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Betty Albo Obeso, A R. Santos, J P. Ponmattam, M Manzur, J L. Goldberg, M L. Bajenaru; Focal Adhesion Kinase (FAK) Is An Important Regulator Of RGC Survival. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2690.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGC) survival and neurite outgrowth are promoted by neurotrophins, and extracellular matrix (ECM) molecules such as laminin. Degradation of laminin in the ECM of RGC in experimental animal models relevant to retinal disease contributes to RGC death. Since integrins are the major ECM receptors, we have recently identified β1 integrin, FAK, Akt, bclxL as important regulators of the integrin survival pathway in RGC, and showed that this pathway is disrupted in animal models of retinal disease. The purpose of this study is to further evaluate the role of Focal Adhesion Kinase (FAK) in RGC survival.
Purified RGC cultures were prepared by immunopanning from retinas of Sprague-Dawley rats at postnatal day 5. They were cultured on poli-D-lysine and/or laminin in 8 well chamber slides. Immunohistochemistry was performed 36 hrs later with specific antibodies, anti-integrin β1, -FAK -phospho-FAK [PY397]. β1 integrin activating antibodies, HUTS-21 (1µg/ml), or isotype control antibodies were added to RGC 12 hours after plating. In several experiments RGCs were preincubated with the FAK inhibitor PP2 (25 µM) overnight, before HUTS-21 addition. RGC viability was assessed 24, 48 hrs later using live cell imaging with an Axiovert Zeiss 200 M microscope and analyzed with Axio Vision 4.7 software. To inhibit expression of FAK freshly purified RGC were transfected by electroporation with SMARTpool FAK siRNA (Dharmacon, Inc) in SCN Nucleofactor solution in an amaxa cuvette using Nucleofactor II (Amaxa). Knockdown of FAK in RGC was tested 24-72 hours later by RT-PCR with specific primers for FAK.
When cultured on laminin RGC exhibited increased survival, and neurite outgrowth. RGC show enhanced focal contacts on laminin versus PDL. These properties correlated with an up-regulation of the integrin survival pathway, increased expression of β1 integrin, and a significant increase in FAK phosphorylation at Tyr 397, essential for FAK activation. Treatment with β1 activating antibodies HUTS-21 of RGC cultured on PDL mimics the effect of laminin, and enhances the RGC survival, and neurite outgrowth. When RGC were incubated with PP2, a FAK inhibitor, both laminin, and HUTS-21 protective effect were abolished. Down-regulation of FAK by siRNA reduces RGC survival.
Our results suggest that FAK is a down-stream regulator in the integrin signaling in RGC and support an important role for FAK in RGC survival.
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