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Featured researches published by Yoshiaki Hirako.


Journal of Biological Chemistry | 1996

Demonstration of the Molecular Shape of BP180, a 180-kDa Bullous Pemphigoid Antigen and Its Potential for Trimer Formation

Yoshiaki Hirako; Jiro Usukura; Yuji Nishizawa; Katsushi Owaribe

The 180-kDa bullous pemphigoid antigen (BP180) is a hemidesmosomal transmembrane glycoprotein comprising interrupted collagen domains in its extracellular part. BP180 is also termed type XVII collagen. But the question of whether it actually takes a collagen-like triple helical conformation in vivo has remained unanswered. Using a monoclonal antibody, we found that a subpopulation of BP180 localizes at the lateral surfaces of corneal basal cells and cultured cells, in addition to the basal surface. This subpopulation of BP180 could be solubilized by 0.5% Triton X-100 and, among examined cell lines, was found to be most abundant in BMGE+H, a bovine mammary gland epithelial cell line. The Triton-soluble fraction of BMGE+H cells was used for characterization. On sucrose gradient centrifugation, the soluble BP180 demonstrated a value of approximately 7 S, and chemical cross-linking experiments revealed a trimer form. The calculated frictional ratio, f/f0 = 2.8, suggests an asymmetric configuration. For further characterization, we purified the soluble BP180 by immunoaffinity column chromatography using an anti-BP180 monoclonal antibody. Rotary shadowing images of the purified BP180 showed a quaver-like molecule consisting of a globular head, a central rod, and a flexible tail. With regard to the primary structure and species comparisons, the central rod, 60-70 nm in length, probably corresponds to the largest collagenous region, forming a collagen-like triple helix, in human form. The globular head and the flexible tail seem to correspond to the cytoplasmic and the interrupted collagenous region, respectively, of the extracellular portions. In conclusion, the present demonstration of the entire configuration of BP180, with a collagen-like trimer in its extracellular part, suggests that BP180 is one of the major components of anchoring filaments.


Journal of Investigative Dermatology | 2009

IgG from Patients with Bullous Pemphigoid Depletes Cultured Keratinocytes of the 180-kDa Bullous Pemphigoid Antigen (Type XVII Collagen) and Weakens Cell Attachment

Hiroaki Iwata; Naoko Kamio; Yumi Aoyama; Yukari Yamamoto; Yoshiaki Hirako; Katsushi Owaribe; Yasuo Kitajima

We have shown that binding of bullous pemphigoid (BP)-patient IgG (BP-IgG) causes the internalization of BP180 from the cell membrane. This study examined whether BP-IgG treatment can deplete cultured keratinocytes of BP180, how it affects cellular levels of alpha6 and beta4 integrins (by western blot analysis using monoclonal antibodies to these antigens), and whether it reduces adhesion of cells to the culture dish (by a vibration detachment assay). All BP-IgG or BP sera with high values of BP180-ELISA from 18 BP patients before and after oral corticosteroid treatment showed dramatically decreased BP180 in cells after 6 hours of BP-IgG stimulation, whereas alpha6 and beta4 integrin levels were not decreased. Even IgG from patients in whom oral corticosteroid had suppressed active blistering could deplete cells of BP180, as long as sera retained a high value of BP180-ELISA. On the other hand, reduction of cell BP180 content increased detachment of cells from the dish. These results suggest that BP-IgG reduces hemidesmosomal BP180 content, weakening the adhesion of hemidesmosomes to the lamina densa. In the presence of BP180 deficiency, inflammation generated by BP180 immune-complex formation might then tear the weakened lamina lucida, and this could lead to generation of the BP-specific split at the lamina lucida.


British Journal of Dermatology | 2001

Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230.

M Leverkus; K Bhol; Yoshiaki Hirako; Hendrikus Pas; C Sitaru; G Baier; Eb Brocker; Marcel F. Jonkman; Ar Ahmed; Detlef Zillikens

Cicatricial pemphigoid is a heterogeneous group of autoimmune subepidermal blistering diseases associated most commonly with autoantibodies to bullous pemphigoid (BP)180 and less frequently with those to laminin 5 or type VII collagen. In addition, a few cases have been described with autoantibodies to the β4 subunit of α6β4 integrin. We describe a patient with extensive disease of ocular, oral, pharyngeal, laryngeal and genital mucous membranes that healed with scarring of conjunctivae. IgG autoantibodies bound to the dermal–epidermal junction on direct immunofluorescence (IF) microscopy and to the epidermal side of 1 mol L−1 NaCl‐split skin on indirect IF microscopy. Our patients circulating IgG recognized a 205‐kDa protein in extracts of 293T cells transfected with the β4 subunit of α6β4 integrin and in the cell extract of DJM‐1 cells. Our patients IgG and IgA autoantibodies also reacted with full‐length BP180 derived from epidermal extracts and the ectodomain of BP180 (LAD‐1) derived from culture supernatant of keratinocytes. In addition, a weak IgG reaction with BP230 was noted. The disease rapidly responded to dexamethasone‐cyclophosphamide pulse therapy, and immunoblot reactivity to both β4 integrin and BP180 decreased according to disease activity.


Gut | 2006

Hepatic stellate cells express synemin, a protein bridging intermediate filaments to focal adhesions

Naoki Uyama; L Zhao; E Van Rossen; Yoshiaki Hirako; Hendrik Reynaert; D H Adams; Z Xue; Z Li; R Robson; Milos Pekny; Albert Geerts

Background and aims: In the liver, stellate cells play several important (patho)physiological roles. They express a broad but variable spectrum of intermediate filament (IF) proteins. The aim of this study was to investigate the expression and functions of the intermediate filament protein synemin in hepatic stellate cells (HSCs). Methods: In isolated and cultured rat HSCs, synemin expression was examined by quantitative reverse transcriptase polymerase chain reaction, western blotting, and immunocytochemistry. Protein–protein interaction between synemin and possible binding partners was investigated by co-immunoprecipitation and confocal microscopy. Results: Expression of synemin was significantly downregulated with increased culture time. In 1-day cultured HSCs, synemin associated with other IF proteins (GFAP, desmin, and vimentin), and with the focal adhesion proteins vinculin and talin, but not with α-actinin or paxillin. Synemin IF and focal adhesion proteins co-localised in long slender processes, but not in the lamellipodia. In human and rat liver tissue, the presence of synemin was investigated by immunohistochemistry. In normal rat and human livers, synemin immunoreactivity was found in HSCs, smooth muscle cells of hepatic arterioles, and nerve bundles in portal tracts, but not in portal fibroblasts. In CCl4-intoxicated rat livers and in human cirrhotic livers, immunoreactivity for synemin in the parenchymal tissue was decreased. Thus synemin was expressed in quiescent HSCs but not in portal fibroblasts; and synemin expression decreased with HSC activation in vivo during chronic liver damage and with HSC activation in culture. Conclusions: Synemin forms heteropolymeric filaments with type-III IF proteins and acts as a bridging protein between IFs and a specific type of focal adhesions.


Cell and Tissue Research | 2003

Characterization of mammalian synemin, an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins.

Yoshiaki Hirako; Hisashi Yamakawa; Yuki Tsujimura; Yuji Nishizawa; Masayo Okumura; Jiro Usukura; Hiroyuki Matsumoto; Kenneth W. Jackson; Katsushi Owaribe; Osamu Ohara

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.


Human Mutation | 2010

Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex

Ken Natsuga; Masashi Akiyama; Hideki Nakamura; Satoru Shinkuma; James R. McMillan; Akari Nagasaki; Cristina Has; Takeshi Ouchi; Akira Ishiko; Yoshiaki Hirako; Katsushi Owaribe; Daisuke Sawamura; Leena Bruckner-Tuderman; Hiroshi Shimizu

Plectin is a cytoskeletal linker protein that has a dumbbell‐like structure with a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS‐MD), and EB simplex with pyloric atresia (EBS‐PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full‐length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS‐MD and three EBS‐PA patients. In EBS‐PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS‐MD, the expression of the N‐ and C‐terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full‐length plectin isoform with residual expression of the rodless plectin isoform leads to EBS‐MD, and that complete loss or marked attenuation of full‐length and rodless plectin expression underlies the more severe EBS‐PA phenotype. These results also clearly account for the majority of EBS‐MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS‐PA PLEC1 mutations are generally outside exon 31. Hum Mutat 30:1–9, 2010.


Dermatology | 1994

A possible cell-biologic mechanism involved in blister formation of bullous pemphigoid : anti-180-kD BPA antibody is an initiator

Yasuo Kitajima; Yoshiaki Hirako; Katsushi Owaribe; H. Yaoita

In this short review, we summarize the results of our recent studies on the effects of anti-bullous-pemphigoid antigen (BPA) antibodies and BP sera on the hemidesmosome in cultured keratinocytes (DJM-1 cells) as examined by immunofluorescence microscopy. The 180-kD and the 230-kD BPAs localized on the basal plasma membrane showed a homogeneously dotted pattern in cells grown with low Ca2+ (0.07 mM), while they formed a peculiar concentric ring or arch (ring/arch) pattern in cells grown with high Ca2+ (1.87 mM). In addition, the 180-kD BPA was distributed also on the lateral/apical cell membrane, and the 230-kD BPA was found in the cytoplasm. The high Ca2+ ring/arch arrangement of BPAs was formed within 3 h after the low-high Ca2+ switch. Anti-180-kD BPA monoclonal antibodies (MAbs) and BP sera, but not anti-230-kD BPA MAbs, which were added into this system, caused the internalization of the 180-kD BPA from the lateral/apical cell membrane and inhibited the formation of the ring/arch pattern. These results suggest that autoantibodies to the 180-kD, but not to the 230-kD, BPAs may directly bind to the antigen on the cell surface of the basal cells and disturb the formation of hemidesmosomes. The 180-kD BPA appears to be an initiator of blister formation.


Microscopy Research and Technique | 1998

Hemidesmosomes and their unique transmembrane protein BP180

Yoshiaki Hirako; Katsushi Owaribe

Hemidesmosomes are adhesion complexes responsible for linking keratin intermediate filaments of stratified and complex epithelia to components of the extracellular matrix such as collagen fibrils. Over the past several years, it has become clear that there are at least five hemidesmosomal proteins, including HD1/ plectin and BP230 as cytoplasmic plaque proteins and integrin α6β4 and BP180 as transmembrane proteins. Among them, BP180 is unique as a transmembrane protein because of its collagenous extracellular domain. Recent biochemical and ultrastructural analyses have revealed its molecular configuration and nature as a major component of anchoring filaments connecting hemidesmosomes to the basement membrane. These results indicate that BP180 is a new type of adhesion receptor. In addition to biochemical analyses of these hemidesmosomal proteins, recent studies on patients with inherited skin blistering diseases and on knockout mice have demonstrated roles in hemidesmosome formation and stabilization, as well as unexpected, novel functions. Microsc. Res. Tech. 43:207–217, 1998.


Journal of Dermatology | 1994

Antibody‐Binding to the 180‐kD Bullous Pemphigoid Antigens at the Lateral Cell Surface Causes Their Internalization and Inhibits Their Assembly at the Basal Cell Surface in Cultured Keratinocytes

Yasuo Kitajima; Yoshiaki Hirako; Katsushi Owaribe; Shunji Mori; Hideo Yaoita

We demonstrated the effects of monoclonal antibodies to the 180‐kD and 230‐kD BP antigens (BPA) and of BP sera on Ca++‐induced formation of hemidesmosomes in cultured human keratinocytes (a cell line, DJM‐1) by immunofluorescence microscopy. Under low Ca++ (0.07 mM) conditions, the 180‐kD and 230‐kD BPAs were distributed homogeneously on the basal plasma membrane, while they formed a peculiar concentric ring or arch (ring/arch) arrangement in high‐Ca++ (1.87 mM) medium. On the other hand, the apical‐lateral cell membrane was stained homogeneously with antibodies to the 180‐kD BPA, but not to the 230‐kD BPA, both in low and high Ca++ media. The low‐high Ca++ switch at first caused disappearance of the antigen from the basal plasma membrane and then formed the high‐Ca++ ring/arch pattern within 3 hrs. In this system, monoclonal antibodies to the 180‐kD and 230‐kD BPAs and the sera from 5 BP patients, 2 pemphigus vulgaris (PV) patients, and 4 normal volunteers were added into the culture media. The addition of anti‐180‐kD BPA antibodies or any BP serum caused the internalization of the 180‐kD BPA from the apical‐lateral cell membrane and inhibited the Ca++‐induced formation of the ring/arch pattern on the basal membrane, possibly by inhibiting the movement of the antigen from the lateral to the basal membrane to form hemidesmosomes. The internalized fluorescence dots were shown to be composed of the 180‐kD BPA and patients IgG, but not of the 230‐kD BPA by double‐immunostaining, suggesting the internalized 180‐kD BPA was from the apical‐lateral membrane, but not from hemidesmosomes. Monoclonal antibodies to the 230‐kD BPA and normal and PV sera did not cause these effects. These results suggest that autoantibodies to the 180‐kD BPA, but not to the 230‐kD BPA, may directly bind the antigen on the cell surface and disturb the formation of hemidesmosomes.


Human Mutation | 2010

Plectin Deficiency Leads to Both Muscular Dystrophy and Pyloric Atresia in Epidermolysis Bullosa Simplex

Ken Natsuga; Satoru Shinkuma; Ken Arita; Hideki Nakamura; Makiko Ohyama; Hitoshi Osaka; Takeshi Kambara; Yoshiaki Hirako; Hiroshi Shimizu

Plectin is a cytoskeletal linker protein which has a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS‐MD), and EBS with pyloric atresia (EBS‐PA). Previous studies have demonstrated that loss of full‐length plectin with residual expression of the rodless isoform leads to EBS‐MD, whereas complete loss or marked attenuation of expression of full‐length and rodless plectin underlies the more severe EBS‐PA phenotype. However, muscular dystrophy has never been identified in EBS‐PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon‐causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient.

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