Background/Purpose:

Krüppel-like factors (Klfs) are known to play key roles in cell cycle regulation, definition of cell fate and regulation of terminal differentiation. There is no information on expression and function of Klfs in cartilage. The objectives of this study were to analyze expression of Klfs in normal and OA cartilage and study their role in regulating chondrocyte functions.

Methods:

Klf expression in normal and OA human cartilage and in developing mouse limb buds was examined by next generation RNA sequencing and qPCR. Klf4 function in normal human chondrocytes and TC28 immortalized chondrocytes was analyzed by gene overexpression and knock down. Overexpression of Klf4 mutants and reporter constructs were used to determine critical motifs for Klf regulation of gene expression.

Results:

Next generation RNA sequencing showed that Klf2, 4, 5, 9, 10 and 15 are significantly suppressed in OA cartilage. Quantitative real time PCR for mouse limb buds showed significantly increased Klf2, 4 and 5 expression from E11.5 to E15.5. Over expression-based screening in the immortalized human chondrocyte cell line TC28 revealed that only Klf4 strongly enhanced Col2A1 and Acan gene expression. Conversely, knockdown of Klf4 resulted in suppression of Col2A1 and Acan genes in human primary chondrocytes. Luciferase reporter assays showed that Klf4 binds to consensus sequences of Col2A1 and Acan promoters and activates their expression. ChIP-qPCR for Col2A1 promoter and Co-IP study revealed that Klf4 could interact with Sox9 directly. Klf4 C-terminus, which contains three tandem zinc fingers, which are critical for this interaction are required for activation of the target genes. Finally, we demonstrated that induction of Klf4 expression could induce Col2A1 and Acan expression in various cell types including dedifferentiated chondrocytes, and the magnitude of upregulation of the target genes was attenuated by reduction of Sox9 expression.

Conclusion:

These results are the first to show that Klf factors have a tissue specific expression in articular cartilage and that OA is associated with dysregulated expression of several Klfs. Among the Klfs, Klf4 expression pattern is most closely linked with chondrogenic markers. Klf4 promotes expression of chondrocyte specific genes, in part through direct interaction with Sox9. Collectively, these findings implicate abnormal expression of Klfs as a novel mechanism of abnormal chondrocyte differentiation and activation in OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/kruppel-like-factors-klfs-novel-transcriptional-regulators-of-articular-chondrocytes-that-are-abnormally-expressed-in-osteoarthritic-cartilage/