Masato Yano

The Endoplasmic Reticulum Stress-C/EBP Homologous Protein Pathway-Mediated Apoptosis in Macrophages Contributes to the Instability of Atherosclerotic Plaques

Objective—To elucidate whether and how the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway in macrophages is involved in the rupture of atherosclerotic plaques. Methods and Results—Increases in macrophage-derived foam cell death in coronary atherosclerotic plaques cause the plaque to become vulnerable, thus resulting in acute coronary syndrome. The ER stress–CHOP/growth arrest and DNA damage-inducible gene-153 (GADD153) pathway is induced in the macrophage-derived cells in atherosclerotic lesions and is involved in plaque formation. However, the role of CHOP in the final stage of atherosclerosis has not been fully elucidated. Many CHOP-expressing macrophages showed apoptosis in advanced ruptured atherosclerotic lesions in wild-type mice, whereas few apoptotic cells were observed in Chop−/− mice. The rupture of atherosclerotic plaques was significantly reduced in high cholesterol–fed Chop−/−/Apoe−/− mice compared with Chop+/+/Apoe−/− mice. Furthermore, using mice that underwent bone marrow transplantation, we showed that expression of CHOP in macrophages significantly contributes to the formation of ruptures. By using primary cultured macrophages, we further showed that unesterified free cholesterol derived from incorporated denatured low-density lipoprotein was accumulated in the ER and induced ER stress-mediated apoptosis in a CHOP-Bcl2-associated X protein (Bax) pathway-dependent manner. Conclusion—The ER stress-CHOP-Bax–mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques.

AIP is a mitochondrial import mediator that binds to both import receptor Tom20 and preproteins.

Most mitochondrial preproteins are maintained in a loosely folded import-competent conformation by cytosolic chaperones, and are imported into mitochondria by translocator complexes containing a preprotein receptor, termed translocase of the outer membrane of mitochondria (Tom) 20. Using two-hybrid screening, we identified arylhydrocarbon receptor–interacting protein (AIP), an FK506-binding protein homologue, interacting with Tom20. The extreme COOH-terminal acidic segment of Tom20 was required for interaction with tetratricopeptide repeats of AIP. An in vitro import assay indicated that AIP prevents preornithine transcarbamylase from the loss of import competency. In cultured cells, overexpression of AIP enhanced preornithine transcarbamylase import, and depletion of AIP by RNA interference impaired the import. An in vitro binding assay revealed that AIP specifically binds to mitochondrial preproteins. Formation of a ternary complex of Tom20, AIP, and preprotein was observed. Hsc70 was also found to bind to AIP. An aggregation suppression assay indicated that AIP has a chaperone-like activity to prevent substrate proteins from aggregation. These results suggest that AIP functions as a cytosolic factor that mediates preprotein import into mitochondria.

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an ∼120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable ∼380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.

Angiopoietin-like Protein 2 Is an Important Facilitator of Inflammatory Carcinogenesis and Metastasis

Chronic inflammation plays important roles at different stages of cancer development, including carcinogenesis, tumor invasion, and metastasis, but molecular mechanisms linking inflammation to cancer development have not been fully clarified. Here, we report that expression of angiopoietin-like protein 2 (Angptl2), recently identified as a chronic inflammation mediator, is highly correlated with the frequency of carcinogenesis in a chemically induced skin squamous cell carcinoma (SCC) mouse model. Furthermore, Angptl2 expression in SCC is highly correlated with the frequency of tumor cell metastasis to distant secondary organs and lymph nodes. When SCC was induced in transgenic mice expressing Angptl2 in skin epithelial cells, epithelial-to-mesenchymal transitions in SCC as well as tumor angiogenesis and lymphangiogenesis were significantly increased, resulting in increased tumor cell metastasis and shortened survival compared with wild-type mice. Conversely, in a chemically induced SCC mouse model, carcinogenesis and metastasis were markedly attenuated in Angptl2 knockout mice, resulting in extended survival compared with wild-type mice. Overall, we propose that Angptl2 contributes to increased carcinogenesis and metastasis and represents a novel target to antagonize these pathologies.

A type I DnaJ homolog, DjA1, regulates androgen receptor signaling and spermatogenesis

Two type I DnaJ homologs DjA1 (DNAJA1; dj2, HSDJ/hdj‐2, rdj1) and DjA2 (DNAJA2; dj3, rdj2) work similarly as a cochaperone of Hsp70s in protein folding and mitochondrial protein import in vitro. To study the in vivo role of DjA1, we generated DjA1‐mutant mice. Surprisingly, loss of DjA1 in mice led to severe defects in spermatogenesis that involve aberrant androgen signaling. Transplantation experiments with green fluorescent protein‐labeled spermatogonia into DjA1−/− mice revealed a primary defect of Sertoli cells in maintaining spermiogenesis at steps 8 and 9. In Sertoli cells of DjA1−/− mice, the androgen receptor markedly accumulated with enhanced transcription of several androgen‐responsive genes, including Pem and testin. Disruption of Sertoli–germ cell adherens junctions was also evident in DjA1−/− mice. Experiments with DjA1−/− fibroblasts and primary Sertoli cells indicated aberrant androgen receptor signaling. These results revealed a critical role of DjA1 in spermiogenesis and suggest that DjA1 and DjA2 are not functionally equivalent in vivo.

Regulatory effects of the mitochondrial energetic status on mitochondrial p66Shc.

Abstract p66Shc promotes apoptosis and controls the intracellular redox balance. A fraction of p66Shc exists within mitochondria, where it oxidizes cytochrome c to form hydrogen peroxide, which in turn induces mitochondrial permeability and apoptosis. However, cells tolerate p66Shc expression and accumulate oxidative damage under normal conditions, implying that the p66Shc functions must be tightly regulated. Here we review available knowledge on the regulation of p66Shc transcription, protein stabilization and post-translational modifications. In addition, we report novel investigations into the role of the mitochondrial import machinery on p66Shc activation, which highlight the energetic status of mitochondria as a crucial determinant of p66Shc function.

CO 2 exchange in a temperate Japanese cypress forest compared with that in a cool-temperate deciduous broad-leaved forest

To examine the characteristics of carbon exchange in coniferous forests, we analysed the seasonal and diurnal patterns of CO2 exchange, as measured using the eddy covariance method, in a Japanese cypress forest in the Kiryu Experimental Watershed (KEW) in central Japan. The net CO2 exchange data during periods of low-friction velocity conditions and during periods of missing data were interpolated. The daily CO2 uptake was observed throughout the year, with maximum values occurring in early summer. Periods of low carbon uptake were seen in late summer owing to high respiratory CO2 efflux. The diurnal and seasonal patterns of daytime CO2 exchange at KEW were compared with those in a cool-temperate deciduous forest of the Tomakomai Experimental Forest (TOEF) in Japan. The environmental differences between evergreen and deciduous forests affected the seasonal patterns of carbon uptake. Although there were great differences in the mean monthly air temperatures between the sites, the mean monthly daytime carbon uptake was almost equal at both sites during the peak growing period. The carbon-uptake values at the same PAR level were greater before noon than after noon, especially at TOEF, suggesting the stomatal regulation of carbon uptake.

Angiopoietin-Related Growth Factor Enhances Blood Flow Via Activation of the ERK1/2-eNOS-NO Pathway in a Mouse Hind-Limb Ischemia Model

Objective—Transgenic mice overexpressing angiopoietin-related growth factor (AGF) exhibit enhanced angiogenesis, suggesting that AGF may be a useful drug target in ischemic disease. Our goal was to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define molecular mechanisms underlying AGF signaling in endothelial cells. Methods and Results—Intramuscular injection of adenovirus harboring AGF into the ischemic limb increased AGF production, which increased blood flow through induction of angiogenesis and arteriogenesis, thereby reducing the necessity for limb amputation. In vitro analysis showed that exposing human umbilical venous endothelial cells to AGF increased nitric oxide (NO) production through activation of an ERK1/2-endothelial NO synthetase (eNOS) signaling pathway. AGF-stimulated eNOS phosphorylation, NO production, and endothelial cell migration were all abolished by specific MEK1/2 inhibitors. Moreover, AGF did not restore blood flow to ischemic hind-limbs of either mice receiving NOS inhibitor L-NAME or eNOS knockout mice. Conclusion—Activation of an ERK1/2-eNOS-NO pathway is a crucial signaling mechanism by which AGF increases blood flow through induction of angiogenesis and arteriogenesis. Further investigation of the regulation underlying AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.

Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106–125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitroimport of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.

Participation of the import receptor Tom20 in protein import into mammalian mitochondria: analyses in vitro and in cultured cells

© 1997 Federation of European Biochemical Societies.