Fig 1.
Activation of the HGF receptor (MET) in primary human airway basal epithelial cells by 3T3-J2 murine embryonic fibroblast feeder cell-conditioned medium.
(A) Receptor tyrosine kinase array analysis of primary human airway basal cells stimulated for 30 minutes with 3T3-J2 feeder cell-conditioned medium. Specific phosphorylation of the hepatocyte growth factor (HGF) receptor (HGFR/MET) at Y1234/Y1235 was observed in cells stimulated with 3T3-J2-conditioned medium both in the presence and absence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor. The three duplicate spots in the top left, top right and bottom left of each array are assay reference spots. This array was performed using one donor cell culture. (B) Schematic representation of MET receptor structure showing relevant phosphorylation sites. Sema = semaphorin; PSI = plexins-semaphorins-integrins; IPT = immunoglobulins-plexins-transcription factor. (C) Western blot confirmation in independent lysates of MET phosphorylation following stimulation of primary human airway basal cells with 3T3-J2 feeder cell-conditioned medium for 30 minutes. Y indicates the presence of 5 μM Y-27632 in the culture medium. The blots shown are representative of experiments performed on three donor cell cultures.
Fig 2.
mHGF is secreted by 3T3-J2 fibroblasts following mitotic inactivation but primary human airway basal cell MET does not mediate important aspects of co-culture expansion.
(A) ELISA quantification of hepatocyte growth factor (HGF) secreted into culture medium by 3T3-J2 feeder cells following mitotic inactivation with mitomycin C. Medium was collected and replaced with fresh medium after 24 and 48 hours (n = 4; mean +/- SEM; ** indicates p<0.01 using a one-way ANOVA with Holm-Sidak’s correction for multiple comparisons). (B) Flow cytometric analysis of EdU uptake in primary human airway basal cells treated with either epithelial culture medium alone (+Y), 3T3-J2-conditioned epithelial growth medium (CM+Y) or the same medium containing 100 nM PF-0421903 (CM+Y+PF), a small molecule MET inhibitor, for 12 hours (CM+Y+PF; n = 4 donor cultures performed in triplicate repeats; mean +/- SEM; * indicates p<0.05 using a one-way ANOVA with Holm-Sidak’s correction for multiple comparisons). (C) Colony-forming efficiency of primary human airway basal cells grown on 3T3-J2 feeder cells in the presence of 5 μM Y-27632 and either 100 nM PF-0421903 or a vehicle control for 10 days (n = 5 donor cultures; ns indicates not significant using a paired t test; representative wells from one donor are shown in images). (D) Colony-forming efficiency of primary human airway basal cells grown on 3T3-J2 feeder cells without EGF or Y-27632 for 10 days. 3 ng/ml or 50 ng/ml mHGF or hHGF was added as indicated (n = 3 donor cultures; * represents p<0.05 and ** represents p<0.01 using a using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare each concentration of HGF to the vehicle control group; representative wells from one donor are shown in images). (E) MTT assay using primary human airway epithelial cells cultured in medium without EGF or Y-27632 for 10 days. 3 or 50 ng/ml mHGF or hHGF was added as indicated (n = 3 donor cultures; ** represents p<0.01 and *** represents p<0.001 using a using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare each concentration of HGF to the vehicle control group). (F) Cell counts comparing primary human airway basal cell number in 3T3-J2 co-culture in medium containing Y-27632 and vehicle protein (3T3+Y+Vehicle) with medium alone (i.e. no feeder cells; Y+Vehicle) and medium plus either 3 ng/ml or 50 ng/ml hHGF. Cells were analysed after 24, 48 or 72 hours. In an additional condition, after 72 hours cells were passaged to the same density as at time 0 and analysed after a further 96 hours (n = 3 donor cultures performed in triplicate repeats; mean +/- SEM; **** indicates p<0.0001 using a two-way ANOVA with Holm-Sidak’s correction for multiple comparisons to compare media compositions with the 3T3+Y group).
Fig 3.
3T3-J2-conditioned medium and hHGF stimulate MET, GAB2 and STAT6 phosphorylation in primary human airway basal cells.
(A) Western blot analysis of MET, GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6) phosphorylation status following stimulation of primary human airway basal cells with basal or 3T3-J2-conditioned medium (CM) for 30 minutes. Y indicates the presence of 5 μM Y-27632 and PF indicates 100 nM PF-04217903. Blots are from one donor cell culture and are representative of experiments performed in three further donor cultures. (B) Western blot analysis of MET, GAB2 and STAT6 phosphorylation status in primary human airway basal cells stimulated with either 3 ng/ml or 50 ng/ml recombinant mouse (mHGF) or recombinant human hepatocyte growth factor (hHGF) for 30 minutes. Blots are from one donor cell culture and are representative of experiments performed in two further donor cultures. (C) Dose-response western blot analysis of HGF-induced STAT6 phosphorylation in primary human airway basal cells stimulated with the indicated amount of hHGF for 30 minutes. This result was replicated in a second donor cell culture.
Fig 4.
HGF-induced STAT6 phosphorylation in primary human airway basal cells occurs independently of GAB2 activity and MET receptor binding and does not result in STAT6 nuclear translocation or transcriptional activity.
(A) Western blot analysis of STAT6 Y641 phosphorylation levels in human airway basal cells treated with either 5 nM non-silencing siRNA or 5 nM anti-GAB2 siRNA. Cells treated with a 0.1% bovine serum albumin (BSA) vehicle control were compared with matched cells stimulated with either 3 ng/ml or 50 ng/ml recombinant human hepatocyte growth factor (hHGF) for 30 minutes. (B) Western blot analysis of STAT6 phosphorylation in the A549 cancer cell line, the A431 cancer cell line and primary human lung fibroblasts following stimulation with either a BSA control or 50 ng/ml hHGF for 30 minutes. This blot is representative of two independent experiments. (C) Western blot analysis of co-immunoprecipitation experiment. Unstimulated A431 cancer cells were compared with matched cells stimulated with 3 or 50 ng/ml hHGF for 30 minutes. MET was immunoprecipitated using Dynabeads and the supernatant retained. STAT6 was not co-immunoprecipitated with MET but was found in the unbound protein fraction. STAT3 binding to MET was used as a positive control. These blots are representative of two experiments in A431 cells and a further experiment in primary human airway basal cells that did not detect HGF-STAT6 binding. (D) Quantification of firefly luciferase STAT6 reporter activity using luminescence in A431 cancer cells treated for 30 minutes, 1 hour or 2 hours with either a vehicle control, 50 ng/ml hHGF or 50 ng/ml recombinant human interleukin-13 (hIL-13). Values were normalised according to expression from a constitutively active renilla luciferase plasmid. No HGF-induced STAT6 activity was seen in three independent experiments. (E) Western blot analysis of STAT6 Y641 phosphorylation status in A431 cancer cells treated with 50 ng/ml hHGF or 50 ng/ml hIL-13 for 30 minutes. Whole cell lysates were obtained using RIPA buffer and compared with independent lysates prepared using a subcellular fractionation kit. These blots are representative of two independent experiments using A431 cells and two further experiments using primary human airway basal cells.