The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model.

Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Iontophoretic transdermal delivery of glycyrrhizin: effects of pH, drug concentration, co-ions, current intensity, and chemical enhancers.

The aim of the present study was to evaluate the feasibility of transdermal delivery of glycyrrhizin, an agent used in the treatment of chronic hepatitis C, by cathodal iontophoresis using Ag/AgCl electrodes in vitro. The effects of donor pH (pH 4-7), concentration of drug (0.025-0.2% (w/v)), concentration of external chloride ions (Cl(-)) (0-133 mM), current strength (0-0.5 mA/cm(2)), and permeation enhancers (urea and Tween 80) on the skin permeability of glycyrrhizin were examined in in vitro skin permeation studies using porcine ear skin as the membrane. The cumulative amount of permeated glycyrrhizin and the steady-state skin permeation flux of glycyrrhizin across porcine skin increased in a pH-dependent manner. The skin permeability of glycyrrhizin was independent of the concentration of drug and competed only with a high external Cl(-) concentration. The skin permeation flux of glycyrrhizin increased with the current (R(2)=0.8955). The combination of iontophoresis and enhancers provided an additive or synergistic effect, and a skin permeation flux of about 60 µg/h/cm(2) was achieved. The plasma concentration of glycyrrhizin in humans, extrapolated from the in vitro steady-state permeation flux across porcine skin, was within the therapeutic level. These results suggest that cathodal iontophoresis can be used as a transdermal drug delivery system for glycyrrhizin using reasonable patch sizes and acceptable levels of current intensity.

Activation of adenosine A1 receptor-induced neural stem cell proliferation via MEK/ERK and Akt signaling pathways.

Adenosine, a modulator of neuronal function in the mammalian central nervous system, exerts a neuroprotective effect via the adenosine A(1) receptor; however, its effect on neural stem cells (NSCs) remains unclear. Because adenosine is released in response to pathological conditions and NSCs play a key role in neuroregeneration, we tested the hypothesis that adenosine is capable of stimulating NSC proliferation. We demonstrated that NSCs dominantly express adenosine A(1) and A(2B) receptors. Adenosine and the adenosine A(1) receptor agonist cyclopentyladenosine (CPA) increased proliferation of NSCs, and this CPA-induced cell proliferation was attenuated by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). CPA also induced phosphorylation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase/ERK kinase (MEK), and Akt, and their phosphorylation was inhibited by DPCPA. In addition, CPA-induced cell proliferation was inhibited by MEK and Akt inhibitors. These results suggest that activation of adenosine A(1) receptor-stimulated proliferation of NSCs occurs via MEK/ERK and Akt signaling pathways.

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish.

BACKGROUND: Death receptors on the cell surface and the interacting cytosolic molecules, adaptors and initiator caspases, are essential as core components of the extrinsic apoptotic signaling pathway. While the apoptotic machinery governing the extrinsic signaling pathway is well characterized in mammals, it is not fully understood in fish. RESULTS: We identified and characterized orthologs of mammalian Fas, FADD and caspase-8 that correspond to the death receptor, adaptor and initiator caspase, from the Medaka fish (Oryzias latipes). Medaka Fas, caspase-8 and FADD exhibited protein structures similar to that of their mammalian counterparts, containing a death domain (DD), a death effector domain (DED) or both. Functional analyses indicated that these molecules possess killing activity in mammalian cell lines upon overexpression or following activation by apoptotic stimuli, suggesting similar pro-apoptotic functions in the extrinsic pathway as those in mammals. Genomic sequence analysis revealed that the Medaka fas (tnfrsf6), fadd and caspase-8 (casp8) genes are organized in a similar genomic structure as the mammalian genes. Database search and phylogenetic analysis revealed that the fas gene, but not the fadd and casp8 genes, appear to be present only in vertebrates. CONCLUSION: Our results indicate that the core components necessary for the extrinsic apoptotic pathway are evolutionarily conserved in function and structure across vertebrate species. Based on these results, we presume the mechanism of apoptosis induction via death receptors was evolutionarily established during the appearance of vertebrates.

Multiple promoters regulate tissue-specific alternative splicing of the human kallikrein gene, KLK11/hippostasin.

The human kallikrein (KLK) family consists of 15 genes located on human chromosome 19q13.4. KLK11/hippostasinis a member of the kallikrein family and is expressed in various tissues. Two types of KLK11 isoforms, isoform 1 and isoform 2, have been predicted from cDNA sequences. Isoform 1 has been isolated from human hippocampus, whereas isoform 2 has been isolated from prostate. However, the regulation and characteristics of these isoforms are unknown. We identified the first three exons (1a, 1b, and 1c) by determining their transcription initiation sites. Exon 1b contained the initiation codon of isoform 2, and noncoding exons 1a and 1c contributed to isoform 1 mRNA. The dual luciferase promoter assay revealed three promoter regions, corresponding to the first exon of each isoform. Reverse transcription and PCR showed that exon 1a was expressed in the hippocampus, thalamus, and non-central nervous system (CNS) tissues, whereas exon 1b was detected only in non-CNS tissues. Exon 1c was observed in both CNS and non-CNS tissues, except for salivary glands. In vitro mutagenesis revealed that the initiation codon for isoform 2 in exon 1b was functional. Isoform 2 had additional hydrophilic amino acids at the amino terminal and was secreted from the neuroblastoma cell line Neuro2a. Isoform 1 fused with green fluorescent protein (GFP) was distributed to cellular processes, whereas isoform 2-GFP was retained in the Golgi apparatus. We suggest that not only alternative splicing but also tissue-specific use of multiple promoters regulate the expression and intracellular trafficking of KLK11/hippostasin isoforms.

A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells.

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.

Green fluorescent protein as a marker in transgenic mice.

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.

In vitro and in vivo transdermal iontophoretic delivery of naloxone, an opioid antagonist.

AIM: The feasibility of transdermal delivery of naloxone, an opioid antagonist, by anodal iontophoresis patches using Ag/AgCl electrodes was investigated. METHODS: To examine the effect of current strength, species variation and drug concentration on skin permeability of naloxone, in vitro skin permeation studies were performed using rat dorsal skin and porcine ear skin as the membrane. To determine in vivo transdermal absorption rate of naloxone, the iontophoretic patch system was applied to the dorsal skin of conscious rat with a constant current supply for 24h. RESULTS: The in vitro steady-state skin permeation flux of naloxone current-proportionally (0-360 µA/cm(2)) increased without significant differences between these two different skin types. The in vitro delivery rate through the porcine skin was found to be independent of the concentration of naloxone hydrochloride dehydrate in the donor patch over the range from 1 to 10% (w/v). In the in vivo pharmacokinetic study, plasma concentrations of naloxone steadily increased and sustained steady-state levels from 4h to 24h after the initiation of current application. In vivo steady-state transdermal absorption rates at 90 and 180 µA/cm(2) were 136 and 305 µg/h/cm(2), respectively. CONCLUSION: These results suggest that the transdermal delivery rates of naloxone by anodal iontophoresis are sufficient for the management of intoxication in opioid-overdosed patients.

In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis.

BACKGROUND: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. CONCLUSIONS: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.

In-vitro and in-vivo transdermal iontophoretic delivery of tramadol, a centrally acting analgesic.

OBJECTIVES: The feasibility of transdermal delivery of tramadol, a centrally acting analgesic, by anodal iontophoresis using Ag/AgCl electrodes was investigated in vitro and in vivo. METHODS: To examine the effect of species variation and current strength on skin permeability of tramadol, in-vitro skin permeation studies were performed using porcine ear skin, guinea-pig abdominal skin and hairless mouse abdominal skin as the membrane. In an in-vivo pharmacokinetic study, an iontophoretic patch system was applied to the abdominal skin of conscious guinea pigs with a constant current supply (250 µA/cm(2)) for 6 h. An intravenous injection group to determine the pharmacokinetic parameters for estimation of the transdermal absorption rate in guinea pigs was also included. KEY FINDINGS: The in-vitro steady-state skin permeation flux of tramadol current-dependently increased without significant differences among the three different skin types. In the in-vivo pharmacokinetic study, plasma concentrations of tramadol steadily increased and reached steady state (336 ng/ml) 3 h after initiation of current supply, and the in-vivo steady-state transdermal absorption rate was 499 µg/cm(2) per h as calculated by a constrained numeric deconvolution method. CONCLUSIONS: The present study reveals that anodal iontophoresis provides current-controlled transdermal delivery of tramadol without significant interspecies differences, and enables the delivery of therapeutic amounts of tramadol.

Heterogeneity of pluripotent marker gene expression in colonies generated in human iPS cell induction culture.

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However, the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4, Sox2, c-Myc, and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog, TDGF, and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction, small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology, gene expression, long-term self-renewal ability, and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation.

The initiator caspase, caspase-10beta, and the BH-3-only molecule, Bid, demonstrate evolutionary conservation in Xenopus of their pro-apoptotic activities in the extrinsic and intrinsic pathways.

Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10beta), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10beta and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10beta and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10beta mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

Transgenic frogs expressing the highly fluorescent protein venus under the control of a strong mammalian promoter suitable for monitoring living cells.

To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.

The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity.

FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.