Inhibiting calcineurin activity under physiologic tonicity elevates anabolic but suppresses catabolic chondrocyte markers.
OBJECTIVE
The physiologic interstitial tonicity of healthy articular cartilage (350-480 mOsm) is lowered to 280-350 mOsm in osteoarthritis (OA). This results in loss of tissue prestress, altered compressive behavior, and, thus, inferior tissue properties. This study was undertaken to determine whether physiologic tonicity in combination with the inhibition of calcineurin (Cn) activity by FK-506 has synergistic effects on human articular chondrocytes and explants in vitro.


METHODS
OA chondrocytes and explants and non-OA chondrocytes were cultured in cytokine-free medium of 280 mOsm or 380 mOsm with or without Cn inhibition by FK-506. Chondrogenic, hypertrophic, and catabolic marker expression was evaluated at the messenger RNA (mRNA), protein, and activity levels.


RESULTS
Compared to OA chondrocytes cultured at 280 mOsm, those cultured at 380 mOsm had increased expression of mRNA for chondrogenic markers (e.g., ∼13 fold for COL2; P < 0.001), and decreased COL1 expression (∼0.5 fold, P < 0.01). Inhibiting Cn activity under physiologic tonicity further enhanced the expression of anabolic markers at the mRNA level (∼50 fold for COL2; P < 0.001, ∼2 fold for AGC1; P < 0.001, and ∼3.5 fold for SOX9; P < 0.001) and at the protein level (∼6 fold for type II collagen; P < 0.001). Cn inhibition suppressed relevant collagenases as well as hypertropic and mineralization markers at the mRNA and activity levels. Expression of aggrecanase 1 and aggrecanase 2 was not influenced by tonicity or FK-506 alone, but the combination suppressed both, by ∼50% (P < 0.05) and ∼40% (P < 0.001), respectively. Generally, similar anabolic and antihypertrophic effects were observed in ex vivo cartilage explant cultures and non-OA chondrocytes.


CONCLUSIONS
Our findings indicate that Cn at physiologic tonicity exerts a superior effect compared to physiologic tonicity or FK-506 alone, increasing anabolic markers while suppressing hypertrophic and catabolic markers. Our data may aid in the development of improved cell-based chondral repair and OA treatment strategies.
University Medical Center Rotterdam, Rotterdam, The Netherlands.
Anna E
AE
van der Windt
Esther
E
Haak
Nicole
N
Kops
Jan A N
JA
Verhaar
Harrie
H
Weinans
Holger
H
Jahr
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology
C508319:ACAN protein, human; D053674:Aggrecans; D015415:Biomarkers; D065095:Calcineurin Inhibitors; D024043:Collagen Type II; D055758:SOX9 Transcription Factor; D019703:Calcineurin; D053509:Matrix Metalloproteinase 13; D016559:Tacrolimus