Cytokine regulation by MAPK activated kinase 2 in keratinocytes exposed to sulfur mustard.

Uncontrolled inflammation contributes to cutaneous damage following exposure to the warfare agent bis(2-chloroethyl) sulfide (sulfur mustard, SM). Activation of the p38 mitogen activated protein kinase (MAPK) precedes SM-induced cytokine secretion in normal human epidermal keratinocytes (NHEKs). This study examined the role of p38-regulated MAPK activated kinase 2 (MK2) during this process. Time course analysis studies using NHEK cells exposed to 200µM SM demonstrated rapid MK2 activation via phosphorylation that occurred within 15 min. p38 activation was necessary for MK2 phosphorylation as determined by studies using the p38 inhibitor SB203580. To compare the role of p38 and MK2 during SM-induced cytokine secretion, small interfering RNA (siRNA) targeting these proteins was utilized. TNF-α, IL-1ß, IL-6 and IL-8 secretion was evaluated 24h postexposure, while mRNA changes were quantified after 8h. TNF-α, IL-6 and IL-8 up regulation at the protein and mRNA level was observed following SM exposure. IL-1ß secretion was also elevated despite unchanged mRNA levels. p38 knockdown reduced SM-induced secretion of all the cytokines examined, whereas significant reduction in SM-induced cytokine secretion was only observed with TNF-α and IL-6 following MK2 knockdown. Our observations demonstrate potential activation of other p38 targets in addition to MK2 during SM-induced cytokine secretion.

Modes of Retinal Cell Death in Diabetic Retinopathy.

Cell death seems to be a prominent feature in the progression of diabetic retinopathy. Several retinal cell types have been identified to undergo cell death in a diabetic environment. Most emphasis has been directed towards identifying apoptosis in the diabetic retina. However, new research has established that there are multiple forms of cell death. This review discusses the different modes of cell death and attempts to classify cell death of retinal cells known to die in diabetic retinopathy. Special emphasis is given to apoptosis, necrosis, autophagic cell death, and pyroptosis. It seems that different retinal cell types are dying by diverse types of cell death. Whereas endothelial cells predominantly undergo apoptosis, pericytes might die by apoptosis as well as necrosis. On the other hand, Müller cells are suggested to die by a pyroptotic mechanism. Diabetes leads to significant Müller cell loss at 7 months duration of diabetes in retinas of diabetic mice compared to non-diabetic, which is prevented by the inhibition of the caspase-1/IL-1ß (interleukin-1beta) pathway using the IL-1 receptor knockout mouse. Since pyroptosis is characterized by the activation of the caspase-1/IL-1ß pathway subsequently leading to cell death, Müller cells seem to be a prime candidate for this form of inflammation-driven cell death. Considering that diabetic retinopathy is now discussed to potentially be a chronic inflammatory disease, pyroptotic cell death might play an important role in disease progression. Understanding mechanisms of cell death will lead to a more targeted approach in the development of new therapies to treat diabetic retinopathy.

siah-1 Protein is necessary for high glucose-induced glyceraldehyde-3-phosphate dehydrogenase nuclear accumulation and cell death in Muller cells.

The translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the nucleus has closely been associated with cell death induction. However, the mechanism of this process has not been completely understood. The E3 ubiquitin ligase siah-1 (seven in absentia homolog 1) has recently been identified as a potential shuttle protein to transport GAPDH from the cytosol to the nucleus. Previously, we have demonstrated that elevated glucose levels induce GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study investigated the role of siah-1 in high glucose-induced GAPDH nuclear translocation and subsequent cell death in retinal Müller cells. High glucose significantly increased siah-1 expression within 12 h. Under hyperglycemic conditions, siah-1 formed a complex with GAPDH and was predominantly localized in the nucleus of Müller cells. siah-1 knockdown using 50 nm siah-1 small interfering RNA significantly decreased high glucose-induced GAPDH nuclear accumulation at 24 h by 43.8 +/- 4.0%. Further, knockdown of siah-1 prevented high glucose-induced cell death of Müller cells potentially by inhibiting p53 phosphorylation consistent with previous observations, indicating that nuclear GAPDH induces cell death via p53 activation. Therefore, inhibition of GAPDH nuclear translocation and accumulation by targeting siah-1 promotes Müller cell survival under hyperglycemic conditions.

Differential regulation of high glucose-induced glyceraldehyde-3-phosphate dehydrogenase nuclear accumulation in Müller cells by IL-1beta and IL-6.

PURPOSE: This study determined the role of the proinflammatory cytokines known to be elevated in the diabetic retina, namely IL-1beta, TNFalpha, and IL-6, in a high glucose-induced nuclear accumulation of GAPDH in retinal Müller cells, an event considered crucial for the induction of cell death. METHODS: With use of the transformed rat Müller cell line (rMC-1) and isolated human Müller cells (HMCs), the authors examined the effect of high glucose (25 mM), IL-1beta, TNFalpha, IL-6, and high glucose (25 mM) plus inhibitors of the caspase-1/IL-1beta signaling pathway on GAPDH nuclear accumulation, which was evaluated by immunofluorescence analysis. RESULTS: High glucose induced IL-1beta, weak IL-6, and no TNFalpha production by rMC-1 and HMCs. IL-1beta (1-10 ng/mL) significantly increased GAPDH nuclear accumulation in Müller cells in a concentration-dependent manner within 24 hours. Further, high glucose-induced GAPDH nuclear accumulation in Müller cells was mediated by IL-1beta. Inhibition of the IL-1 receptor using an IL-1 receptor antagonist (IL-1ra; 50 ng/mL) or inhibition of IL-1beta production using a specific caspase-1 inhibitor (YVAD-fmk; 100 microM) significantly decreased high glucose-induced GAPDH nuclear accumulation. In contrast, IL-6 (2 ng/mL) had a strong protective effect attenuating high glucose and IL-1beta-induced GAPDH nuclear accumulation in Müller cells. TNFalpha (1-10 ng/mL) did not have any effect on GAPDH nuclear accumulation. CONCLUSIONS: These results revealed a novel mechanism for high glucose-induced GAPDH nuclear accumulation in Müller cells through production and autocrine stimulation by IL-1beta. The protective role of IL-6 in high glucose- and IL-1beta-induced toxicity indicates that changes in the balance of these cytokines might contribute to cellular damage mediated by elevated glucose levels.