Figure 3.

Mitogen-activated protein kinase signalling mediates EGFR-induced activation of PTHrP gene expression. (A) Epidermal growth factor receptor and ERK phosphorylation was reduced by EGFR TKI treatment in both RWGT2 and HARA cells. Upper bands are from a Western blot of conA-Sepharose precipitated proteins probed first with the generic phosphotyrosine monoclonal antibody 4G10, then stripped and reprobed with an EGFR antibody. Lower bands are from a Western blot of 30 μg of protein probed first with a phospho-ERK 1/2 antibody, then stripped and reprobed with an antibody to ERK 1/2 and finally stripped and reprobed with an antibody to β-actin (not shown). C (control) indicates cells treated with DMSO vehicle for 6 h. EGFRI indicates cells treated with PD (1 μM) for 6 h. This experiment was repeated twice. (B) A MEK inhibitor significantly reduced basal PTHrP/GAPDH mRNA ratios in both cell lines. RWGT2 and HARA cells were treated with 1 μM DMSO (C), PD (EGFRI, 1 μM) or PD98059 (MEKI, 10 μM) for 6 h. The mRNA was harvested from four independent cultures and analysed. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). For the RWGT2 experiments: MEKI, P=0.045; EGFRI, P=0.03; for HARA experiments: MEKI, P=0.04; EGFRI, P=0.04. (C, D) A MEK inhibitor blunted EGFR ligand-induced increases in PTHrP/GAPDH mRNA ratios in both cell lines. Cells were preincubated with 1 μM DMSO (C), PD (EGFRI, 1 μM) or PD98059 (MEKI, 10 μM) for 1 h and then stimulated with 100 ng ml⁻¹ EGF (+EGF) or 1 μg ml⁻¹ AREG (+AREG) for 6 h. B represents untreated cells in all experiments. Panels B–D represent four replicates of samples with all experiments repeated twice. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). (C) EGF EGFRI, P=0.01; MEKI, P=0.05; AREG EGFRI, P=0.02; MEKI, P=0.053 not significant. (D) EGF EGFRI, P=0.001; MEKI, P=0.046; AREG EGFRI, P=0.049; MEKI, P=0.03. (E) Basal reporter gene activity from a PTHrP-P3 construct was reduced by a dominant-negative Raf construct in both the RWGT2 and HARA cells. RLU represents relative luciferase unit as defined in the Materials and methods. Co-transfection of the PTHrP reporter gene with the empty vector is indicated by pcDNA3, a dominant-negative Ras construct (−ras) a dominant-negative Raf construct (−raf). Values in all panels represent the mean of four samples from individual cultures±s.e.m. These experiments were repeated three times with cells derived from independent passages with similar results. Two-tailed Student's t-test. ^*P<0.05 relative to C (control). RWGT2: ras, P=0.019; raf, P=0.033; HARA: ras P=0.02.