Figure 7.

Basal ERK activity in NHKs is dependent upon proteolytic release of ErbB ligands. (A and B) GF-deprived NHKs were preincubated in fresh basal M154 for 90 min at 37°C in fresh basal M154 medium containing DMSO control (indicated by minus signs above the autoradiographs), 40 μM GM6001 (A), or 50 μM MMP-2/MMP-9 inhibitor II (B) and then stimulated with 1 ng/ml EGF for 10 min at 37°C, or left untreated. After lysate preparation and Western blotting (20 μg protein/lane), replicate blots were decorated with the antibodies indicated to the right of the autoradiographs. The results shown are from a single experiment and are representative of at least four independent experiments for A and two independent experiments for B. (C) GF-deprived NHKs were preincubated in fresh basal M154 containing 40 μM GM6001 or DMSO control for 90 min at 37°C, followed by stimulation with 1 ng/ml EGF or PBS control for 10 min. Conditions are indicated above the autoradiographs. Cell lysate (150 μg) was subjected to immunoprecipitation for ErbB1 as described in Materials and Methods. An isotype control was negative (unpublished data). Eluted proteins were detected by Western blotting with the primary antibodies indicated to the right of the autoradiographs. The results shown are representative of two independent experiments yielding similar results. (D) GF-deprived NHKs were grown to 40% confluence in complete M154 medium and then preincubated for 24 h in basal M154 containing 0.1% DMSO (NHK-CM) or 40 μM GM6001 [NHK(GM)-CM]. The CM was then collected and 4 ml was applied without concentration for 10 min to 60-mm dishes of HaCaT cells grown to 40% confluence, then maintained in serum-free DMEM for 24 h to induce quiescence (Iordanov et al., 2002). Controls included untreated quiescent HaCaT cells (no Tx), treatment with 100 ng/ml for 10 min (EGF), NHK-CM to which 40 μM GM6001 was added just before addition to HaCaT cells (NHK-CM/GM), and preincubation of HaCaT cells with 1 μM PD158780 for 1 h before addition of CM (denoted by the plus sign above the autoradiogram). Application of fresh basal M154 medium, rather than conditioned medium, to the HaCaT cells produced no increase in ERK phosphorylation (unpublished data). Note the reduction in ERK phosphorylation when GM6001 is added to NHKs during the 24-h medium-conditioning period, but not when it is added to the NHK-CM just before exposure to HaCaT cells. Lanes 1–4 are from one experiment and lanes 5–7 are from another. The no treatment controls from the first and second experiments both revealed similarly low levels of ERK phosphorylation. Only the no treatment control from the first experiment is shown in the figure.