FIGURE 4.

IGF-1/IGFR requirement for glucose-activated RUNX2 activity. A, lanes 1–4, glucose- and serum-starved HBME were treated with 5 mm glucose for 4 h in the presence or absence of neutralizing antibody to IGFR (0, 0.5, and 1.0 μg/ml) or epidermal growth factor receptor (1 μg/ml). Nuclear extracts were examined for RUNX2 DNA binding activity by EMSA. Lanes 6–10, cells were exposed to neutralizing IGF-1 (0.5, 5, and 15 μg/ml) antibody and analyzed for RUNX2 DNA binding activity. Lanes 11–14, cells were treated with the IGFR kinase inhibitor NVP-ADW742 (150 and 300 nm), and RUNX2 DNA binding was determined by EMSA. B, cells were treated with the MEK inhibitor U0126 (lanes 4 and 5) or the ERK inhibitors 76 (lanes 6, 9, and 10), 99 (lane 7), or 101 (lanes 8, 11, and 12) for 4 h, and nuclear extracts were resolved on EMSA gels. C, cells were treated with the indicated PKCβ-specific (lanes 4 and 5) or general calphostin C (lanes 6–9) PKC inhibitors for 4 h prior to extraction of nuclear proteins and analysis of RUNX2 DNA binding by EMSA.