**8. Conclusions**

Hepatic regeneration provides a good model for studying the mechanisms controlling cell proliferation and the ways in which they might be modified. This last aspect is very important in liver diseases and transplantation.

It is well known that cell duplication is delayed after hepatectomy for 12 h in the rat and this is not due to the operation effect as shown by the sham operated rats in which this may only be justified for the first four hours.

Much attention has been paid to the possible factors that may be activated during this period. Three hypotheses were made: the original one supposed that there was a single humoral factor, the second concerned the activation of a pathway involving many components and the more recent one, the activation of multiple pathways; the latter is the one most accepted today (Fausto 2006).

The cytokine pathway is activated in the first phase of liver regeneration which stimulates quiescent hepatocytes, growth factors then override a restriction point in G1 the entrance into cycle being associated with Rb phosphorylation, increased expression of the Rb family member p 107 together with cyclins D, E and A that form cdk4/cyclin D and cdk2/ cyclin E complexes (Menjo et al. 1998, Albrect et al. 1998).

The events preceding the entrance into the cell cycle were intensively studied , but less attention was dedicated to events inside the nuclei that favour the initiation of S-phase and G2/M transition. The presence of lipids in chromatin represents a component that appears essential for the two events. The activation of phospholipases causes a transient increase of DAG due to the hydrolysis of PLI followed by a more consistent peak due to the hydrolysis of PC. At the same time, the decrease in SM, due to an increase of activity of SMase, causes an increase in the free cholesterol fraction thus favouring DNA duplication.

The cyclin A complex is activated in parallel and the cells progress to the S phase.

When the S-phase is near completion, SM synthase increases the SM fraction and the cholesterol free fraction decreases. At the same time, cyclin B1 complex is activated thus favouring the cells' transit from G2 to M. Therefore, it is clear that all external stimuli that may favour liver regeneration can act only through the modification of the chromatin components of which lipids seem to have an important role. They may be also independent from external stimuli and may function as an internal balance. In fact, the increase in DAG due to PC hydrolysis stimulates SMase with the liberation of ceramide that may stimulate reverse sphingomyelin synthase to form new PC so favouring the liberation of DAG. On the other hand, the increase in SM reduces the ceramide present.

This internal clock appears to control cell activity favouring proliferation, differentiation when the cells remain in Go or apoptosis when the ceramide present is transformed into sphingosine.

It is clear from this that the role of phospholipids must be considered for a role in cell duplication regulation favouring the regeneration process in liver.

#### **9. References**

72 Liver Regeneration

However, the complexing of cyclin A with p32cdk2 kinase increased up to 22h i.e. the end of S-phase. After 18h, SM synthase activity increased with the increase in SM reducing the

Cyclin B complexing with p34cdc2 increased its activity so favouring the transition of G1-M phase. The phosphoinositides also decrease during S-phase leading to an inhibition of DNA synthesis through an increase in the activity of dephosphorylating enzymes (York and Majerus 1994). The small fraction of cyclin A complexed with p32 cdc2 is inactivated by phosphorylation, the cells progressing to G2-M (Splewak & Thorgeirsson 1997). The complex B-p34cdc2 also decreases followed by a second peak between 38-44 h post-

Hepatic regeneration provides a good model for studying the mechanisms controlling cell proliferation and the ways in which they might be modified. This last aspect is very

It is well known that cell duplication is delayed after hepatectomy for 12 h in the rat and this is not due to the operation effect as shown by the sham operated rats in which this may only

Much attention has been paid to the possible factors that may be activated during this period. Three hypotheses were made: the original one supposed that there was a single humoral factor, the second concerned the activation of a pathway involving many components and the more recent one, the activation of multiple pathways; the latter is the

The cytokine pathway is activated in the first phase of liver regeneration which stimulates quiescent hepatocytes, growth factors then override a restriction point in G1 the entrance into cycle being associated with Rb phosphorylation, increased expression of the Rb family member p 107 together with cyclins D, E and A that form cdk4/cyclin D and cdk2/ cyclin E

The events preceding the entrance into the cell cycle were intensively studied , but less attention was dedicated to events inside the nuclei that favour the initiation of S-phase and G2/M transition. The presence of lipids in chromatin represents a component that appears essential for the two events. The activation of phospholipases causes a transient increase of DAG due to the hydrolysis of PLI followed by a more consistent peak due to the hydrolysis of PC. At the same time, the decrease in SM, due to an increase of activity of SMase, causes

When the S-phase is near completion, SM synthase increases the SM fraction and the cholesterol free fraction decreases. At the same time, cyclin B1 complex is activated thus favouring the cells' transit from G2 to M. Therefore, it is clear that all external stimuli that may favour liver regeneration can act only through the modification of the chromatin components of which lipids seem to have an important role. They may be also independent

an increase in the free cholesterol fraction thus favouring DNA duplication.

The cyclin A complex is activated in parallel and the cells progress to the S phase.

CHO free fraction and hence the activity of cyclinA.

hepatectomy corresponding to the second mitotic peak.

important in liver diseases and transplantation.

be justified for the first four hours.

one most accepted today (Fausto 2006).

complexes (Menjo et al. 1998, Albrect et al. 1998).

**8. Conclusions** 


Possible Roles of Nuclear Lipids in Liver Regeneration 75

D'Santos, C.S., Clarke, J.H. & Divecha, N. (1998). *Phospholipid signalling in the nucleus. Een* 

Exton, J.H. (1990). Signaling through phosphatidylcholine breackdown. Biol. Chem. 265, 1-

Fausto, N., Campbel J.S. & Riehte, K. J (2006). *Liver regeneration*. Hepathology, 43,

Futerman, A.H., Stieger, B., Hubbard, A.L. & Pagano, R. (1990). *Spingomyelin synthesis in rat* 

Gahan P.B. (1965a). *Histochemical evidence for the presence of lipids on the chromosomes of animal* 

Gahan, P.B. (1965b). *The possible presence of aldehydes and carbohydrates in chromosomes and* 

Gahan, P.B., Bartlett, R., Cleland, L., & Olsen, K., (1974). *Cytochemical evidence for the presence* 

Gahan, P.B., Viola Magni, M.P. & Cave, C.F. (1987). *Chromatin and nucleolar phospholipids*.

Gomez-Munoz, A., Waggoner, D.W., O'Brien, L. & Brindley, D.N., (1995). *Interaction of* 

Kent, C. (1990). *Regulation of phosphatidylcholine biosynthesis Prog*. Lipid Res. 29, 87-105. Kim, M. Y., Linardic, C., Obeid, L. & Hannun, Y. (1991). *Identification of sphingomyelin* 

*interferon. Specific role in cell differentiation.* Biol. Chem. 266, 484-489. 25 Koval, M. and Pagano, R.E. (1991). *Intracellular transport and metabolism of shingomyelin.* 

*niveau du chromosome.* Comptes Rend. Acad. Sci. 244, 1827-1829.

*chromosome*. Comptes Rend. Acad. Sci. 246, 1098-1101.

Irvine, R.F. (2003). *Nuclear lipid signalling*. Nat. Rev. Mol. Cell Biol. 4, 349-360

Idelman, S. (1957). *Existence d'un complexe lipides-nucléoprotéines à groupements sulfhydridés au* 

Idelman, S. (1958a). *Localisation du complexe lipiedes-protéines å sulfhydrylés au sein du* 

Idelman, S. (1958b). *Démasquage des lipids du chromosome géant des glandes salivaires de* 

Jayadev, S., Linardic, C. M. & Hannun, Y. A. (1994). *Identificationof arachidonic acid as a* 

Jeckel, D., Karrenbauer, A., Birk, R. & Schmidt, R.R. (1990). *Sphingomyelin is synthesized in the* 

La Cour, L.F., Chayen, J. & Gahan, P.B. (1958). *Evidence for lipid material in chromosomes.* Exp.

*Choronome par difestion enzymatique des proteins.* Comptes Rend. Acad. Sci. 246, 3282-

*mediator of sphingomyelin hydrolysis in response to tumor necrosis factor alpha*. J. Biol.

*ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and* 

*turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-*

*of phospholipids on human chromosomes.* Histochem. J. 6, 219-222.

*phospholipase D activity.* J.Biol.Chem.270,26318-26325.

1436, 202-232.

545-563 [8]

Chem. 265, 8650-8657.

*cells.* Exp. Cell Res.39,136-144.

*interphase nuclei*. Histochemie 5, 289-296.

Basic Appl. Histochem. 31, 343-353.

Biochim. Biophys. Acta 1082, 113^125.

4.

3286.

Chem.. 269, 5757-5763.

Cell Res. 14, 469-474.

*cis Golgi.* FEBS Lett. 261, 155^157.

*DAG uit het leven van de inositide signalering in de nucleus.* Biochim. Biophys. Acta.

*liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus.* J. Biol.


Alessenko, A. & Burlakova, E.B. (2002). *Functional role of phospholipids in nuclear events*.

Alessenko, A.& Chatterjee, S. (1995). *Neutral sphingomyelinase: localization in rat liver nuclei and involvement in regeneration/proliferation*. Mol. Cell. Biochem. 143, 169-174. Baldassare, J.J., Jarpe, M.B., Alferes, L. & Raben, D.M. (1997). *Nuclear translocation of RhoA* 

Ballou, L. R., Chao, C. P., Holness, M. A., Barker, S. C., & Raghow, R. (1992). *Interleukin-1-*

Bjerve, K.S. (1971). *The Ca(2+) stimulated incorporation of choline into microsomal lecithin* 

Berg, N.O. (1951). *A histological study of masked lipids; stainability, distribution and functional* 

Bresnick, E, (1971). *Regenerating liver an experimental model for study of growth*. Methods

Buchner, K. (1995). *Protein kinase C in the transduction of signals toward and within the cell* 

Cave, C.F. & Gahan, P.B. (1971). *A cytochemical and autoradiographic investigation of nucleolar* 

Chayen, J., La Cour, L.F. & Gahan, P.B. (1957). *Uptake of benzopyrene by chromosomal* 

Chayen, J., Gahan, P.B. & La Cour, L.F. (1959a). *The masked lipids of nuclei. Quart*. J.Microscop.

Chayen, J., Gahan,P.B. & La Cour, L.F. (1959b). *The nature of a chromosomal phospholipid*

Cocco, L., Martelli, A.M., Gilmour, R.S., Ognibene, A., Manzoli, F.A. & Irvine, R.F. (1988).

Cocco, L., Martelli, A.M., Gilmour, R.S., Rhee, S.G. & Manzoli, F.A. (2001). *Nuclear* 

Diringer, H., Marggraf, W.D., Koch, M.A. & Anderer, F.A. (1972). *Evidence for a new* 

Divecha, N., Banfic, H.& Irvine, R.F. (1991). *The polyphosphoinositide cycle exists in the nuclei of* 

Dressler, K. A., Mathias, S., and Kolesnick, R. N. (1992). *Tumor necrosis factor-alpha activates* 

*phospholipase C and signalling*. Biochim. Biophys. Acta 1530, 1-14.

*translocation of proteinkinase C to the nucleus*. EMBO J. 10, 3207-3214.

*Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulinlike growth factor I*. Biochem. Biophys. Res. Commun.

*biosynthetic patway of sphingomyelin in SV 40 transformed mouse cells.* Biochem.

*Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces* 

*the sphingomyelin signal transduction patway in a cell-free system* Science 255, 1715-

Chayen, J.& Gahan, P.B. (1958). *Lipid components in nucleohistone*. Biochem. J. 69, 49P.

*mediates the mitogen-induced activation of phospholipasem D involved in nuclear envelope* 

*mediated PGE2\production and sphingomyelin metabolism. Evidence for the regulation of cycloossigenase gene expression by sphingosine and ceramide.* J. Biol. Chem. 267, 20044-

Bioelectrochemistry 58, 13-21.

20050.

Sci. 100, 3

154, 1266-1272.

1718.

*signal transduction*. J. Biol. Chem. 272, 4911-4914.

*variations*. Acta Pathol. Microbiol Scand Suppl 90:1-192.

*subspecies in vitro*. FEBS Lett. 17, 14-16.

*nucleus*. Eur. J. Biochem. 288, 211-221.

*phospholipids*. Caryologia 23, 303-312.

*phospholipids*. Nature 180, 652-65

Quart. J. Microscop Sc.100,325

Biophys. Res. Commun. 47, 1345^1351.

Cancer Res., 6,347-397


Possible Roles of Nuclear Lipids in Liver Regeneration 77

Rogue, P., Labourdette, G., Masmoudi, A., Yoshida, Y., Huang, F. L., Huang, K. P., Zwiller,

Santi, P., Zini, N., Santi, S., Riccio, M., Guiliani Piccarei, G., De Pol, A. & Maraldi N.M.

Scribney, M. & Kennedy, E.P. (1958). *The enzymatic synthesis of sphingomyelin.* J. Biol. Chem.

Song, M. & Rebel, G. (1987). *Rat liver nuclear lipids. Composition and biosynthesis.* Basic Appl.

Slife, C.W., Wang, E., Hunter, R., Wang, S., Burgess, C., Liotta, D.C. & Merill, A.M. (1989).

Spangler, M., Coetzee, M.L., Katlyl, S.L., Morris, H.P. & Ove, P. (1975). *Some biochemical* 

Splewak Rinaudo, J.A. &.Thorgeirsson S.S. (1997). *Detection of a tyrosine -phosphorilated* 

Stetten, D. (1941). *Biological relationship of choline, ethanolamine and related compounds*.

Sun, B., Murray N.R. & Field A.P. (1997). *A role for nuclear phosphatidylinositolspecific* 

Tata, J.R., Hamilton, M.J. & Cole, R.D. (1972). *Membrane phospholipids associated with nuclei* 

Tsugane, K., Tamiya-Koizumi, K., Nagino, M., Nimura, Y. & Yoshida, S. (1999). *A possible* 

Ullman, M.D. & Radin, N.S. (1974). *The enzymatic formation of spingomyelin from ceramide and* 

Upreti, G.C., DeAutmno, R.J. & Wood, R. (1983). *Membrane lipids of hepatic tissue. II.*

van Meer, G. & Burger, K.N. (1992). *Sphingolipid trafficking-sortedout?* Trends Cell Biol.

Viola Magni, M.P., Gahan, P.B. & Pacy, J. (1985a). *Phospholipid in plant and animal chromatin*.

Viola Magni, M.P., Gahan, P.B., Albi, E., Iapoce, R. & Gentilucci, P.F. (1985b). *Chromatin phospholipids and DNA synthesis in hepatic cells*. Bas. Appl. Histochem. 29, 253-259. Viola Magni, M.P., Gahan, P.B., Albi, E., Iapoce, R. & Gentilucci, P.F. (1986). *Phospholipids in* 

*chromatin: incorporation of in different subcellular fraction of hepatocytes.* Cell Biochem.

Stillman, B., (1996). *Cell cycle control of DNA replication*. Science 274, 1659-1661

*lecithin in the mouse liver*. J. Biol. Chem. 249, 1506^ 1512.

Phospholipids from subcellular fractions of liver and hepatoma

*phospholipase C in the G2/M transition.* J:Bio.Chem:272,26313-26317

*isozyme type II*. J. Biol. Chem. 265, 4161-4165.

233, 1315-1322.

10377

301-309

67, 231-346

31, 8-17.

11,332-337

Funct. 4, 283-288.

Cell Biochem. Funct. 3, 71-78.

Histochem. 31, 377-387.

Res. 35, 3145-3145.

J.Biol..Chem. 138,437-438

*enzymes in rat hepatoma cells*. Int.J.Oncol.18,165-174

J., Vincendom, G., & Malviya, A. N. (1990). *Rat liver nuclei protein kinase C is the* 

(2001). *Increased activity and nuclear localizationof inositol lipid signal transduction* 

*Free sphingosine formation from endogenous substrates by a liver plasma membrane system with a divalent cation dependence and a neutral pH optimum*. J.Bio. Chem. 264, 10371-

*characteristics of rat liver and Morris hepatoma nuclei and nuclear membranes.* Cancer

*form of Cyclin A during liver regeneration*. Cell Growth and Differentiation 8)

*and chromatin: melting profile, template activity and stability of chromatin*. J. Mol. Biol.

*role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver*. J. Hepatol.


Loyer ,P., Gialse,D., Carlou, S., Baffet, G., Meyer, L. & Guguen-Guillouzo, C. (1994).

Luskey, K.L. (1988). *Regulation of cholesterol synthesis:mechanism for controlof HMG-CoA* 

Manzoli, F.A., Capitani, S., Mazzotti, G., Barnabei, O. & Maraldi, N.M. (1981). *Role of* 

Marinetti, G.V., Erbland, J., Witter J.F., Petix, J. & Stoltz, E. (1958). *Metabolic patways of lysolecithin in a soluble rat-liver system*. Biochim.Biophys. Acta 30, 223-226 Martelli, A.M., Bortul, R., Tabellini, G., Aluigim, M., Peruzzi, D., Bareggi, R., Narducci, P. &

Martelli, A. M., Billi, A.M., Manzoli, L., Faenza, I., Aluigi, M., Falconi, M., De Poi, A.,

Micheli, M., Albi, E., Leray, C. & Viola Magni, M.P. (1998). *Nuclear sphingomyelin protects* 

Moir, D.R., Mountag-Lowy, M. & Goldman, R. D. (1994). *Dynamic properties of nuclear* 

Neri, L.M., Ricci, D., Carini, C., Marchisio, M., Capitani, S. & Bertagnolo, V. (1997). *Changes of nuclear PI-PLC gamma 1 during rat liver re generation.* Cell Signal.9,353-362 Okazaki, T., Bell, R. M. & Hannun, Y. A. (1989). *Spingomyelin turnover induced by vitamin D3* 

Okazaki, T., Bielawska, A., Bell, R.M. & Hannun, Y.A. (1990). *Role of ceramide as a lipid* 

Pagano, M., Pepperfcok, R., Verde, F., Ansorge, W. & Draetta, G. (1992). *Cyclin A is required* 

Rao, K.N. (1986). *Regulatory aspects of cholesterol metabolismin cells with dfifferent degrees of* 

Reszka, A.A., Halasy-Nagy, J. & Rodan, G.A. (2001). *Nitrogenbisphosphonate block* 

Riboni, L., Bassi, R., Sonnino, S. & Tettamanti, G. (1992). *Formation of free sphingosine and* 

*laminins:laminin B is associated with sites of DNA replication.* J. Cell Biol. 125, 1201-

*mediator of 1-alpha-25 dihydroxyvitamin D3-induced HL-60 cell differentiation*. J. Biol.

*retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation*. Mol.

*ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture.* FEBS

*(MAP) kinase dependent serine phosphorylation.* FEBS Lett 486, 230-236 Menjo, M., Ikeda K. & Nakanishi, M. (1998). *Regulation of G1 cyclin-dependent kinases in liver* 

*regeneration.* J. Gastroenterol Hepattol 13,100-105

*RNA from RNase action.* FEBS Lett. 431, 443-447.

*in HL-60 cells. Role in cell differentiation.* J. Biol. Chem. 22.

*at two points in the human cycle.* EMBO J. 11,961-971.

*liver regeneration*. J.Biol:Chem. 269, 2491-2500

*reductase.Recent Prog*. Horm. Res., 44, 35-51.

Adv. Enzyme Regul. 20, 247-262.

*metabolism.* FEBS Lett. 505, 1-6.

Chem. 265, 15823-15831. 5441.,

Pharmacol. 59, 193-202.

Letters 300, 188-192

*replication*. Toxicol.pathol. 14, 430-437.

1212.

*Expression and activation of cdks (1and 2) and cyclins in the cell cycle progression during* 

*chromatin phsopholipids on template availability and ultrastructure of isolated nuclei.* 

Cocco, L. (2001). *Re-examination of the mechanisms regulating nuclear inositol lipid* 

Gilmour, R.S. & Cocco, I. (2000). Insulin *selectively stimulates nuclear phosphoinositidespecific phospholipase C (PI-PLC) beta1 activity through a miogeno activated protein* 


**Matrix Restructuring During Liver** 

Naomi Sobukawa1 and Kimie Asanuma-Date1 *1Graduate School of Advanced Sciences and Humanities, and Glycoscience Institute, Ochanomizu University,* 

Haruko Ogawa1, Kotone Sano2,

*Japan* 

**of the Matrix Glycoprotein Vitronectin** 

**Regeneration is Regulated by Glycosylation** 

*2Faculty of World Heritage, Department of Liberal Arts, Cyber University, Tokyo,* 

There are three major approaches for regenerative medicine. The most innovative approach among them is: induction of target cells from various stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) and implantation of them to regenerate the organ. The second approach is: in vitro tissue regeneration that involves preparation of artificial tissue by combining human cells with scaffolding biomaterials and growth factors. The third is: promotion of self-regeneration through controlling the repair activity of each tissue, which most organisms do naturally, is a more fundamental approach, but it will also be important in cell therapy to regulate tissue organization after induction of

Because tissue homeostasis depends on spatially and temporally controlled expression of multifunctional adhesive glycoproteins and receptors, many studies have examined the changes of expression of extracellular matrix (ECM) molecules during tissue remodeling, inflammation and invasion by cancer cells (DeClerck, Y.A., et al. 2004; Seiffert, D. 1997; Kato, S., et al. 1992; Hughes, R.C. 1997) on the one hand. On the other hand, there is increasing evidence that glycosylations post-translationally modulate various biological phenomena by altering the activity and specificity or the stability of glycoproteins through the biosignaling functions of oligosaccharides (Varki, A. 1993; Varki, A., et al., 2009). During tissue remodeling, the glycosylated ECM molecules are different from those of normal tissue owing to the changes in the expression of many proteins that are responsible for glycan synthesis (Dalziel, M., et al. 1999). However, the glycan modulation of most glycoproteins

When the three big lobes of a liver are excised, the remaining liver recovers its former mass and function within about two weeks in humans or 7 to 10 days in rats (Diehl, A.M. and Rai, R.M. 1996). ECM degradation occurs in the early stage of this process, followed by biosynthesis of the matrix, cell proliferation, and cell differentiation. During this process, many glycosyl transferases (Bauer, C.H., et al. 1976; Serafini-Cessi, F. 1977; Okamoto, Y., et

that are involved in tissue remodeling has remained unknown.

**1. Introduction** 

differentiation.

York, J.D. & Majerus, P.W. (1994). *Nuclear phosphatidylinositols decrease during S-phase of the cell cycle in HeLa cells.* J. Biol. Chem. 269, 7847-7850 **5** 
