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Transforming growth factor-β as a differentiating factor for cultured smooth muscle cells

The aim of the present study was to determine whether the development of supercontractile smooth muscle cells, contributing to the nonspecific hyperreactivity of airways in asthmatic patients, is due to transforming growth factor (TGF)-β. In cultured smooth muscle cells starved by removal of 10% foetal bovine serum for 7 days, growth arrest was seen; 30% became elongated and demonstrated super contractility. Study of conditioned medium suggested that the differentiating factor was TGF-β. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out on conditioned medium from the arrested cells. Two protein bands were identified as matrix metalloproteinase (MMP)-2 and TGF-β1. To determine second messenger signalling by SMAD2, Western blotting and confocal microscopy were employed. Conditioned medium from arrested cultures showed the presence of MMP-2 and TGF-β1, as revealed by SDS–PAGE; 68- and 25-kDa bands were seen. Differentiation was confirmed by upregulation of marker proteins, smooth muscle type myosin heavy chain and myosin light chain kinase. Confirmation was obtained by downregulating these proteins with decorin treatment, which reduces the levels of active TGF-β and an adenoviral dominant-negative vector coding for a mutated type II TGF-β-receptor. Activation of second messenger signalling was demonstrated immunocytochemically by the presence of phosphorylated SMAD2 and SMAD4. Transforming growth factor-β is likely to be the differentiating factor responsible for the development of these supercontractile smooth muscle cells. The development of such cells in vivo after cessation of an asthmatic attack could contribute to the nonspecific hyperreactivity of airways seen in patients.