Adenoviral overexpression of Smad-7 and Smad-6 differentially regulates TGF-beta-mediated chondrocyte proliferation and proteoglycan synthesis.
OBJECTIVE
To assess if various biological responses to transforming growth factor-beta (TGF-beta) in chondrocytes are differentially regulated by Smad-6 and Smad-7.


METHODS
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-beta-induced PAI-1 and aggrecan mRNA upregulation was studied via quantitative RT-PCR. The effect of Smad-6 and -7 overexpression on TGF-beta-induced chondrocyte proliferation was studied via DNA quantification, whereas TGF-beta-induced proteoglycan (PG) synthesis was studied by 35S-sulfate incorporation.


RESULTS
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-beta-stimulated chondrocyte proliferation, although proliferation was not completely abolished. Smad-7 overexpression, however, completely antagonized the TGF-beta effect on proliferation. Smad-6 overexpression had no effect on TGF-beta-induced PAI-1 expression, while overexpression of Smad-7 completely blocked this TGF-beta effect. Additionally, overexpression of Smad-7, but not Smad-6, totally prevented TGF-beta-induced PG synthesis on the mRNA and protein levels.


CONCLUSIONS
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-beta-mediated biological responses such as proliferation and PG synthesis, while overexpressed Smad-6 had no or only a partial inhibitory effect on TGF-beta activity. We conclude that in chondrocytes distinct TGF-beta activities are differentially regulated by Smad-6 and Smad-7.
Scharstuhl
A
A
Experimental Rheumatology and Advanced Therapeutics, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Diepens
R
R
Lensen
J
J
Vitters
E
E
van Beuningen
H
H
van der Kraan
P
P
van den Berg
W
W
D000256:Adenoviridae / Q000235:genetics; D053674:Aggrecans; D000818:Animals; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D016326:Extracellular Matrix Proteins; D005822:Genetic Vectors; D037181:Lectins, C-Type; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051905:Smad6 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology
C508320:Acan protein, mouse; D053674:Aggrecans; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D017395:Plasminogen Activator Inhibitor 1; D011509:Proteoglycans; D012333:RNA, Messenger; D051905:Smad6 Protein; C497803:Smad6 protein, mouse; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta