FIG. 4.

(A) MG63 human osteosarcoma and MC3T3-E1 mouse osteoblasts were treated with 30 ng/ml IGF-1, 100 ng/ml IGFBP-5, or vehicle for 10 minutes. Cell lysate was used for determination of total and phosphorylated ERK-1/2 using specific antibodies. Three replicates were used for each treatment. Both IGF-1 and IGFBP-5 increased ERK-1/2 phosphorylation. (B) MC3T3-E1 mouse osteoblasts were treated with 30 ng/ml IGF-I, 100 ng/ml IGFBP-5, or vehicle under serum-free conditions. Two, 10, and 60 minutes after treatment, cells were lysed, and cell lysate was used for determination of total and phosphorylated ERK-1/2 levels. Three replicates were used for each treatment. IGFBP-5, like IGF-1, increased phosphorylation levels of ERK-1/2 in a time-dependent manner. (C) Levels of total and phosphorylated ERK-1/2 in IGFBP-5–treated cultures were quantitated by densitometric scanning of bands and are expressed as percentage of vehicle-treated control for data shown in B. Values are mean ± SD of three replicates per group. ^Ap < 0.05 vs. control. (D) Effects of IGFBP-4 pretreatment on IGFBP-5–induced increase in ERK phosphorylation. MC3T3-E1 cells were pretreated for 60 minutes with 300 ng/ml IGFBP-4 or vehicle before the addition of IGFBP-5. Ten minutes later, total and phosphorylated ERK-1/2 levels were determined by Western immunoblot analysis. IGFBP-5 increased ERK-1/2 phosphorylation in the presence of IGFBP-4. (E) Effects of RASSF1C siRNA on IGFBP-5–induced ERK-1/2 phosphorylation. MC3T3-E1 cells were incubated with transfection medium containing vehicle or siRNA258. Twenty-four hours after transfection, the media were changed to serum-free and incubated for 24 h before the addition of the vehicle or 100 ng/ml IGFBP-5. Ten minutes later, phosphorylated and total levels of ERK-1/2 were determined by Western immunoblot using specific antibodies. RASSF1C siRNA blocked IGFBP-5–induced increase in ERK-1/2 phosphorylation.