PMC

HGF and c-Met expression is developmentally regulated. (A) Dissociated hippocampal neurons were lysed at the indicated DIV and probed by immunoblot against HGF, phospho-c-Met (Y1230/1234/1235), c-Met, or tubulin as a loading control. Molecular masses in kDa are shown on the left. The ratio of pixel densities of each protein relative to tubulin was calculated for each time point from three independent blots and the means ± SD are shown. (B) Dissociated hippocampal neurons at 4 DIV were fixed and immunostained against HGF, c-Met, or phospho-c-Met (Y1230/1234/1235), and then labeled with Alexa 488-conjugated goat anti-rabbit or mouse IgG. Scale bar, 30 μm. (C) Dissociated hippocampal neurons at 4 DIV were fixed and triple-labeled with use of antibodies against c-Met, MAP2, and Alexa 555-conjugated phalloidin to stain actin as described in Materials and Methods. Scale bar, 30 μm.

HGF treatment increases phosphorylation of Akt and GSK-3β. (A) Cultured hippocampal neurons at 4 DIV were pre-incubated with the indicated concentrations of PHA-665752 or an equal volume of DMSO for 2 hr. The cells were then incubated for 10 min with 50 ng/ml HGF and lysed. Fifty μg of protein was separated by SDS-PAGE and probed with antibodies against phospho-Akt (T308), Akt, phospho-GSK-3β (S9), GSK-3β, or α-tubulin as a loading control. Relative phosphorylation changes of Akt and GSK-3β were quantitatively analyzed from three independent trials. Error bars represent ± SD. (B) Dissociated hippocampal neurons transfected with empty pmU6pro plasmid or indicated shRNAs plasmids against c-Met at 1 DIV were lysed at 4 DIV. The lysates were analyzed by immunoblot as described above. Relative phosphorylation changes of Akt and GSK-3β were quantitatively analyzed with blots from three independent trials. Error bars represent SEM. *, P<0.05. Con, Control; Scrm, Scrambled.

Inhibition of c-Met suppresses MAP2 expression and increases its phosphorylation. (A) Dissociated hippocampal neurons incubated for 24 hr without or with 50 ng/ml HGF, 50 ng/ml HGF plus 0.25 or 1 μM PHA-665752, with only 1 μM PHA-665752, or first treated for 12 hr with 1 μM PHA-665752 and then another 12 hr with 50 ng/ml HGF were lysed and probed against phospho-MAP2 (T1620/1623), MAP2, and α-tubulin. The staining intensity of bands from three independent trials was quantified relative to α-tubulin and graphed as mean ± SD. (B) Immunoblots against phospho-MAP2 (T1620/1623) and MAP2 were conducted after equalizing the amount of MAP2 protein in each lane. The relative band intensity of phospho-MAP2 was quantitatively analyzed from three independent experiments. Error bars represent SEM. *, P<0.05. (C) Dissociated hippocampal neurons were transfected with shRNAs against c-Met or scrambled shRNA at 1 DIV and lysed at 4 DIV. The relative amount of phospho-MAP2 (T1620/1623) to total MAP2 was quantified from three independent immunoblots. Error bars represent SEM. *, P<0.05. Con, Control; Scrm, Scrambled.

GSK-3β inhibition decreases MAP2 phosphorylation and increases dendrite elongation similar to HGF. (A) Hippocampal neurons were incubated for 6 hr without or with 50 ng/ml HGF, 50 ng/ml HGF plus 10 μM SB415286 or 20 μM SB415286, or 10 μM SB415286. The cell lysates were probed against phospho-MAP2 (T1620/1623) and MAP2 after equalizing the amount of MAP2 protein. The relative phosphorylation change of MAP2 was quantitatively analyzed with blots from three independent trials. Error bars represent ± SD. (B) Neurons treated as above were fixed and immunostained with anti-MAP2 antibody and then labeled with Alexa 568-conjugated goat anti-mouse IgG. The numbers indicate the concentration of SB415286 in μM. Scale bar, 30 μm. SB, SB415286. (C and D) Primary dendrite number and average dendrite length were quantitatively analyzed from 40 neurons derived from three independent experiments. Error bars represent SEM. *, P<0.05.

Inhibitors that reduce Akt activity increase phosphorylation of MAP2 by GSK-3β and suppress dendrite elongation in response to HGF. (A) Hippocampal neurons were incubated for 6 hr without or with 50 ng/ml HGF, 50 ng/ml HGF plus 20 μM LY294002, 20 μM LY294002, 50 ng/ml HGF plus 2.5 μM Akt inhibitor X, or 2.5 μM Akt inhibitor X. The cells were lysed, and the lysates were probed against phospho-GSK-3β (S9), GSK-3β, and α-tubulin as a loading control. The relative phosphorylation of GSK-3β was quantitatively analyzed with blots from three independent trials. Error bars represent ± SD. (B) Hippocampal neurons were treated as described above and probed against phospho-MAP2 (T1620/1623) after equalizing the amount of MAP2 protein. Relative phosphorylation of MAP2 was quantitatively analyzed with blots from three independent trials. Error bars represent ± SD. (C) Neurons treated as above were fixed and immunostained with anti-MAP2 antibody and labeled with Alexa 568-conjugated goat anti-mouse IgG. The numbers refer to the concentration of inhibitors in μM. Scale bar, 30 μm. LY, LY294002; Inh X, Akt inhibitor X. (D and E) Primary dendrite number and average dendritic length were quantitatively analyzed from 40 neurons derived from three independent experiments. Error bars represent SEM. *, P<0.05.

Knock-down of c-Met by shRNA alters dendrites. (A) RT-PCR against c-Met and GAPDH was conducted with total RNA isolated at 4 DIV from hippocampal neurons transfected with control, c-Met shRNAs, or scrambled shRNA plasmids at 1 DIV. Ethidium bromide stains of the PCR products were scanned and densitometric analysis was performed with Image J software. Results were quantified from three independent experiments. Error bars represent ± SD. *, P<0.05. (B) Hippocampal neurons were transfected with the indicated shRNA plasmids at 1 DIV and lysed at 4 DIV. One-hundred μg of protein was probed by immunoblot against c-Met or α-tubulin. The sum of both bands of c-Met was normalized to α-tubulin expression. Error bars show ± SD. *, P<0.05. (C) Neurons transfected with EGFP and the indicated c-Met shRNAs, scrambled shRNA, or empty control plasmid were immunostained with mouse anti-c-Met and chicken anti-MAP2, and then Alexa 568-conjugated goat anti-mouse IgG and Cy5-conjugated goat-anti chicken IgG. Scale bar, 30 μm. (D–F) Primary dendrite number, total dendrite length per cell, and average dendritic length were quantitatively analyzed with MAP2-stained images from three independent experiments. n=40 cells/condition. Error bars represent SEM. *, P<0.05. Con, Control; Scrm, Scrambled.

Exogenous HGF treatment induces phosphorylation of c-Met and elongation of primary dendrites. (A) Hippocampal neurons at 4 DIV were pre-incubated with either the indicated concentrations of PHA-665752, a specific c-Met inhibitor, or an equal volume of DMSO for 2 hr, and treated with 50 ng/ml HGF for 10 min and then lysed. Fifty μg of protein was separated by SDS-PAGE and probed with antibodies against phospho-c-Met (Y1230/1234/1235), c-Met, or α-tubulin as a loading control. Changes in phosphorylation and expression of c-Met relative to α-tubulin were quantified from three independent immunoblots and are shown as mean ± SD. (B) Cultured neurons were treated with the indicated doses of PHA-665752 and HGF for 24 hr, and cell viability was tested with use of a Cell Counting Kit-8 as described in Materials and Methods. Error bars denote SEM. *, P<0.05. (C) Dissociated hippocampal neurons at 3 DIV were either untreated or treated for 24 hr with 50 ng/ml HGF (HGF), 50 ng/ml HGF plus 1 μM PHA-665752 (HGF+PHA1), or only 1 μM PHA-665752 (PHA1), fixed, immunostained against MAP2, and viewed by confocal microscopy. Scale bar, 30 μm. (D–F) The numbers of MAP2-positive primary dendrites directly emerging from cell somas were quantified, and the total length of primary dendrites and average length of each dendrite were measured using Image J software. n=40 neurons from three independent experiments. *, P<0.05.