The immunosuppressant FK506 promotes development of the chondrogenic phenotype in human synovial stromal cells via modulation of the Smad signaling pathway.
OBJECTIVE
To assess the effect of the immunosuppressant FK506 on chondrogenic differentiation of human synovial stromal cells (hSSCs).


METHODS
hSSCs were isolated from synovium of the knee joint and 2x10(5) cells were subjected to pellet culture in chondrogenic culture medium for 3 weeks with or without growth factors [bone morphogenetic protein 2 (BMP2) or transforming growth factor beta(1) (TGFbeta(1))] and +/- addition of FK506 in chondrogenic culture media was evaluated. Chondrogenesis was assessed by the size of the pellet, the production of proteoglycans, and messenger RNA (mRNA) levels for chondrogenic markers. Furthermore, levels and intracellular location of phosphorylated Smad proteins related to BMP signaling and TGFbeta signaling were evaluated following exposure to FK506.


RESULTS
FK506 enhanced the differentiation of hSSCs toward a chondrogenic phenotype in a dose-dependent manner associated with increases in glycosaminoglycan synthesis and increased mRNA levels for chondrogenic marker genes. Additionally, FK506 further enhanced chondrogenesis of synovial stromal cells (SSCs) induced by BMP2 and TGFbeta(1), also in a dose-dependent manner. Notably, phosphorylation of Smad1/5/8 and Smad3 was significantly increased by FK506. Also, the ratio of nuclear translocation to cytoplasmic levels of phosphorylated Smad1/5/8 and Smad3 were increased following exposure of SSCs to FK506. Moreover, inhibition of Smad signaling significantly abrogated FK506-induced chondrogenic differentiation of SSCs.


CONCLUSIONS
This study demonstrated that FK506 promotes chondrogenic differentiation of hSSCs likely via impact on Smad signaling pathways. With further optimization, FK506 could potentially be a unique therapeutic tool to promote cartilage repair in clinical situations, as well as enhance development of tissue engineered cartilage in vitro.
Tateishi
K
K
Department of Orthopaedics, Osaka University Graduate School of Medicine, Osaka, Japan.
Higuchi
C
C
Ando
W
W
Nakata
K
K
Hashimoto
J
J
Hart
D A
DA
Yoshikawa
H
H
Nakamura
N
N
D000328:Adult; D002358:Cartilage, Articular / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use*; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D051785:Smad Proteins / Q000187:drug effects* / Q000235:genetics; D017154:Stromal Cells / Q000187:drug effects*; D016559:Tacrolimus / Q000627:therapeutic use; D053773:Transforming Growth Factor beta1 / Q000187:drug effects / Q000235:genetics
D007166:Immunosuppressive Agents; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D016559:Tacrolimus