Systematic analysis of the TGF-β/Smad signalling pathway in the rhabdomyosarcoma cell line RD

摘要

Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth and differentiation, whose actions are highly cell type specific. To study the role of the TGF-β1 autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD, an attempt was made to establish a framework for the expression of several components of TGF-β1/Smad signalling pathway at the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis in RD cells compared with the normal myoblasts. Higher exogenous concentration of TGF-β1 was necessary to reach a growth-inhibition effect, whereas TGF-β1 downregulated the expression of myosin heavy-chain mRNA at lower concentrations than that was required for growth inhibition. Treatment with TGF-β1 significantly decreased the number of sarcomeric actin and myosin-expressing cells. In this study, we have shown that RD cells displayed higher expression of TβRI, TβRII, Smad2 and Smad4 at both the mRNA and protein levels than myoblasts. Smad3 and Smad7 mRNA were expressed at higher level in RD cells than in myoblasts. The staining patterns of TβR and Smads suggest that they may transduce different TGF-β1 signalling in RD cells than in myoblasts. TGF-β1 signalling induced a rapid relocation of Smad2 to the nucleus; in contrast, Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. These studies suggest that signalling from the cell surface to the nucleus through Smad proteins is a required component of TGF-β1-induced cell response in RD cells. The RD cell line is a suitable model to study the TGF-β autocrine loop involved in growth and differentiation of RMS.