Regulatory dynamics of Nanog in chondrocyte dedifferentiation: role of KLF4/p53 and p38/AKT signaling

Abstract

Homeobox protein Nanog, a member of the transcription factor family, plays a crucial role in maintaining the pluripotency and self-renewal of embryonic stem cells. Due to its diverse activities, Nanog has been identified in multiple cell types, including embryonic stem cells (ESCs) and cancer stem cells (CSCs). However, its molecular mechanism in chondrocytes remains unclear. In this study, we explored the effects of Nanog on chondrocytes and its interaction with chondrocyte-specific proteins. Chondrocytes were transfected with a Nanog cDNA vector, resulting in reduced expression of the chondrogenic markers Type II collagen and SOX9, as confirmed by western blot, RT-PCR, and immunofluorescence. Following siRNA transfection, the dedifferentiation effect of Nanog was reversed, restoring Type II collagen and SOX9 expression. We also discovered that the mechanism by which Nanog affects chondrocytes is closely linked to p53 and KLF4. Overexpression of KLF4 induced the phosphorylation of p53, and phospho-p53 directly inhibited Nanog expression. Moreover, the p53 activator Nutlin-3 A accelerated Nanog degradation, while the p53 inhibitor Pifithrin-α stabilized Nanog. Stabilized Nanog continued to promote chondrocyte dedifferentiation. Additional experiments were performed to identify the signaling pathways involved in Nanog-induced chondrocyte dedifferentiation. Our results showed that Nanog overexpression in chondrocytes significantly impacted the p38 kinase and AKT signaling pathways. Inhibition of p38 and AKT with SB203580 and LY294002 reduced Nanog expression and partially restored Type II collagen levels. Conversely, activation with anisomycin(ANS) and 740 Y-P enhanced Nanog expression, further reducing Type II collagen levels. To investigate Nanog's role in early development in vivo, we injected Nanog expression vectors into zebrafish embryos. The injected zebrafish exhibited structural defects in craniofacial cartilage, confirming Nanog's involvement in chondrocyte differentiation. These findings suggest that Nanog induces chondrocyte dedifferentiation, and this process can be modulated via the p53/KLF4 and p38/AKT pathways.

Keywords: AKT signaling; Dedifferentiation; KLF4; Nanog; Type II collagen; p38 kinase; p53.