Tomoka Takatani

Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells

In hypertonic environment, taurine accumulates in cells via activation of TauT (taurine transporter) as an adaptive regulation. Recent studies revealed that TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway regulated the expression of various molecules which protect cells against hypertonic stress. In the present study, we investigated the osmoregulatory mechanisms of TauT expression. TauT was up-regulated at both functional and transcriptional levels in HepG2 under hypertonic condition. The TonE site was identified in the promoter region of TauT gene. Reporter gene assay revealed that promoter activity was increased under hypertonic conditions, whereas deletion or mutation of TonE sequence abolished the induction of the promoter activity in response to hypertonicity. By using the reporter gene plasmids containing a TonE site of TauT promoter (p2xTonE-Luc), it was demonstrated that a TonE site was sufficient for the hypertonicity-mediated activation of TauT promoter. Importantly, co-transfection of TauT promoter gene plasmid with wild-type TonEBP expression vector enhanced promoter activity under isotonic conditions, whereas dominant-negative TonEBP abrogated the TauT promoter activity induced by hypertonicity. Finally, treatment with taurine prevented HepG2 cells from cell death induced by hypertonic medium. These findings suggested that induction of TauT by hypertonicity is mediated by the activation of the TonE/TonEBP pathway and confers resistance to hypertonic stress.

Taurine renders the cell resistant to ischemia-induced injury in cultured neonatal rat cardiomyocytes.

Taurine is found in very high concentration in the mammalian heart. Because chronic myocardial taurine loss produces myocardial injury, the effects of taurine supplementation on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia. Neonatal rat heart cells were cultured for 24–72 h in a sealed flask, a condition that leads to simulated ischemia characterized by a decrease in the pH and oxygen content of the medium and a catabolite accumulation. The consequences of altered medium taurine on cellular apoptosis and necrosis were then evaluated. Exposure of cardiomyocytes to medium containing high extracellular concentrations of taurine (20 m M) significantly elevated intracellular taurine levels, reduced p53 content, and enhanced cellular Bcl-2 content. In the absence of taurine treatment, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation by 24–72 h. Based on DNA ladder analysis and the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. CPK was lost from some of the cells during simulated ischemia. In contrast to the untreated ischemic cells, the cells that were incubated in medium supplemented with taurine exhibited significantly less ischemia-induced necrosis and apoptosis. The data suggest that taurine renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of taurine may be related to the elevation in cellular Bcl-2 content.

Signals through gp130 upregulate Wnt5a and contribute to cell adhesion in cardiac myocytes

Glycoprotein 130 (gp130), a common receptor of IL‐6 family cytokines, plays critical roles in cardiac functions. Here, we demonstrate that the stimulation of gp130 with leukemia inhibitory factor (LIF) promoted cell adhesion in a cadherin‐dependent manner in cultured cardiomyocytes. Wnt5a was upregulated by the stimulation of gp130 with IL‐6 family cytokines, accompanied by N‐cadherin protein upregulation. Wnt5a was not induced by LIF in cardiomyocytes expressing dominant‐negative STAT3. Ablation of Wnt5a by antisense cDNA inhibited LIF‐induced cell adhesion. Collectively, signals through gp130 upregulate Wnt5a through STAT3, promoting the N‐cadherin‐mediated cell adhesion.

The transport mechanism of metallothionein is different from that of classical NLS-bearing protein

A nuclear localization signal (NLS) has been detected in several nuclear proteins. Classical NLS‐mediated nuclear pore targeting is performed by using the cytosolic factors, importin α and importin β, whereas nuclear translocation requires the small GTPase, Ran. In the present study, we demonstrated that nuclear localization of metallothionein (MT) differs from that of classical NLS‐mediated substrates. In digitonin‐permeabilized BALB/c3T3 cells, biotinylated MT was localized in the nucleus in the presence of ATP and erythrocyte cytosol in the same manner as for SV40 large T NLS‐conjugated allophycocyanin (APC‐NLS). Under ATP‐free conditions, nuclear rim‐binding was observed in both transport substrates. Rim‐binding of labeled MT was competitively inhibited by the addition of an excess amount of unlabeled MT. Different elution profiles were observed for the localization‐promoting activities of MT in the cytosol compared to those of APC‐NLS. Furthermore, nuclear localization of MT was determined to be a wheat germ agglutinin‐insensitive, GTPγS‐sensitive, and anti‐Ran antibody‐sensitive process. Green fluorescent protein‐metallothionein (GFP‐MT) fusion protein was also localized in the nucleus in the stable transformant of CHL‐IU cells. These results strongly suggest that the targeting by MT of the nuclear pore is mediated by cytosolic factor(s) other than importins and that MT requires Ran for its nuclear localization. J. Cell. Physiol. 185:440–446, 2000.

Molecular mechanisms of cardioprotection by taurine on ischemia-induced apoptosis in cultured cardiomyocytes.

An integral part of the pathogenesis of heart failure is myocyte loss. The traditional explanation for myocyte loss was cell necrosis but there has been a surge of evidence affirming the role of apoptosis in the genesis of heart failure (Garg et al., 2005). Evidence for apoptotic cell death was shown in clinical cases of myocardial infarction, as well as in rabbit, rat, and mouse models of continuous ischemia or ischemia/reperfusion (Garg et al., 2005). It has been shown that the mitochondrial pathways participate in apoptosis induced by ischemia (Garg et al., 2005). Taurine (2-aminoethanesulfonic acid), the -amino acid, is one of the factors that regulates the degree of apoptosis during ischemia (Roysommuti et al., 2003; Schaffer et al., 2003). However, little is known about the cytoprotective signalling pathways mediate this response. We have previously reported that isolated neonatal cardiomyocytes become resistant to ischemia-induced apoptosis when exposed to medium containing 20 mM taurine (Takahashi et al., 2003). In this study, the interaction between taurine and mitochondria-mediated apoptosis is investigated in a newly developed simulated ischemia model utilizing isolated cardiomyocytes that are incubated with medium containing and lacking taurine and then sealed within cultured flasks (Takahashi et al., 2003, Takatani et al., 2004a,b).

Minoxidil attenuates ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes

The effects of minoxidil (a mitochondrial K+ATP channel opener) on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia, since mito-chondrial K+ATP channel openers have been suggested to be involved in the mechanisms of cardioprotective action against ischemia/reperfusion injury. In the absence of minoxidil, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation within 24 to 72 hours. Based on the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. Myocytes treated with 5 μM of minoxidil failed to alter the degree of ischemia-induced CPK loss for 48 to 72 hours. However, minoxidil treatment prevented the loss of beating function in many of the ischemic cells, and attenuated the decline in intracellular ATP content after a 48-hour ischemic incubation. The number of nuclear fragmentation was significantly reduced in minoxidil-treated cells after a 72-hour ischemic insult compared with untreated ischemic cells. This effect was blocked by the mitochondrial K+ATP channel antagonist 5-HD. The data suggest that minoxidil renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of minoxidil appear to be related to the opening of mitochondrial K+ATP channels.

Cellular characterization of taurine transporter in cultured cardiac myocytes and nonmyocytes.

Taurine is by far the most abundant free amino acid in the mammalian heart, comprising in excess of 50% of the total free amino acid pool1,2. Although its physiological function remains undefined, taurine exhibits an extensive cardiovascular pharmacology. Carnivores depend to a large extent on taurine obtained through the diet, which must be transported across cell membranes to accumulate in the heart. The taurine transporter, which belongs to a gene family that encodes Na+-and Cl--coupled transporters2,3has been cloned from mammalian tissues and cells3–10. Although numerous cell types are present in the heart, cardiomyocytes and cardiac fibroblasts are the predominant cell type in the neonatal rat heart. There are no known studies showing the detailed characterization of the taurine transporter in cardiac cells. The present study was undertaken to clarify the physiological characteristics of the cardiac taurine transporter using cultured myocytes and nonmyocytes prepared from neonatal rats.