Nobuyo Higashi

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum ‐associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin‐null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Cell–cell junctions between mammalian (human and rat) hepatic stellate cells

To investigate intercellular junctions between mammalian hepatic stellate cells, we examined cultured human and rat hepatic stellate cells at the ultrastructural and molecular levels. Intercellular junctions between cultured human stellate cells, which developed irrespective of the type of culture substratum, were detected by transmission electron microscopy. On the basis of their characteristic ultrastructure, these junctions were identified in cultured human hepatic stellate cells as adherens junctions but not as tight junctions, desmosomes, or gap junctions. N-cadherin, α-catenin and β-catenin, and p120ctn were detected by Western blotting in rat stellate cells as molecular components of the intercellular adhesive structures. Immunofluorescence for pan-cadherin, α-catenin, and β-catenin were also detected in cultured human stellate cells. Moreover, pan-cadherin and β-catenin were co-localized at the contact regions between the cultured human stellate cells. These data suggest that the junctional adhesion between the stellate cells can be formed both in vivo and in vitro. Thus, hepatic stellate cells may participate in the structural organization of the cells in liver lobules through the formation of intercellular adherens junctions. This is the first description of the presence of cell–cell junctions between hepatic stellate cells in mammals at the fine structural and molecular levels.

Intralobular Distribution of Vitamin A-Storing Lipid Droplets in Hepatic Stellate Cells with Special Reference to Polar Bear and Arctic Fox

We examined the liver of adult polar bears, arctic foxes, and rats by gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, hematoxylin-eosin staining, staining with Massons trichrome, Ishii and Ishiis silver impregnation, and transmission electron microscopical morphometry. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. These results indicate that the hepatic stellate cells of the polar bears and arctic foxes possess heterogeneity of vitamin A-storing capacity in their liver lobules.

3-D structure of extracellular matrix regulates gene expression in cultured hepatic stellate cells to induce process elongation.

HSCs showed myofibroblast-like shapes when cultured on polystyrene surface or on type I collagen-coated surface, whereas HSCs cultured on type I collagen gel were induced to elongate cellular processes, suggesting that HSCs recognize 3-D structure of extracellular type I collagen fibrils and change their morphology and function. In this study we examined the differentially regulated gene expression by extracellular matrix (ECM) components by PCR-differential display (PCR-DD) analysis followed by cloning and FASTA homology search, and identified the mRNA species as a transcription factor SP1, breast cancer resistant protein (BCRP), dystonin, and KAP3B. Regulation of dystonin and KAP3B expression was confirmed by RT-PCR analysis. Thus, cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression, and HSCs not only produce various ECM components but also change their morphology and gene expression in response to ECM components adhering to the cells.

Decreased Capacity for Vitamin A Storage in Hepatic Stellate Cells for Arctic Animals

Under physiological conditions, hepatic stellate cells store 80 % of the total vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm, and regulate both transport and storage of vitamin A [1-3]. It has been demonstrated that animal or human individuals exposed to drugs such as methadone, prednisone and phenobarbital, antiepileptics [4], and xenobiotics like the environmental contaminants DDT, PCD and dioxins have dramatic changes in their retinoid metabolism and function. Recent data demonstrate that top predators among Svalbard mammals and birds like polar bear, arctic fox and glaucous gull accumulate relatively large amounts of persistent organic pollutants [5]. Since it has been reported that vitamin A accumulates at near toxic doses in some arctic predators [6] and recent data suggest that PCB and DDT may reduce the threshold for vitamin A-toxicity [7], an increasing accumulation of persistent organic pollutants might eventually precipitate vitamin A-toxicity in these animals. To elucidate the possibility of vitamin A-related toxicity in arctic predators, we have performed a systematic characterization of the hepatic vitamin A-storage, which is the best index of the vitamin A-status, in mammals of the Svalbard archipelago.

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.

Intercellular Adhesive Structures Between Stellate Cells – An Analysis in Cultured Human Hepatic Stellate Cells

To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro.