GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.
Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is a potent hematopoietic cytokine. In the present study, we examined whether GM-CSF is neuroprotective in retinal ganglion cells (RGCs). First, we studied the expression of GM-CSF and the GM-CSF-alpha-receptor in rat and human retina and in RGC-5 cells. Then, RGC-5 cells were incubated with apoptosis-inducing agents (e.g., staurosporine, glutamate and NOR3). The cell death was assessed by Live-Death-Assays and apoptosis-related-proteins were examined by immunoblotting. In addition, the expression of phosphorylated ERK1/2-pathway-proteins after incubation with GM-CSF and after inhibiting MEK1/2 with U0126 was analyzed. To assess the in vivo-effect, first staurosporine or GM-CSF plus staurosporine was injected into the vitreous body of Sprague-Dawley rats. In a second axotomy model the optic nerve was cut and GM-CSF was injected into the vitreous body. In both models, the RGCs were labeled retrogradely with either Fluoro-Gold or 4-Di-10-Asp and counted. As a first result, we identified GM-CSF and the GM-CSF-alpha-receptor in rat and human retina as well as in RGC-5 cells. Then, in the RGC-5 cells GM-CSF counteracts induced cell death in a dose-and time-dependent manner. With respect to apoptosis, Western blot analysis revealed a decreased Bad-expression and an increased Bcl-2-expression after co-incubation with GM-CSF. Concerning signaling pathways, incubation with GM-CSF activates the ERK1/2 pathway, whereas inhibition of MEK1/2 with U0126 strongly decreased the phosphorylation downstream in the ERK1/2 pathway, and the antiapoptotic activity of GM-CSF in vitro. Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye.
Schallenberg
Maurice
M
Department of Experimental Ophthalmology, School of Medicine, University Eye Hospital Münster and Interdisciplinary Centre for Clinical Research (IZKF), Domagkstrasse 15, D-48149 Münster, Germany.
Charalambous
Petar
P
Thanos
Solon
S
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005901:Glaucoma / Q000201:enzymology* / Q000473:pathology; D018698:Glutamic Acid / Q000633:toxicity; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D006898:Hydroxylamines / Q000633:toxicity; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D056347:Neuroanatomical Tract-Tracing Techniques; D009570:Nitriles / Q000494:pharmacology; D009574:Nitro Compounds; D020221:Optic Nerve Injuries / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D019311:Staurosporine / Q000633:toxicity; D051025:bcl-Associated Death Protein / Q000378:metabolism
C494821:Bad protein, rat; D002070:Butadienes; D006898:Hydroxylamines; D009570:Nitriles; D009574:Nitro Compounds; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; C113580:U 0126; D051025:bcl-Associated Death Protein; D018698:Glutamic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C061872:FK 409; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020930:MAP Kinase Kinase Kinases; D019311:Staurosporine