ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine-and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1⌬N, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 ؊/؊ ) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 ؊/؊ ) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.

Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H 2 O 2 and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H 2 O 2 . JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage.

ABSTRACT. The Wnt-β-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in embryonic development. It is tempting to speculate that the Wnt-4-β-catenin pathway contributes to the recovery from acute renal failure (ARF). This study used an in vivo model of ARF rats to clarify the significance of the Wnt-4-β-catenin pathway in ARF. ARF was induced by clamping the rat left renal artery for 1 h. At 3, 6, 12, 24, 48, and 72 h after reperfusion, whole kidney homogenate and total RNA were extracted for examination by Western blot analysis and real-time RT-PCR. Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 h and 6 to 24 h after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and co-expressed with aquaporin-1, GM130, and PCNA. Cyclin D1 and cyclin A were expressed at 24 to 48 h after reperfusion. In addition, the overexpression of Wnt-4 and β-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Taken together, these data suggest that the Wnt-4-β-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Wnt-4-β-catenin pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. E-Mail: yterada.kid@tmd.ac.jp

The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.

Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11␤-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease.

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Abstract. While the serine/threonine protein kinase Akt has attracted attention as a mediator of survival (anti-apoptotic) signal, the regulation and function of the PI3-kinase-Akt pathway in mesangial cells is not well known. To explore the significance of the PI3-kinase-Akt pathway, this study used PI3-kinase inhibitors (Wortmannin and LY294002) and recombinant adenoviruses encoding a dominant-active mutant of Akt (AxCAmyrAkt) and a dominant-negative mutant of Akt (AxCAAkt-AA) in cultured rat mesangial cells. Apoptotic signals were measured by nucleosomal laddering of DNA, caspase 3 assay, and cell death detection ELISA. The PI3 kinase inhibitors and dominant-negative mutant of Akt increased the apoptotic signals in the presence of platelet-derived growth factor (PDGF), while the dominant-active mutant of Akt prevented apoptosis induced by a serum-free medium. In separate exper-

Abstract. The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. The hypothesis in this study was that Ets-1 is re-expressed during regeneration after acute renal failure (ARF) and plays a key role in the transcriptional regulation of cyclin D1 and the cell cycle progression in renal tubular cells. For clarifying the significance of Ets-1 in ARF, a rat ARF model in vivo and LLC-PK1 cells as an in vitro model were used. After the left rat renal artery was clamped for 1 h, the whole kidney homogenate was examined and total RNA was extracted at 6, 12, 24, 48, and 72 h after reperfusion by Western blot analysis and real-time reverse transcription-PCR. Ets-1 mRNA and protein expression were strongly increased at 6 to 24 h after the ischemia, respectively. The expression of hypoxia-inducible factor-1␣ was increased dramatically as early as 6 h after ischemia-reperfusion and decreased at 48 and 72 h after ischemia-reperfusion. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with proliferating cell nuclear antigen (PCNA). Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 and 6 h in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. The Ets-1 induction by hypoxia was abolished by the transfection of dominant-negative hypoxia-inducible factor-1␣. A gel shift assay demonstrated that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition. Overexpression of Ets-1 did not significantly change the caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell-cycle progression of renal tubules in ARF. The Ets-1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.