Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.     

Depletion of Jab1 inhibits proliferation of pancreatic cancer cell lines

Jab1 overexpression is observed in many human cancers, but its physiological significance remains to be investigated. We reduced the level of Jab1 expression in pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 by the RNA interference and found that Jab1-knockdown resulted in impaired cell proliferation and enhanced apoptosis regardless of the genotype of the tumor suppressor p53. This growth inhibition was rescued by the introduction of siRNA-resistant mouse Jab1 cDNA. Jab1-knocked-down cells expressed a higher level of c-myc, and additional depletion of c-myc rescued cells from Jab1-knockdown-mediated growth suppression. Thus, Jab1 overexpression contributes to pancreatic cancer cell proliferation and survival. Jab1 could be a novel target in cancer therapy.     

Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.     

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.     

NADP(+)-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Critical Role of Heparin Binding Domains of Ameloblastin for Dental Epithelium Cell Adhesion and Ameloblastoma Proliferation

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.The extracellular matrix provides structural support for cells and regulates cell proliferation, migration, differentiation, and apoptosis for tissue development and homeostasis (1). The extracellular matrix also plays a crucial role in pathological processes and diseases, such as wound healing, tumorigenesis, and cancer development (2, 3). AMBN (ameloblastin), also known as amelin and sheathlin, is a tooth-specific extracellular matrix and the most abundant non-amelogenin enamel matrix protein (4–6). AMBN is expressed primarily by ameloblasts, which are differentiated from the oral ectoderm and form a polarized single cell layer underlying the enamel matrix. In a previous study, we created Ambn-null mice and demonstrated that AMBN is required for cell attachment and polarization and for maintaining the differentiation state of ameloblasts and is essential for enamel formation (3). Overexpression of Ambn in transgenic mice causes abnormal enamel crystallite formation and enamel rod morphology (7). These results suggest that enamel formation and rod morphology are influenced by temporal and spatial expressions of AMBN and imply that the AMBN gene locus may be involved in the etiology of a number of cases of undiagnosed hereditary amelogenesis imperfecta (8). Further, it was reported that recombinant AMBN enhances pulpal wound healing and reparative dentine formation following pulpotomy procedures, suggesting that it functions as a signal molecule in epithelial-mesenchymal interactions (9).We previously reported that about 20% of Ambn-null mice developed an odontogenic tumor of dental epithelium origin in the buccal vestibule of the maxilla (3). The epithelial cells of odontogenic tumors express enamel matrix proteins, including AMEL (amelogenin), ENAM (enamelin), and TUFT (tuftelin), but not AMBN, indicating that AMBN deficiency is probably the primary cause of tumorigenesis seen in those mice. An ameloblastoma appearing in the jaw is the most frequently encountered odontogenic tumor and is characterized by benign but locally invasive behavior with a high rate of recurrence. Since abnormal proliferation and growth of ameloblastoma cells easily destroys surrounding bony tissues, wide excision is required to treat this disorder. It is also reported that ameloblastomas rarely metastasize to other parts of the body, such as the lungs and regional lymph nodes (10, 11). Associations of AMBN mutations were reported in ameloblastomas, adenomatoid odontogenic tumors, and squamous odontogenic tumors (12). These results suggest that AMBN regulates odontogenic tumor formation.In the present study, we investigated the mechanism of AMBN in dental epithelial cell adhesion and ameloblastoma proliferation. We found that AMBN has heparin binding domains, which are essential for AMBN binding to dental epithelial cells. We demonstrate that overexpression of recombinant AMBN inhibits proliferation of human ameloblastoma cells. This inhibition requires the heparin binding sites of AMBN and is accompanied by dysregulation of Msx2, p21, and p27. These results suggest that AMBN suppresses ameloblastoma cell proliferation by regulating cellular signaling through the heparin binding domains.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Host range of obligate intracellular bacterium Parachlamydia acanthamoebae

The obligate intracellular bacterium Parachlamydia acanthamoebae is a potential human pathogen, but the host range of the bacteria remains unknown. Hence, the growth of P. acanthamoebae Bn₉ in protozoa (Tetrahymena, Acanthamoeba, Dictyostelium) and mammalian cells (HEp‐2, Vero, THP‐1, PMA‐stimulated THP‐1, Jurkat) was assessed using an AIU assay which had been previously established by the current authors. P. acanthamoebae grew in Acanthamoeba but not in the other cell types. The growth was also confirmed using DAPI staining, FISH and TEM. These results indicate that the host range of P. acanthamoebae is limited.