Counterregulatory roles of GLI2 and GLI3 in osteogenic differentiation via Gli1 expression

The GLI1/GLI2/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 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, and constitutively GLI3R-producing (cGLI3R) cells, and expressed GLI1, GLI2, and GLI3 constructs in these cells. The results demonstrate at the cellular level that GLI2 and GLI3R counterregulate osteogenic differentiation via activating and repressing 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 Gli1 itself and Gli2, constituting a GLI1-GLI2 positive feedback loop.