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KMID : 0578319960060030325
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Molecules and Cells
1996 Volume.6 No. 3 p.325 ~ p.333
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Expression and Functional Analysis of the 66-kDa, Protein, a Matrix Assembly Receptor of Fibronection during Myogenesis
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Moon, Kyeong-Yeop
Kook, Seung Hyi/song, Woo Keun/Kwon, Hyockman/Chung, Chin Ha/Kang, Man-Sik
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Abstract
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Previously, we have identified a 66-kDa protein on the surface of chick myoblasts that has a binding activity toward the N-terminal 29-kDa fragment of fibronectin [Moon, K Y., Shin, K S., Song, W. K, Chung, C. H., Ha, D. B., and Kang, M. S. (1994) J. Biol. Chem. 269, 7651- 7657]. In this report, we describe the changes in expression and cellular localization of the 66-kDa protein, fibronectin, and α5 integrin during myogenesis. The effects of an anti-66-kDa antibody on myoblast differentiation were also studied. Immunofluorescence staining of myoblasts with an anti-66-kDa protein antibody revealed dot-like circular aggregates in which the 66-kDa protein colocalized with α5 integrin and fibronectin. These aggregates are proposed to be initiation sites of fibronectin matrix assembly in early myoblasts. As myoblasts became elongated, the dot-like aggregated pattern progressively diminished and the 66-kDa protein and fibronectin redistributed into long fibrillar structures located along the cell periphery. These long fibrillar structures did not contain α5 integrin. Instead, the cells stained at this stage with an anti-α5 integrin antibody showed small spikes in the adhesion plaques. As the cells further matured, the 66-kDa protein and the fibronectin matrix totally disappeared, whereas α5 integrin was still present in myotubes. These results suggest that the 66-kDa protein is responsible for the initiation and elongation of fibronectin matrix assembly, whereas α5 integrin is only involved in the initiation of fibronectin matrix assembly. Immunoblotting analysis showed that the expression of the 66-kDa protein and fibronectin dramatically decreased during myogenesis and then completely disappeared in the late stage of myogenesis. The disappearance of fibronectin matrices is closely correlated to the decrease in the 66-kDa protein. Furthermore, the anti-66-kDa protein antibody suppressed the accumulation of fibronectin into the extracellular matrix and promoted the differentiation of myoblast in concentration- and treatment time-dependent manners. These results suggest that the 66-kDa protein may regulate myoblast differentiation by controlling the incorporation of fibronectin into the extracellular matrix.
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