Keiko Tamiya-Koizumi

A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver

BACKGROUND/AIMS Portal vein branch ligation induces apoptosis of hepatocytes in the ligated lobes in rat liver. Sphingomyelin degradation was studied during the process to evaluate its possible involvement in apoptosis in vivo. METHODS DNA scissions were detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and an agarose gel electrophoresis of DNA. Using both ligated and non-ligated lobes, we measured activities of sphingomyelin degradation enzymes and contents of their products in purified nuclei and plasma membrane. RESULTS DNA fragmentation was detectable in the ligated lobes at 90 min after the portal vein branch ligation by gel electrophoresis. At 15 h after the ligation, 27% of hepatocytes became TUNEL-positive. Prior to the onset of apoptosis, the activity of neutral sphingomyelinase increased in the nuclei of hepatocytes in ligated lobes (30 min after the ligation). The increase in sphingomyelinase paralleled its reaction product, ceramide. This was followed by the elevation of ceramidase activity in nuclei (60 min after the ligation) in association with an increase of its reaction product, sphingosine. Activities of these two enzymes and their products increased for at least 90 min. These changes were not observed in nuclei of the non-ligated lobes, or in the plasma membranes from either ligated or non-ligated lobes. CONCLUSIONS These results, specific to the liver where apoptosis is being generated, suggest that nuclear sphingomyelin breakdown with an accumulation of ceramide and/or sphingosine in nuclei may induce the apoptosis of hepatocytes in vivo.

Inhibition of DNA primase by sphingosine and its analogues parallels with their growth suppression of cultured human leukemic cells

Sphingosine is a potent inhibitor of a mammalian DNA primase in vitro (Simbulan et al., Biochemistry 33, 9007‐9012, 1994). Here we measured the inhibition of DNA primase in vitro by 9 sphingosine‐analogues with respect to RNA primer synthesis and DNA primase‐dependent DNA synthesis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells. Sphingosine, phytosphingosine and N, N‐dimethylsphingosine strongly inhibited the activity of purified calf thymus DNA primase, and also inhibited the growth of human leukemic cell line HL‐60, exerting strong cytotoxicity. Dihydrosphingosine and cis‐sphingosine, which showed more subtle inhibition of DNA primase in vitro, moderately inhibited the cell growth in vivo and caused cell death. In contrast, N‐acyl‐, N‐octyl‐, and N‐acetylsphingosine (ceramides) showing little inhibition of DNA primase suppressed cell growth only slightly. HL 60 cell was arrested at Go/G1 phase by exogenously added sphingosine. From these results, it is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis.

Transcription factor specificity protein 1 (Sp1) is the main regulator of nerve growth factor‐induced sphingosine kinase 1 gene expression of the rat pheochromocytoma cell line, PC12

Sphingosine kinase (SPHK) is known to exert an anti‐apoptic role in various cells and cell lines. We previously reported that human brain is rich in SPHK1 ( Murate et al. 2001 ). After showing a high expression of SPHK1 in rat brain, we examined the gene expression mechanism using nerve growth factor (NGF) ‐stimulated rat PC12 cells. With RT–PCR, we found that both rat brain and PC12 utilized exon 1d mostly out of eight untranslated first exons. NGF induced an increase in SPHK enzyme activity and protein about double those in PC12 cells, and NGF‐induced SPHK1 mRNA was three times higher than in the control. The minimal 5′ promoter was determined, and TrkA specific inhibitor K252a inhibited the NGF‐induced promoter activity of SPHK1. The truncation or mutation of putative transcription factor‐binding motifs revealed that one specificity protein 1 (Sp1) binding motif of the 5′ region of exon 1d is prerequisite. Electrophoresis mobility shift assay confirmed the promoter analysis, indicating increased Sp1 protein binding to this motif after NGF treatment. Chromatin immunoprecipitation assay also showed the binding of Sp1 and the promoter region in vivo. These results suggest the signal transduction pathway from NGF receptor TrkA to transcription factor Sp1 protein binding to the promoter Sp1‐like motif in NGF‐induced rat SPHK1 gene expression.

A novel phospholipase A2 associated with nuclear matrix: stimulation of the activity and modulation of the Ca2+ dependency by polyphosphoinositides

Neutral phospholipase A2 activity, which hydrolyzed phosphatidylcholine and phosphatidylethanolamine with the same efficiency, was identified in the nuclear matrix prepared from purified nuclei of rat ascites hepatoma cells (AH 7974). The enzyme activity was optimal at pH 7.0 and required Ca2+ absolutely. Concentrations of Ca2+ for a maximal and a half-maximal activation were 1.10(-2) and 1.10(-3) M, respectively, and little activity was detected at Ca2+ concentrations lower than 1.10(-5) M. Addition of acidic phospholipids markedly stimulated the enzyme activity, and further, lowered the minimum Ca2+ concentration required for activation. In particular, the polyphosphoionositides phosphatidylinositol 4-monophosphate and 4,5-diphosphate were most effective. These two polyphosphoinositides lowered the Ca2+ concentration required for half-maximal activation to 10(-5) M and dramatically stimulated the activity at that Ca2+ concentration (greater than 30-fold). The neutral phospholipase A2 activity such as characterized in the present study was very low in the other subcellular fractions including mitochondria, microsome, plasma membrane and cytosol.

Up-regulation of Acid Sphingomyelinase during Retinoic Acid-induced Myeloid Differentiation of NB4, a Human Acute Promyelocytic Leukemia Cell Line

All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor α (RARα) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation ofASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5′-upstream flanking region of human ASMase gene (−519/+300) conjugated with theluciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RARα or the PML/RARα hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5′-end (−519/−485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence.

Nuclear ADP-ribosylation Factor (ARF)- and Oleate-dependent Phospholipase D (PLD) in Rat Liver Cells INCREASES OF ARF-DEPENDENT PLD ACTIVITY IN REGENERATING LIVER CELLS

Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol produced from exogenous radiolabeled phosphatidylcholine in the presence of butanol. In nuclear lysates from either rat liver or ascites hepatoma AH 7974 cells, the PLD activity was markedly stimulated by a recombinant ADP-ribosylation factor (rARF) in the presence of the guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and phosphatidylinositol 4,5-bisphosphate. ATP and phorbol-12-myristate 13-acetate had no synergistic effect on this PLD activity. On the other hand, the nuclear PLD was stimulated by unsaturated fatty acids, especially by oleic acid. The ARF-dependent nuclear PLD activity was increased in the S-phase of the regenerating rat liver after partial hepatectomy and also was much higher in AH 7974 cells than in the resting rat liver. In contrast, the levels of the oleate-dependent PLD activity remained constant throughout the cell cycle in liver regeneration. The intranuclear levels of the stimulating proteins of the nuclear PLD activity, e.g. ARF, RhoA, and protein kinase Cδ increased in the S-phase of the regenerating liver. These results suggested that the nuclear ARF-dependent PLD activity may be associated with cell proliferation.

Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin.

Mg(2+)-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between -148 and -42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.

Interaction of DNA polymerases with phospholipids

Effects of various phospholipids on the in vitro reactions of eukaryotic DNA polymerases alpha, beta and gamma were tested systematically. When phospholipids were added directly to the reaction mixture, neither stimulation nor inhibition was produced. However, when phospholipids were preincubated with enzymes in the absence of template-primer, some of them showed strong inhibition. Cardiolipin strongly inhibited the reactions of all three DNA polymerases and also of terminal deoxynucleotidyl transferase. Phosphatidylinositol selectively inhibited the reaction of DNA polymerase gamma. Phosphatidic acid moderately inhibited DNA polymerase alpha and strongly inhibited DNA polymerase gamma. The inhibition of DNA polymerase gamma by cardiolipin was nearly competitive with template-primer. Since the inhibition was reversed by the addition of 0.05% Triton-X 100 during preincubation, the phospholipid might interact with enzyme protein at the hydrophobic region in competition with template-primer. These results suggest a possible involvement of phospholipids in DNA replication in mitochondria and in nucleus through interaction with DNA polymerase.

ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1‐phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all‐trans retinoic acid (ATRA)‐induced differentiation of a human neuroblastoma cell line, SH‐SY5Y. ATRA reduced CERK mRNA and protein levels. Over‐expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA‐induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5′‐promoter of CERK. Truncation and mutation study suggests that ATRA‐responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between −40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP‐TFI), RARα, and RXRα, respectively. DNA pull‐down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP‐TFI and siRNA transfection of these genes revealed that COUP‐TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co‐repressors as well as three transcription factors. These results suggest that COUP‐TFI was the ATRA‐responsive suppressive transcription factor of CERK gene transcription.

Growth-associated changes in fatty acid compositions of nuclear phospholipids of liver cells.

To know the possible relationships between nuclear phospholipids and cell proliferation, we have extensively analyzed phospholipids extracted from the nuclei of rat hepatic cells at various growth states. The content of phospholipid in nuclei as well as its composition was similar among liver cells tested, i.e., the regenerating rat livers (28 h, post-hepatectomy), sham-operated or non-treated control livers, and rat ascites hepatoma, AH7974 cells. In contrast, the fatty acid compositions of phospholipids differed from each other among these cells. At the 2-position of phospholipids in the regenerating liver nuclei at 28 h after partial hepatectomy, 18:1 (oleic acid) increased transiently at the expense of 20:4 (arachidonic acid) and 22:6 (docosahexaenoic acid), compared with those in the sham-operated control nuclei. This change in fatty acid composition was commonly observed throughout all phospholipids analyzed, i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). On the other hand, the change at 1-position was rather limited: in the regenerating liver nuclei (28 h), 18:1 increased only in PC at the expense of 18:0 (stearic acid). The similar and more marked deviation at the 2-position was observed with AH7974 nuclei it contained approximately 2-times more of 18:1 in PC, PE and PI than regenerating liver nuclei (28 h), and the decreased levels of 20:4 and/or 22:6. It should be noted that there were significant differences in the fatty acid compositions of PE and PS between sham-operated and non-treated controls. So, the sham-operated rat is the appropriate control for proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)