengthening and formation of axons and dendrites.

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