ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes.
BACKGROUND
TGF-beta is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-beta action has been implicated in cartilage diseases such as osteoarthritis. TGF-beta signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-beta receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-beta signaling and responses in human chondrocytes.


METHODS
ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-beta receptors was determined by affinity labeling/immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-beta1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin.


RESULTS
Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-beta receptor system, associating with ALK5, type II TGF-beta receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-beta-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-beta1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-beta-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes.


CONCLUSIONS
Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-beta signaling and function in these cells.
Finnson
Kenneth W
KW
Division of Plastic Surgery, Department of Surgery, McGill University, Montreal, Quebec, Canada.
Parker
Wendy L
WL
ten Dijke
Peter
P
Thorikay
Midory
M
Philip
Anie
A
D030301:Activin Receptors, Type II / Q000235:genetics / Q000378:metabolism*; D000368:Aged; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051898:Smad1 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D051902:Smad5 Protein / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D018125:Receptors, Transforming Growth Factor beta; C497399:SMAD1 protein, human; C497790:SMAD3 protein, human; C497797:SMAD5 protein, human; D051898:Smad1 Protein; D051900:Smad3 Protein; D051902:Smad5 Protein; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human