FIGURE 3.

Aldose reductase inhibits RUNX2 activity in response to hyperglycemia. A, HBME cells were serum- and glucose-starved before incubation with glucose (5 or 25 mm) and the AR inhibitor (ARi) alrestatin. Triangles indicate increasing doses of alrestatin as follows: 0.1, 1.0, 10, and 20 μm. After 4 h, nuclear extracts were analyzed for RUNX2 DNA binding using EMSA. Bars below each lane indicate the relative band density of the specific RUNX2-shifted band. B, specific siRNA targeting AR or scrambled siRNA oligonucleotides (25 nm) were transfected into HBME cells grown in the presence of 5 or 25 mm glucose. Cells were harvested and replated for 24 h prior to extraction of total protein for AR immunoblotting. C, cells grown in 5 mm glucose were transfected with siRNA (scrambled) or siRNA (AR) and replated in complete medium for 24 h prior to overnight starvation and treatment with the indicated doses of glucose (0, 5, and 25 mm) for 4 h. RUNX2 DNA binding activity was determined by EMSA as before. Relative densities of the RUNX2-shifted bands are indicated. Representative results from a single EMSA gel are marked with dashed lines for clarity of analysis. Similar results were obtained from three separate determinations.