Abstract
Purpose: :
To study high mobility group box 1 (HMGB1) which is a non-histone chromosomal protein implicated in a variety of biologically important processes, including transcription, differentiation, development, glaucomatous neurodegeneration, regeneration and inflammation.
Methods: :
We focused on the proteomic analysis of HMGB-1 protein during retinal ganglion cell (RGC) degeneration and regeneration. 2-D-SDS-PAGE profiles from normal and glaucomatous rat retina, regenerating and non-regenerating rat retina after optic nerve crush and lens injury were compared, respectively. Western blots were performed to verify these results. RGC-5 cells were incubated either in presence or absence of HMGB1 protein. The supernatant of the medium was collected to examine the cytokine level within the medium. The cleaved caspase 3, Bcl-2, Bad and pBad expression of HMGB1 treated RGC-5 were examined by Western blot. RGC-5 cells and explanted retina were incubated in a pressure chamber and the HMGB1, Bcl-2, Bax, TLR4 and RAGE receptor expression was measured with Western blot and RT-PCR, respectively. The supernatant of the medium was collected to examine the HMGB1 concentration.
Results: :
HMGB1 was differentially expressed throughout the experiments. In glaucomatous retina the HMGB1 expression was strongly up-regulated compared to normal retina. When the retina regenerates after optic nerve crush and lens injury, expression of HMGB1 is nearly abolished. The Cytokine Array revealed a down-regulation of CNTF and up-regulation of TNF-α and VEGF. Western blot analysis of HMGB1 treated RGC revealed an increased cleaved caspase 3 activity and a decreased Bcl-2 and pBad expression while the Bad expression remains unaffected. Incubation under pressure showed an increased HMGB1, Bax and TLR4 expression and decreased RAGE receptor expression on mRNA and protein level, respectively. The Bcl-2 expression remains unaffected. The HMGB1 concentration in the medium was increased.
Conclusions: :
Our data suggest that HMGB1 promotes inflammation and participates in the pathogenesis of neurodegenerative disease of the eye by mediating apoptosis in retinal ganglion cells. HMGB1 may play a key role in ongoing damage of retinal ganglion cells after an initial injury.
Keywords: ganglion cells • proteomics • apoptosis/cell death