Masumi Akita

Constitutively Activated ALK2 and Increased SMAD1/5 Cooperatively Induce Bone Morphogenetic Protein Signaling in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice.

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(-)]. At 4 and 8 weeks, VC levels in brains from VC(-) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(-) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.

Age-related development of the arrangement of connective tissue fibers in the lamina propria of the human vocal fold.

A scanning electron microscopic study was made on the morphological changes occurring with age in collagen and elastic fibers in the lamina propria of the human vocal fold. We obtained the specimens from 32 autopsy cases ranging from 20 gestational weeks to 22 postnatal years and submitted them to digestion treatments with 10% sodium hydroxide and 90% formic acid. The vocal folds in fetuses and neonates consisted of sparse and dense areas of collagen and elastic fibers, and the vocal ligament was not found. In subjects 5 years of age, a deep dense area was found in the anterior and posterior maculae flavae, and longitudinal fibers were noted between the maculae. A structure of superficial versus deep layers appeared in children older than 10 years of age. The layered structure of the lamina propria was complete around 17 years of age. The development of the layered structure and the maturity of the fibers appeared to reflect the complexity of phonatory function during adolescence.

The proton pump inhibitor inhibits cell growth and induces apoptosis in human hepatoblastoma

PurposeIn normal physiology, a vacuolar-type proton pump (V-ATPase) maintains an intracellular acid microenvironment in lysosome, endosome, and other endomembrane systems. Cancer cells overexpress V-ATPase compared with normal cells, and disturbances of the acid environment are thought to significantly impact the cancer cell infiltration and growth. Bafilomycin A1 (Baf-A1) is a specific inhibitor of the proton-pump inhibitor (PPI) V-ATPase. Neoplastic cells are reportedly more sensitive to Baf-A1 than normal cells, and the difference between the susceptibility to Baf-A1 in normal cells and that in cancer cells may become a target in the cancer therapy. With this in mind, we used cells of hepatoblastoma, the cancer type accounting for 80% of all childhood liver cancers, to investigate the effects of Baf-A1 as an inducer of cancer cell apoptosis and inhibitor of cancer cell reproductionMethods and resultsElectron microscopy showed significant morphological change of the hepatoblastoma cells of the Baf-A1-treated group compared with hepatoblastoma cells of the Baf-A1-free group. The rate of the apoptotic cell increased, and cell reproduction was inhibited. Moreover, the analysis of hepatoblastoma cells using the gene Chip gene expression analysis arrays showed that three of the 27 V-ATPase-related transcripts (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the Baf-A1-treated cells than in the Baf-A1-free cells. In normal human hepatic cells, on the other hand, the inhibition of cell growth of the Baf-A1-treated cells was negligible compared to that of the cells without Baf-A1 treatment. The result of apoptotic cell detection by morphological observations and flow cytometry revealed that Baf-A1 inhibits hepatoblastoma cellular reproduction by inducing apoptosis. On the other hand, the Baf-A1-conferred inhibition of cell growth was negligible in normal human hepatocytesConclusionThe V-ATPase inhibitor Baf-A1 has been proven to selectively inhibit the reproduction and induce the apoptosis of hepatoblastoma cells without adversely influencing normal hepatic cells. With these effects, V-ATPase inhibitors may hold promise as therapeutic agents for hepatoblastoma. Given that three V-ATPase-related genes (ATP6V0D2, ATP6V1B1, and ATP6V0A1) were more weakly expressed in the hepatoblastoma cells of the Baf-A1-treated group than in the Baf-A1-free cells, drug development targeting V-ATPase gene of hepatoblastomas is expected.

A unique mutation of ALK2, G356D, found in a patient with fibrodysplasia ossificans progressiva is a moderately activated BMP type I receptor

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.

Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Ultrastructure of the lamina propria of the human vocal fold.

We studied three-dimensional arrangement of collagen fibers and elastic fibers in the human vocal fold by scanning electron microscopy after digesting cellular elements and collagen fibers with formic acid treatment and cellular elements and elastic fibers with 10% sodium hydroxide. The superficial layer consisted of clusters of collagen fibers and fine elastic fibers running straight or coiled. The intermediate layer consisted of thick bundles of collagen and elastic fibers running longitudinally and fine coiled elastic fibers. The deep layer consisted of coil elastic fibers and dense collagen bundles. Collagen fibers may have a role of maintaining the organization of vocal folds even during vibration, and elastic fibers act to rapidly restore the vocal folds to their original form. We also studied the distribution of oxytalan fibers in vocal folds by aldehyde-fucusin staining. Oxytalan fibers were distributed throughout the connective tissue of the vocal folds, and a large number of fibers was present just under the epithelial basement membrane and around the muscle fibers. If these fibers are damaged and lose their functions, vibration of the vocal folds will be impaired.

Unilateral anomalous left common carotid artery; a case report.

An anomaly of the left common carotid artery was observed in a Japanese male cadaver during an anatomy class at the Saitama Medical School in 1995. The superior thyroid, lingual and facial arteries arose from the common carotid artery, and the posterior auricular, maxillary and superficial temporal arteries arose from the common carotid artery by a common trunk. The occipital and ascending pharyngeal arteries arose from the internal carotid artery. The left carotid body (glomus caroticum) was observed to be slightly below the lingual artery, behind the common carotid artery, and it was located at the level of the intervertebral disk between C2 and C3 or at the same level as the right carotid body. The carotid body was richly innervated by a branch of the glossopharyngeal nerve and by a plexus of sympathetic fibers from the vagus and glossopharyngeal nerves. We assumed that the artery above the level of the carotid body was the internal carotid artery; there was no specific external carotid artery and all branches of the external carotid artery arose from the internal carotid artery.

ROLES OF MACROPHAGES IN PROGRAMMED CELL DEATH AND REMODELING OF TAIL AND BODY MUSCLE OF XENOPUS LAEVIS DURING METAMORPHOSIS

Abstract Examination was made of the involvement of macrophage phagocytosis in programmed cell death of tail and body muscle of the frog, Xenopus laevis, during metamorphosis by electron microscopy and immunohistochemical analysis. Electron microscopic observation revealed that macrophages were often found to be present in body and tail muscles at the most active stage of metamorphosis and to actively phagocytose apoptotic muscle fragments. Developmental changes in macrophages were examined using the macrophage-specific antibody, HAM56. Macrophages initially appeared in the early climax stage (stage 59), when the triiodothyronine (T3) level was high, increased rapidly during the process of muscle cell death, and assumed their greatest number at the late climax stage (stage 63/64). They decreased after stage 65/66, with a decrease in T3. Distribution and change in the number of macrophages were the same as those of muscle apoptotic bodies (sarcolytes) during metamorphosis, which suggests an interactive mechanism between macrophages and dying muscle cells. For clarification of this, study was made of the expression of HAM 56 antigens that were X. laevis homologs of mouse attachmin, non-specific adhesion proteins in macrophages. The expression of HAM56 antigens in macrophages was found to increase with macrophage phagocytosis at the late climax stage, thus, macrophage differentiation would appear to take place during metamorphosis and HAM56 antigens may be essential for macrophage–dying muscle cell interactions.

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver

 Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.