ABSTRACT
The GLI1, GLI2 and GLI3 transcription factors mediate Hedgehog (Hh) signaling, which is crucial for bone development. During intramembranous ossification, mesenchymal stem cells (MSCs) are directly differentiated into osteoblasts. Under basal and Hh pathway-stimulated conditions, primary cilia play essential roles in proteolytic processing of GLI3 to its repressor form (GLI3R) and in activation of GLI2. Although previous studies in mice have suggested that Gli1 expression depends on GLI2 and GLI3, coordinated roles of GLI1, GLI2 and GLI3 in osteogenic differentiation are not fully understood at the cellular level. From the MSC line C3H10T1/2, we established Gli2-knockout (KO) and Gli3-KO cells, as well as constitutively GLI3R-producing (cGLI3R) cells, and expressed GLI1, GLI2 and GLI3 constructs in these cell lines. The results demonstrate at the cellular level that GLI2 and GLI3R counterregulate osteogenic differentiation via activation and repression of Gli1 expression, respectively; GLI3R, which results from GLI3 processing requiring protein kinase A-mediated phosphorylation, downregulates expression of Gli2 as well as Gli1; and GLI1 upregulates expression of Gli1 itself and Gli2, constituting a GLI1–GLI2 positive feedback loop.
Footnotes
Author contributions
Conceptualization: Y.T., Y.I., S.Y., H.-W.S., Y.K., K.N.; Formal analysis: Y.T., Y.I.; Funding acquisition: Y.I., Y.K., K.N.; Investigation: Y.T., Y.I.; Resources: S.Y.; Supervision: H.-W.S., Y.K.; Visualization: Y.T., Y.I.; Writing – original draft: K.N.; Writing – review & editing: Y.T., Y.I., S.Y., H.-W.S., Y.K., K.N.
Funding
This work was supported in part by grants from the Japan Society for the Promotion of Science (JSPS; grant numbers 20H04904 and 24K02022 to K.N., and 21H02427 and 22H05539 to Y.K.). Y.I. was supported by a JSPS Research Fellowship (grant number 22J20116).
Data availability
All relevant data can be found within the article and its supplementary information.