Takehisa Ishii

3β-Hydroxysteroid-Δ24 Reductase Is a Hydrogen Peroxide Scavenger, Protecting Cells from Oxidative Stress-Induced Apoptosis

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

Differential regulation of IgA production by TGF-β and IL-5: TGF-β induces surface IgA-positive cells bearing IL-5 receptor, whereas IL-5 promotes their survival and maturation into IgA-secreting cells☆

Transforming growth factor beta (TGF-beta) and IL-5 have been shown to augment IgA production by LPS-stimulated murine B cells. We investigated the effect of TGF-beta on the expression of surface Ig-isotype and IL-5 receptor on LPS-stimulated B cells. TGF-beta increased the proportion of both surface IgA-positive (sIgA+) B cells and sIgG2b+ B cells and enhanced IgA and IgG2b production by LPS-stimulated B cells. TGF-beta synergized with IL-5 only for IgA production of the seven Ig-isotypes and in combination with IL-5 caused a significant increase in the proportion of sIgA+ B cells up to 17.4%. In contrast, IL-5 decreased the proportion of sIgG2b+ B cells and sIgG3+ B cells and inhibited the production of IgG2b and IgG3 by LPS-stimulated B cells. About 50% of sIgA+ cells induced by TGF-beta expressed IL-5 receptor. They secreted peak levels of IgA and seemed to maintain long viability in the presence of IL-5; whereas TGF-beta had the opposite effects on sIgA+ B cells and down-regulated the IL-5 receptor expression. These results indicate that TGF-beta increases the number of sIgA(+)- and IL-5 receptor-positive B cells which respond to IL-5 giving rise to IgA-secreting cells and also support the notions that TGF-beta preferentially induces switching to sIgA+ B cells and IL-5 induces the maturation of postswitch sIgA+ B cells into IgA-secreting cells in a stepwise fashion.

The decrease in total collagen fibers in the liver by hepatocyte growth factor after formation of cirrhosis induced by thioacetamide.

Liver cirrhosis is an inveterate disease accompanying fibrosis, hepatocyte damage, and liver dysfunction. In this study, the therapeutic effects of recombinant human hepatocyte growth factor (rhHGF) on liver cirrhosis were examined in rats administered thioacetamide (TAA). Repeated administration of TAA for 10 weeks to rats induced liver cirrhosis with collagen nodes and pseudo-lobe generation, a condition that was pathologically similar to that in humans. Administration of rhHGF after the formation of liver cirrhosis markedly decreased the incidence of pathological fibrosis and the degree of fibrosis as measured by a computed image analysis system. Continuous administration of rhHGF by infusion pump was more effective than bolus administration. Northern blotting analysis showed that rhHGF reduced mRNA levels of procollagen alpha2(I), alpha1(IV), and transforming growth factor-beta1 (TGF-beta1) that were stimulated in the TAA-treated liver. The labeling index of hepatocytes increased following administration of rhHGF in this model. These observations suggest that the pathological recession of liver fibrosis is the result of the reduction of TGF-beta1 and collagen synthesis and, in part, of the stimulation of mitosis of hepatocytes directly by rhHGF and indirectly by TGF-beta1 reduction in the cirrhotic liver. These results demonstrate the usefulness of rhHGF as a therapeutic agent in liver cirrhosis.

Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.

The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.

DEVELOPMENTAL CHANGES IN DISTRIBUTION OF DEATH-ASSOCIATED PROTEIN KINASE MRNAS

Death‐associated protein kinase (DAP‐kinase) is Ca2+/calmodulin‐dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon‐γ‐induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP‐kinase, the tissue distribution and developmental changes in mRNA expression of DAP‐kinase were investigated by Northern blot and in situ hybridization analyses. DAP‐kinase mRNA was predominantly expressed in brain and lung. In brain, DAP‐kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP‐kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP‐kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP‐kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP‐kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP‐kinase and neuronal cell death. J. Neurosci. Res. 58:674–683, 1999.

MORPHOLOGIC ALTERATION OF HEPATOCYTES AND SINUSOIDAL ENDOTHELIAL CELLS IN RAT FATTY LIVER DURING COLD PRESERVATION AND THE PROTECTIVE EFFECT OF HEPATOCYTE GROWTH FACTOR

BACKGROUND Fatty liver grafts are considered to be one of the main factors of primary nonfunctioning graft in transplantation. We investigated here, the hepatic damage during cold preservation in a rat fatty liver model by ultrastructural observation, and examined the effect of human recombinant hepatocyte growth factor (hrHGF) on amelioration of the cold-preserved graft condition. METHODS Wistar rats were fed a choline-deficient diet (CDD) for 7 days. Livers were stored in cold University of Wisconsin (UW) solution for 0, 4, and 24 hr. We evaluated the ultrastructural alteration of the hepatocytes, sinusoidal architecture, and endothelial cells (SECs) by scanning and transmission electron microscopy. Ex vivo, we measured alanine aminotransferase (ALT) in first effluent as an index of hepatocyte injury and the hyaluronic uptake rate (HUR) as that of SEC damage. We injected hrHGF into rats fed CDD for 7 days through the portal vein and also added it to the UW solution to determine whether or not the agent ameliorated the hepatic damage in cold-preserved fatty livers. RESULTS In rats fed CDD for 7 days, the lesion occupied by fat deposits appeared to enlarge with the duration of cold preservation leading to the disarrangement of sinusoidal architecture. Furthermore, sinusoidal endothelial damage, in which gaps, blebs, microvilli, and sinusoid denudation were detected, appeared to be more severe in these livers than in the corresponding control livers. ALT significantly increased in the 4-hr cold-preserved livers of rats fed CDD for 7 days. HUR decreased with 4-hr cold preservation and/or with CDD feeding. Administration of hrHGF prevented the expansion of fatty droplets and reduced SEC injury as detected by morphological observations. Increase of ALT in first effluent was inhibited to about one fourth the level observed in the 4-hr cold-preserved livers of rats fed CDD. Moreover, HUR significantly increased with the pretreatment of hrHGF. CONCLUSION The hepatic injury in both hepatocytes and SECs in cold-preserved fatty liver graft developed more rapidly and severely than in the corresponding controls and demonstrated a protective effect of hrHGF.

Internalization and degradation of hepatocyte growth factor in hepatocytes with down-regulation of the receptor/c-Met

Hepatocyte growth factor (HGF) promotes proliferation of cultured hepatocytes by its interaction with cell surface receptors. In this paper, we examined the metabolic fate of HGF using hepatocytes. Kinetic analysis using [125I]HGF showed that 40% of surface‐bound HGF was rapidly internalized in hepatocytes within 30 min at 37°C. Under these conditions, the amount of HGF‐bound c‐Met, the high‐affinity receptor, decreased from the cell surface. Furthermore, the internalized HGF was degraded and released from the cells. These results indicate that cell surface‐bound HGF is internalized and degraded by the receptors, including c‐Met, on hepatocytes.

Protective effect of hepatocyte growth factor against degeneration of the retinal pigment epithelium and photoreceptor in sodium iodate-injected rats.

Purpose: To investigate the possible protective effect of hepatocyte growth factor (HGF) against degeneration of photoreceptors and retinal pigment epithelium (RPE) in vivo. Methods: Sprague-Dawley (SD) rats received an intravitreal injection of HGF in the right eye. The left eye was injected with vehicle as a control. Two days after the intravitreal injections, rats were administered 40 mg/kg of sodium iodate (NaIO3) intravenously. Scotopic ERGs were elicited by different stimulus intensities with a maximum luminance of 0.84 log cds/m2. To evaluate RPE function, the azide response was evoked by intravenous injection of 0.1 mg sodium azide. These electrophysiological measurements were conducted on days 4, 7, 14, and 28 after the NaIO3 injections. After recording ERGs or azide response, animals were sacrificed for quantification of the histological change and immunohistochemical analysis using antibodies against RPE 65. Results: The threshold for the scotopic b-wave was significantly lower in HGF-treated eyes than in untreated control eyes (p < 0.005), and maximum b-wave amplitudes (Vbmax) were significantly larger in HGF-treated eyes (p < 0.05) across all experimental time points after NaIO3 injection. Azide response amplitudes were significantly larger in the HGF-treated eyes than in the untreated eyes (p < 0.05). The structure of the outer retina was preserved to a greater degree in the HGF-treated eyes than in the untreated eyes (p < 0.05). Immunohistochemical analysis demonstrated that irregular alignment of the outer nuclear layer was confined to the retinal area that was not stained with RPE 65. Conclusions: Our results indicated that an intravitreal injection of HGF provided significant protection against degeneration of the photoreceptor and RPE induced by systemic administration of NaIO3. This suggests that HGF could be used as a therapeutic agent for degeneration of photoreceptors as well as RPE.

Characterization of Monoclonal Antibodies against Human Parvovirus B19

Eleven hybridoma cell lines producing mouse monoclonal antibodies (mAbs) against human parvovirus B19 were established. Their specificity was as follows. Approximately 5% of fetal erythroid cells inoculated with B19 reacted with all the mAbs and with anti‐B19 positive human serum, but not with negative serum by indirect double immunofluorescence staining. All the mAbs recognized both VP‐1 (84 kDa) and VP‐2 (58 kDa) capsid proteins of B19 virions propagated in vitro and in vivo by Western blotting, and immunoprecipitated B19 virions.

Effects of recombinant human hepatocyte growth factor on the proliferation and function of porcine hepatocytes.

A porcine hepatocyte based bioartificial liver (BAL) is still insufficient to replace liver transplantation. In this experiment, to strengthen the performance of a BAL, the effect of human recombinant hepatocyte growth factor (rhHGF) on the proliferation and function of xenogeneic porcine hepatocytes was studied. Isolated porcine hepatocytes were seeded at various densities (5 x 10(3) to 8 x 10(4) cells/well) on a collagen coated 96 well plate in Dulbeccos modified Eagles medium (DMEM) with 10% FCS. After 4 hours, the medium was changed to DMEM with added insulin and dexamethasone. Subsequently, rhHGF was added at various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 ng/ml) and cultured for an additional 24, 48, and 72 hours, respectively. The proliferation of porcine hepatocytes in response to rhHGF reached a plateau at 2.5 ng/ml at 24 hours and subsequently decreased. The levels of porcine albumin vs protein present in the supernatant increased when cultured at high cell density. In conclusion, rhHGF was found to stimulate proliferation of porcine hepatocytes at low cell density and low concentration. rhHGF can also increase albumin synthesis at higher cell density, thus indicating its potential use in a more satisfactory porcine hepatocyte based BAL.