PI15, a novel secreted WNT-signaling antagonist, regulates chondrocyte differentiation

ABSTRACT

Purpose/Aim of Study

During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation.

Materials and Methods

ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays.

Results and Conclusions

shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.

KEYWORDS:

• PI15
• protease inhibitor 15
• chondrogenesis
• WNT
• LRP6

Acknowledgments

We are grateful to all the members of the Molecular Signaling & Biochemistry labs at Kyushu Dental University for their insightful advice and assistance over the course of this work. HK is a grateful recipient of the Japan Student Services Organization graduate scholarship program.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/03008207.2024.2349818.

Additional information

Funding

This work was supported by grants from the Novartis Japan Foundation for the Promotion of Science (WNA); the Japan Society for the Promotion of Science KAKEN 19K24152 (WNA), 21K10078(SK), 21H03144(SK and WA), 22KK0141(SK and WA); the Fukuoka Foundation for Sound Health Cancer Research Fund (WNA); the Kumamoto University Program for Joint Usage/Research Center for Developmental Medicine IMEG (SK and WA); and the YMFG Regional Enterprise Support Foundation (SK).