Abstract
Purpose::
To confirm that spontaneously immortalized MIO-M1 human Muller glia cell line is equipped to respond to FGF9 and to explore possible interaction of FGF9 and PKA pathways by using this cell line.
Methods::
Expression of FGF9 and its cognate receptors in MIO-M1 was investigated by immunocytochemical stainings using anti-FGF9, anti-FGFR2 and anti-FGFR3. Activation of FGF9 signal transduction pathway was studied by western blot analysis of lysates from FGF9 treated MIO-M1 cells using anti-pERK antibody. To asses the effect of PKA on FGF9 dependent ERK activation, cells were treated with 8-bromo-cAMP or H89 prior to growth factor induction. Changes in phosphorylated ERK levels as a result of the stimuli were observed, and results were normalized to the internal ERK levels.
Results::
We have shown that expression of FGFR2 and FGFR3 were diffuse across the cell surface with intense labeling within the nucleus. No FGF9 expression was detected in these cells. Upon FGF9 induction of MIO-M1 cells, pERK levels doubled within 10 min and returned to basal levels by 30 min. The prior PKA inhibitor treatment resulted in an increase of pERK levels and the signal attenuation, evident at 30 min in cells subjected to FGF9 induction alone, was no longer observed. When PKA was activated prior to FGF9 treatment, overall pERK levels decreased, while the phosphorylation pattern with respect to FGF9 treated cells did not seem to be altered.
Conclusions::
Our data indicate that MIO-M1 cells are equipped with the components of signal transduction to respond to FGF9 stimuli in a similar fashion to primary Muller glia cells. We suggest that prior PKA activation may play function in fine-tuning of FGF9 signal transduction at downregulation phase in MIO-M1 cells.
Keywords: Muller cells • growth factors/growth factor receptors • signal transduction