Hirofumi Sawai

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1β converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells.

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1.

Current status and perspectives in ceramide-targeting molecular medicine.

Ceramide is not only structurally but also functionally a key molecule in diverse kinds of sphingolipids. In the past decade, ceramide has been shown to be of crucial significance in several cell functions including apoptosis, cell growth, senescence, and cell cycle control. Among them, the role of ceramide in apoptosis induction has extensively been studied, and ceramide-targeting molecular medicine for apoptosis-based diseases such as malignant tumors, atherosclerosis and neurodegenerative disorders appears to come out to the clinical field. We here describe the recent advances in research of ceramide-mediated apoptosis signaling. We also show the relation of ceramide level through regulation of ceramide-related enzymes (sphingomyelinase, ceramidase, sphingomyelin synthase and glucosylceramide synthase) with diseases such as cancer, leukemia, bacterial infections, AIDS, Alzheimers disease, atherosclerosis, diabetes mellitus and atopic dermatitis. The strategies to construct the ceramide-targeting medicine for intractable diseases such as cancer and leukemia are discussed.

Differential effects of caspase inhibitors on TNF-induced necroptosis.

TNF has been reported to induce caspase-independent necroptosis in the presence of Z-VAD-fmk, a pan-caspase inhibitor. We examined whether necroptosis was induced by caspase inhibitors other than Z-VAD-fmk. TNF-induced necroptosis was detected in the presence of Z-DEVD-fmk, which is commonly used as a caspase-3-specific inhibitor, but not in the presence of Z-Asp-CH2-DCB, which was reported to be a pan-caspase inhibitor. TNF-induced caspase-3 activity was completely inhibited by Z-VAD-fmk, Z-DEVD-fmk, or Z-Asp-CH2-DCB. Although TNF-induced proteolytic activation of procaspase-3 was completely prevented by Z-VAD-fmk or Z-DEVD-fmk, the partial proteolysis of procaspase-3 was induced in the presence of Z-Asp-CH2-DCB. Furthermore, although TNF-induced proteolytic activation of procaspase-8 was completely inhibited by Z-VAD-fmk or Z-DEVD-fmk, the partial proteolysis of procaspase-8 to the p43/41 intermediate and p18 active fragment was detected in the presence of Z-Asp-CH2-DCB. The cleavage of RIP1, which plays a crucial role in TNF-induced necroptosis and is cleaved by caspase-8, was completely inhibited by Z-VAD-fmk or Z-DEVD-fmk, whereas the partial degradation of RIP1 was detected in the presence of Z-Asp-CH2-DCB. These results suggest that the partial activation of caspase-8 in the presence of Z-Asp-CH2-DCB may suppress TNF-induced necroptosis via the cleavage of RIP1, and also suggest that Z-Asp-CH2-DCB, but not Z-DEVD-fmk, may be used as a caspase-3-specific inhibitor in cells.

Characterization of TNF-induced caspase-independent necroptosis

Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin.

Augmentation of TNF-induced osteoclast differentiation by inhibition of ERK and activation of p38: Similar intracellular signaling between RANKL- and TNF-induced osteoclast differentiation

Abstract Research in osteoclast differentiation has been greatly advanced since the identification of receptor activator of nuclear factor-κB ligand (RANKL) as osteoclast differentiation factor. The mechanisms of RANKL-induced osteoclast differentiation have been extensively investigated. Mitogen-activated protein kinases (MAPKs) were shown to play crucial roles in RANKL-induced osteoclast differentiation. RANKL-induced osteoclast differentiation was enhanced by inhibition of extracellular signal-regulated kinase (ERK), whereas it was suppressed by inhibition of p38 MAPK. It was reported that tumor necrosis factor (TNF), a major proinflammatory cytokine, induced osteoclast differentiation independently of RANKL. A report showed that inhibition of p38 suppressed TNF-induced osteoclast differentiation, whereas inhibition of ERK did not augment TNF-induced osteoclast differentiation. In this study we reevaluated the roles for MAPKs in TNF-induced osteoclast differentiation. In contrast with the previous report, pretreatment of mouse monocytic RAW264 cells with MAPK/ERK kinase (MEK) inhibitors including PD98059 and U-0126 augmented TNF-induced osteoclast differentiation. Furthermore, we found that U-0126 was more effective in augmentation of osteoclast differentiation than PD98059. Western blot analysis showed that U-0126 inhibited ERK phosphorylation and enhanced p38 phosphorylation, whereas PD98059 inhibited both ERK and p38 phosphorylation. SB203580, a p38 inhibitor, suppressed TNF-induced osteoclast differentiation, and inhibited p38 phosphorylation whereas it augmented ERK phosphorylation. These results demonstrate that ERK inhibition and p38 activation play crucial roles in both RANKL- and TNF-induced osteoclast differentiation.

Sphingosine‐induced c‐jun expression: differences between sphingosine‐ and C2‐ceramide‐mediated signaling pathways

Sphingolipids such as ceramide and sphingosine are putative intracellular signal mediators in cell differentiation, growth inhibition and apoptosis. Previously, we reported that C2‐ceramide induced c‐jun expression in apoptosis of human leukemia HL‐60 cells. Here we report that sphingosine also induced c‐jun expression in apoptosis of HL‐60 cells. Sphingosine‐induced c‐jun expression was stimulated by H‐89, a protein kinase A inhibitor, whereas C2‐ceramide‐induced c‐jun expression was inhibited by protein kinase C inhibitors. Furthermore, H‐89 potentiated sphingosine‐induced but not C2‐ceramide‐induced growth inhibition. These results suggest that sphingosine and C2‐ceramide might induce c‐jun expression and apoptosis in distinct signaling pathways.

Characterization of C2-ceramide-resistant HL-60 subline (HL-CR): involvement of PKC δ in C2-ceramide resistance

We have established a C2-ceramide-resistant HL-60 subline (HL-CR). HL-CR cells were resistant not only to C2-ceramide but also to various anticancer drugs. HL-CR cells did not respond to differentiation-inducing reagents including 1alpha,25-dihydroxyvitamin D(3), retinoic acid, and 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA induced apoptosis in HL-CR cells much slower than in parental HL-60 cells. As it was reported that PKC isozymes were involved in C2-ceramide-induced apoptosis, we investigated the role of PKC isozymes in C2-ceramide resistance in HL-CR cells. The protein level of PKC delta was lower in HL-CR cells than in parental HL-60 cells, whereas the levels of PKC alpha, betaI, epsilon, and zeta were rather higher in HL-CR cells than in parental cells. Translocation of PKC delta from membrane to cytosol was induced by C2-ceramide in HL-CR cells as well as in wild-type HL-60 cells. Furthermore, overexpression of PKC delta in HL-CR cells potentiated C2-ceramide- and TPA-induced apoptosis and growth inhibition. These results suggest a role for ceramide in apoptosis and differentiation in HL-60 cells, and also suggest that PKC delta might be involved in ceramide- and TPA-induced apoptosis.

Direct effects of estrogen on differentiation and apoptosis of osteoclasts

Abstract Osteoclasts play a crucial role in bone resorption. Since osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites, the investigation on osteoclasts is very important to the field of orthodontics. It is well known that estrogen has the protective effect on bone. However, the mechanisms by which estrogen prevents bone loss remain to be elucidated. Although estrogen was recently reported to induce apoptosis of osteoclasts, the precise mechanisms of estrogen-induced osteoclast apoptosis remained controversial with regard to whether estrogen affects osteoclasts directly or not. Here we investigated whether estrogen directly induces differentiation and apoptosis of osteoclasts in vitro using mouse monocytic RAW264 cells differentiated into osteoclasts by RANKL. It was observed that estrogen inhibited RANKL-induced osteoclast differentiation of RAW264 cells in a dose-dependent manner. Estrogen suppressed p38 phosphorylation while it enhanced ERK phosphorylation induced by RANKL, suggesting that modulation of MAPK signaling may be involved in inhibition of osteoclast differentiation by estrogen. Next, it was shown that estrogen dose-dependently augmented caspase-3 activation in osteoclasts differentiated from RAW264 cells by RANKL, demonstrating that estrogen directly enhanced apoptosis of osteoclasts. Estrogen-induced caspase-3 activation was attenuated by ICI 182,780, suggesting that the effects of estrogen on osteoclast apoptosis is mediated through estrogen receptors. Thus, these results suggest that estrogen may directly inhibit differentiation and induce apoptosis of osteoclasts.

Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis.

We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)-induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak.