Abstract
The immunoproteasome is upregulated by disease, oxidative stress, and inflammatory cytokines, suggesting an expanded role for the immunoproteasome in stress signaling that goes beyond its canonical role in generating peptides for antigen presentation. The signaling pathways that are regulated by the immunoproteasome remain elusive. However, previous studies suggest a role for the immunoproteasome in the regulation of PTEN and NF-κB signaling. One well-known pathway upstream of NF-κB and downstream of PTEN is the Akt signaling pathway, which is responsible for mediating cellular survival and is modulated after optic nerve crush (ONC). This study investigated the role of retinal immunoproteasome after injury induced by ONC, focusing on the Akt cell survival pathway. Retinas or retinal pigment epithelial (RPE) cells from wild type (WT) and knockout (KO) mice lacking either one (LMP2) or two (LMP7 and MECL-1) catalytic subunits of the immunoproteasome were utilized in this study. We show that mRNA and protein levels of the immunoproteasome subunits are significantly upregulated in WT retinas following ONC. Mice lacking the immunoproteasome subunits show either a delayed or dampened apoptotic response as well as altered Akt signaling, compared to WT mice after ONC. Treatment of the RPE cells with insulin growth factor-1 (IGF-1) to stimulate Akt signaling confirmed that the immunoproteasome modulates this pathway, and most likely modulates parallel pathways as well. This study links the inducible expression of the immunoproteasome following retinal injury to Akt signaling, which is important in many disease pathways.
Highlights
Regulation of cell signaling includes the degradation of proteins involved in the signaling cascade, and the main mediator of this process is the proteasome
optic nerve crush (ONC) produces a partial axotomy of the optic nerve, which results in retinal injury, quick apoptotic death, and loss of retinal ganglion cells (RGC) in a wild type (WT) animal [21,42]
The ratio of p-S6K1/S6K1 kinase expression revealed similar kinetics in an increase followed by decrease of expression for WT, L2, and L7M1-retinal pigment epithelial (RPE) cells indicating that the temporal differences observed upstream were not translated downstream through mammalian target of rapamycin (mTOR) signaling, similar to the lack of effect on mTOR signaling after ONC (Fig 7)
Summary
Regulation of cell signaling includes the degradation of proteins involved in the signaling cascade, and the main mediator of this process is the proteasome. The optic nerves of wild-type (WT) mice and mice deficient in one (lmp2-/-) or two (lmp7-/- and mecl1-/-) subunits of the immunoproteasome were crushed and the resulting injury was monitored through evaluation of ganglion cell layer (GCL) density and apoptosis, as well as whole retinal expression of proteins in the PTEN/Akt pathway. In order to assess the role of the immunoproteasome in the PTEN/Akt pathway without additional inputs, retinal pigment epithelial (RPE) cells isolated from the mice were treated with IGF-1 This current work is the first to assess the role of the immunoproteasome in a discrete injury to a limited number of specific retinal neurons via optic nerve injury, and provides new insight into the cellular stress response after ONC
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