To assess if various biological responses to transforming growth factor-β (TGF-β) in chondrocytes are differentially regulated by Smad-6 and Smad-7.

Adenoviral overexpression of Smad-6 or -7 mRNA in a chondrocyte cell line was determined via semi-quantitative RT-PCR and protein overexpression was studied by immunocytochemistry. Furthermore, the effect of Smad-6 and -7 overexpression on TGF-β-induced PAI-1 and aggrecan mRNA upregulation was studied via quantitative RT-PCR. The effect of Smad-6 and -7 overexpression on TGF-β-induced chondrocyte proliferation was studied via DNA quantification, whereas TGF-β-induced proteoglycan (PG) synthesis was studied by ³⁵S-sulfate incorporation.

Adenoviral transfection of chondrocytes with Smad-6 and -7 resulted in strong upregulation of Smad-6 and -7 mRNA levels, respectively. Immunocytochemistry showed overexpression of Smad-6 and -7 proteins in both the nucleus and cytoplasm. Smad-6 overexpression significantly inhibited TGF-β-stimulated chondrocyte proliferation, although proliferation was not completely abolished. Smad-7 overexpression, however, completely antagonized the TGF-β effect on proliferation. Smad-6 overexpression had no effect on TGF-β-induced PAI-1 expression, while overexpression of Smad-7 completely blocked this TGF-β effect. Additionally, overexpression of Smad-7, but not Smad-6, totally prevented TGF-β-induced PG synthesis on the mRNA and protein levels.

Adenoviral transfection of chondrocytes with Smad-6 and -7 resulted in strong upregulation of Smad-6 and -7 mRNA and protein levels. Furthermore, overexpression of Smad-7 in chondrocytes totally inhibited important TGF-β-mediated biological responses such as proliferation and PG synthesis, while overexpressed Smad-6 had no or only a partial inhibitory effect on TGF-β activity. We conclude that in chondrocytes distinct TGF-β activities are differentially regulated by Smad-6 and Smad-7.