EGFR Signaling Suppresses Osteoblast Differentiation and Inhibits Expression of Master Osteoblastic Transcription Factors Runx2 and Osterix

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF-like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real-time RT-PCR and promoter reporter assays revealed that EGF-like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR-dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR-induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co-repressors HDAC4 and 6 and over-expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down-regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage.