Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and β‐catenin–related pathways

Abstract

Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long‐segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF‐β3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF‐β3 induction. hUCMSCs were preconditioned with 1 µM of KGN for 3 d, sequentially pelleted, and incubated with TGF‐β3 for 28 d. Then, the expression of chondrogenesis‐ and ossification‐related genes was evaluated by immunohistochemistry and RT‐PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and β‐catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up‐regulated in hUCMSCs. Furthermore, the KGN‐preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene (SOX9, aggrecan, and collagen II) expression and reduced expression of ossifie genes (collagen X and MMP13) compared with hUCMSCs treated with TGF‐β3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up‐regulation of JNK phosphorylation and a suppression of β‐catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt‐related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the β‐catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN‐preconditioned hUCMSCs and TGF‐β3 encapsulated electrospun poly (l‐lactic acid‐co‐ε‐caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed β‐catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF‐β3 induction might be preferable for cartilage engineering strategies using MSCs.—Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X. Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and β‐catenin—related pathways. FASEB J. 33, 5641–5653 (2019). http://www.fasebj.org

Inhibition of Rac1 activity by NSC23766 prevents cartilage endplate degeneration via Wnt/β‐catenin pathway

“…introduced to further confirm the interaction between the Rac1 and Wnt/β-catenin pathway in rat EPCs. As shown in Figure 5A 30 And the result of immunofluorescence staining of collagen II was in accordance with the mRNA results ( Figure 5B,C).…”

“…Moreover, Wnt/b‐catenin signalling inhibitor and activator were introduced to further confirm the interaction between the Rac1 and Wnt/β‐catenin pathway in rat EPCs. As shown in Figure A, real‐time PCR analysis showed that Wnt/b‐catenin signalling inhibitor XAV‐939 (10 μmol/L) could mimic the effects of NCS23766, which increased the expression of aggrecan and collagen II and decreased the expression of MMP13 and ADAMTS5 after IL‐1β stimulation, while Wnt/b‐catenin signalling activator SKL2001 (40 µmol/L) could rescue the effects of NCS23766 . And the result of immunofluorescence staining of collagen II was in accordance with the mRNA results (Figure B,C).…”

Transforming growth factor-beta stimulates human bone marrow-derived mesenchymal stem/stromal cell chondrogenesis more so than kartogenin

“…While TGF-β1 or KGN alone did not yield positive outcomes, the authors found that a combination of TGF-β1, BMP-7 and KGN worked synergistically to drive rat BMSC chondrogenesis 7 . A potentially more relevant study published in 2019 reported that preconditioning of human umbilical cord-derived mesenchymal stromal cells in medium supplemented with 1 µM KGN enhanced their chondrogenic response to TGF-β3 21 . Similar to our observations with human BMSC, treatment of human umbilical cord-derived mesenchymal stromal cells with KGN alone was not chondrogenic.…”

Kartogenin mediates cartilage regeneration via stimulating the IL-6/Stat3-dependent proliferation of cartilage stem/progenitor cells

“…[21,22] Another study also demonstrated that KGN could increase type-I collagen synthesis in the dermis of BALB/C mice in vivo through activation of the smad4/smad5 signaling. [23,24] Furthermore, some other studies have demonstrated that KGN may also stimulate proliferation of several stem cells types originated from none-cartilage tissues. [25] Whether the proliferation of MSC or chondrogenic progenitor cells also plays a significant role in cartilage regeneration remains unknown.…”