Abstract
Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.
Highlights
§ Present address: Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662
It will be shown that this inhibition is mediated directly by interfering with the signaling cascade triggered upon binding of TGF- to its transducing receptors. These findings demonstrate that the responsiveness of myoblasts to TGF-, an inhibitor of skeletal muscle differentiation, is directly modulated by decorin expression
Because decorin binds TGFϪ, a strong inhibitor of skeletal muscle differentiation, we reasoned that the down-regulation of decorin expression would affect skeletal muscle differentiation by modulating TGF- activity
Summary
Trizol LS, LipofectAMINE, Dulbecco’s modification of Eagle’s minimal essential medium, CEE, horse serum, FCS, Opti-MEM I, Hanks’ balanced salt solution, G418, and human FGF-2 were obtained from Life Technologies, Inc. Wizard plus maxipreps and prime-a-gene labeling system dual-luciferase reporter assay system, pGL3 basic vector, and pRL were from Promega, Madison, WI. The day the cells were rinsed twice with Hanks’ balanced salt solution and cultured in normal growth medium. For transfection with TGF-inducible luciferase reporter construct (3TP-Lux), the cells were plated in growth medium 1 day before transfection at a density of 8000 cells/cm in 60-mm plates. The cells were washed twice with Hanks’ balanced salt solution and incubated for 2 days in growth medium followed by 1 day in differentiation medium or 30 h in differentiation medium containing TGF-1, TGF-2 (0 –1.0 ng/ml), or FGF-2 (0 –30 ng/ml). DNA and Protein Determination—DNA [43] and protein [44] were determined in aliquots of cell extracts as described
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