Akinobu Hayashi

Elastin expression is up-regulated by retinoic acid but not by retinol in chick embryonic skin fibroblasts

Effects of retinoid derivatives (retinol and retinoic acid) on elastin expression and cell proliferation in chick embryonic skin fibroblasts were studied. Retinoic acid inhibited cell proliferation one half of control at the concentration 10(-5) M. Retinoic acid exhibited stimulatory effect on elastin synthesis with a maximum stimulation of 2.0-fold at the concentration of 10(-6) M for 24 h treatment. Elastin level detected by Western blot analysis in the pooled conditioned medium was compatible with the increase of elastin synthesis by retinoic acid treatment. Comparable increase in elastin mRNA level was observed by retinoic acid treatment. Retinol showed no significant effects on either cell proliferation or elastin expression. The results indicate that retinoic acid was potentially active and retinol was inactive for modulation of cell proliferation and elastin expression.

In vitro degradation of tropoelastin by reactive oxygen species.

Abstract The effects of reactive oxygen species (ROS) on elastin molecules (tropoelastin) were studied in vitro. ROS generated by ultraviolet A and hematoporphyrin rapidly degraded tropoelastin within 5 min. Their degradative activity was inhibited by the addition of NaN 3 . Treatment of tropoelastin with copper sulfate/ascorbic acid resulted in degradation of tropoelastin producing fragments of molecular weight 45, 30 and 10 kDa within 30 min. The degradation of tropoelastin was partially blocked by the addition of mannitol. ROS induced by the xanthine/xanthine oxidase system also degraded tropoelastin within 6 h. The degradation was blocked by catalase but not by superoxide dismutase (SOD). ROS generated by copper-ascorbate seems to be unique in that it cleaves relatively specific sites of the tropoelastin molecule. Thus ROS may play a degradative role in elastin metabolism which may cause the elastolytic changes or the deposition of fragmented elastic fibers in photoaged skin or age-related elastolytic disorders.

Melanogenesis, biosynthetic phenotype of fibronectin and collagen, and migrating activity in cloned B16 mouse melanoma cells

Three clones, melanotic (M3), amelanotic (A4 and A7) cells were isolated from B16 F10 mouse melanoma cell line. These clones exhibited different biosynthetic activities of fibronectin and collagen: A4 clone showed relatively active synthesis of both collagen and fibronectin, and A7 clone exhibited most active fibronectin synthesis, whereas no significant synthesis of these molecules was observed in M3 clone. No significant difference in growth rate was observed in these three clones. Migrating activities measured by basement membrane matrix-coated dishes were greater in A7 clone than in A4 and M3 clones. Messenger RNA levels of collagen and fibronectin paralleled collagen and fibronectin synthesis in these clones whereas tyrosinase mRNA level was unaltered between melanotic (M3) and amelanotic (A4 and A7) cells. These results indicate that B16 melanoma cells have heterogeneous cell populations consisting of different biosynthetic and metastatic properties. These clones may provide good tools for studying the relationship between the phenotypes of melanogenesis biosynthesis of extracellular macromolecules and migrating activity of melanoma cells.

Stimulation of elastin expression by minoxidil in chick skin fibroblasts

Minoxidil inhibited the proliferation of embryonic skin fibroblasts during the growth phase but not during the stationary phase. Minoxidil stimulated elastin synthesis two-fold in a dose-dependent manner at a concentration of 1 mM during the stationary phase. The stimulation of elastin synthesis paralleled a comparable increase in elastin mRNA level. These results suggest that the stimulation of elastin expression by minoxidil in skin fibroblasts was controlled at the elastin mRNA level and also suggest that its elastinstimulating effect is not related to the suppressive effect on cell proliferation. Minoxidil appears to be a potent stimulator for elastin expression in skin fibroblasts.

Presence of elastin-related 45-kDa fragment in culture medium: specific cleavage product of tropoelastin in vascular smooth muscle cell culture

Significant amount of 45-kDa polypeptide was found to be present in the cultured medium of chick aortic smooth muscle cells. The polypeptide as well as tropoelastin (65-kDa) reacted with monoclonal antibody for tropoelastin. Pulse-chase experiments revealed that the relative density of the 45-kDa polypeptide to tropoelastin increased with chase periods. Partially purified radioactive tropoelastin (65-kDa) was converted to a 45-kDa polypeptide fragment in the test tube. The processing from the 65- to the 45-kDa polypeptide in the test tube was inhibited by ethylenediaminetetraacetic acid but not by N-ethylmaleimide and aminophenylmethylsulfonyl fluoride. These results indicate that the 45-kDa fragment is a degradation product of tropoelastin and that processing is mediated by enzymatic cleavages with metal proteinase. Fully hydroxylated tropoelastin treated with ascorbic acid was more resistant to the enzymes than underhydroxylated tropoelastin with scorbutic condition, suggesting that the structural stability of tropoelastin is also involved in the processing rate.

A novel gelatinolytic enzyme secreted by amelanotic cells isolated from B16 melanoma cell line

Mouse B16 melanoma cells are of heterogenous populations and consist of melanotic and amelanotic cells. Two clones, M3 (melanotic) and A7 (amelanotic), were screened from B16 melanoma cells using a microscope, and gelatinolytic activities in these clones were studied with zymography. Gelatinolytic enzyme was found in the cultured medium of A7 cells, but not in M3 cells. The enzymes with apparent molecular weights of 70, 140 and 280 kDa were detected and found to form complexes: a dimer or more than dimer. Their enzyme activities were not inhibited by ethylenediaminetetraacetic acid. The optimal pH of this enzyme was found to be at neutral pH. Preferential expression of this novel gelatinolytic enzyme in amelanotic melanoma will be related to tumorigenic and metastatic phenotype of amelanotic melanoma.

Ascorbic acid reduces tropoelastin levels in culture medium of chick skin fibroblasts

Ascorbic acid reduced tropoelastin levels in the culture medium of embryonic chick skin fibroblasts to one-third or one-fourth of their original value in a dose- and exposure time-dependent manner under conditions in which collagen synthesis was stimulated 1.8-fold. The steady-state level of elastin mRNA remained essentially constant, whereas the collagen mRNA content increased in proportion to the increase in collagen synthesis with ascorbic acid treatment. These results suggest that the reduction of tropoelastin levels is not controlled by the elastin mRNA level but, rather, is related to the posttranslational modification of the tropoelastin molecule.

Partial characterization of a unique 84-kDa polypeptide stimulated by ascorbic acid in skin fibroblasts

Ascorbic acid stimulated the synthesis of a noncollagenous 84 kDa polypeptide by up to 3.5-fold in primary cultures of chick skin fibroblasts. Stimulation was observed with concentrations ranging 0.4 to 50μg/ml and was dose-dependent within the concentrations up to 10μg/ml during 24 h treatment under conditions in which procollagen synthesis was increased. This protein was found to be translated with a molecular size of 84 kDa in an in vitro cell-free translation experiment, indicating that the 84 kDa polypeptide was a primary translate. The stimulation was accompanied by a 1.7-fold increase in translational activity of the mRNA for this polypeptide. Its synthesis rapidly reduced when the cells were serially passaged until the third subcultivation, whereas procollagen synthesis remained essentially constant. These results indicate that the stimulation of this unique 84 kDa polypeptide by ascorbic acid was modulated at least in part at a translational level and its synthesis appears to be related to in vitro cellular aging.