A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Revisited role of TRAF2 and TRAF2 C-terminal domain in endoplasmic reticulum stress-induced autophagy in HAP1 leukemia cells.

The scaffold protein Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) has been reported to play a key role in the endoplasmic reticulum (ER) stress-induced activation of c-Jun N-terminal Kinase (JNK) and hence autophagy. Autophagy is a highly conserved catabolic process, whose dysregulation is involved in the pathogenesis of various human diseases, including cancer. We investigated the involvement of TRAF2 in autophagy regulation in the human leukemic HAP1 cell line, under both basal and ER stress conditions. In TRAF2-knockout HAP1 cell line (KO), the basal autophagic flux was higher than in the parental cell line (WT). Moreover, tunicamycin-induced ER stress stimulated JNK activation and autophagy both in WT and KO HAP1. On the other hand, re-expression of a TRAF2 C-terminal fragment (residues ,310-501), in a TRAF2-KO cellular background, rendered HAP1 cells unable to activate both JNK and autophagy upon ER stress induction. Of note, this apparent dominant negative effect of the C-terminal fragment was observed even in the absence of the endogenous, full-length TRAF2 molecule. Furthermore, the expression of the C-terminal fragment resulted in both protein kinase B (AKT) pathway activation and increased resistance to the toxic effects induced by prolonged ER stress conditions. These findings indicate that TRAF2 is dispensable for the activation of both JNK and autophagy in HAP1 cells, while the TRAF2 C-terminal domain may play an autonomous role in regulating the cellular response to ER stress.