Receptor-interacting protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. Here, we report that the RIPK3 expression level is negatively regulated by CHIP (carboxyl terminus of Hsp70-interacting protein; also known as STUB1) E3 ligase-mediated ubiquitylation. Chip(-/-) mouse embryonic fibroblasts and CHIP-depleted L929 and HT-29 cells exhibited higher levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNF (also known as TNFα). These phenomena are due to the CHIP-mediated ubiquitylation of RIPK3, which leads to its lysosomal degradation. Interestingly, RIPK1 expression is also negatively regulated by CHIP-mediated ubiquitylation, validating the major role of CHIP in necrosome formation and sensitivity to TNF-mediated necroptosis. Chip(-/-) mice (C57BL/6) exhibit inflammation in the thymus and massive cell death and disintegration in the small intestinal tract, and die within a few weeks after birth. These phenotypes are rescued by crossing with Ripk3(-/-) mice. These results imply that CHIP is a bona fide negative regulator of the RIPK1-RIPK3 necrosome formation leading to desensitization of TNF-mediated necroptosis.

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Dry eye syndrome (DES) is a disorder of the eye due to tear deficiency or excessive evaporation that causes damage to the eye and is associated with discomfort and dryness. 11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is an enzyme that converts inactive cortisone to active cortisol. Recently, 11β-HSD1 has been expressed in human and rodent eyes and has been recognized as a target of glaucoma. In this study, the therapeutic effects and underlying mechanisms of topical carbenoxolone, an 11β-HSD1 inhibitor, were investigated in benzalkonium chloride (BAC)-treated human conjunctival epithelial cells and a rat DES model. In the in vitro study, carbenoxolone dose-dependently inhibited cell death and 11β-HSD1 activity in BAC-treated human conjunctival epithelial cells. For the in vivo study, carbenoxolone or a solvent was administered to the BAC-induced DES model twice daily. BAC-treated rat eyes showed significant increases in ocular surface damage, a reduction of tears, decrease corneal thickness, corneal basement membrane destruction, apoptosis in the conjunctival epithelium, and expression of pro-inflammatory cytokines (TNF-α and IL-6) and 11β-HSD1. These effects of BAC were reversed by topical carbenoxolone treatment. These results demonstrate that carbenoxolone can prevent DES by inhibiting pro-inflammatory cytokine expression and cell death of the corneal and conjunctival epithelium via inhibition of both 11β-HSD1 activity and expression in the eyes of BAC-treated rats. It is suggested that topical 11β-HSD1 inhibitors may provide a new therapeutic window in the prevention and/or treatment of DES.

Lipids are important signaling molecules regulating biological processes under normal and diseased conditions. Although p53 mutation is well-known for causing cancer, the relationship between p53-related tumorigenesis and altered lipid profile is unclear. We profiled differences in lipid expressions in liver, lung, and kidney in p53 knockout (KO) mice by high-speed quantitative analysis of 320 lipids (399 species identified) using nanoflow ultrahigh performance liquid chromatography−tandem mass spectrometry (nUPLC-MS/MS). Lung tissues were most severely affected by the lack of p53 gene, as shown by significant reduction (24−44%, P < 0.05) in total phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), diacylglycerol (DG), and triacylglycerol (TG), and significant increases (30−50%) in phosphatidylserine (PS), phosphatidylinositol (PI), and monohexosylceramide (MHC). MHC levels increased in all tissues. Dihexosylceramide (DHC) level decreased only in kidney tissue. Most PI, PS, and phosphatidic acid (PA) species showing significant increases contained a saturated acyl chain (18:0) in lung and liver tissues. Neutral glycerolipids (16:0/22:0-DG and most TGs with saturated and monounsaturated acyl chains) decreased 2−4-fold in the liver tissue. Our results suggest that the lack of p53 and altered lipid profiles are closely related, but as their changes vary from one tissue to another, the lipid alterations are tissue-specific.

Tumour suppressor p53 is known to be associated with the maintenance of mitochondrial functional properties in the skeletal muscles. As deactivation or mutation of p53 can affect the synthesis of lipids, investigating the relationship between p53-related energy generation metabolism and perturbation of lipid profile is critical. In this study, 329 lipid species (among 412 identified species) in two different skeletal muscle tissues (the gastrocnemius and soleus) from p53 knockout (KO) mice were quantitatively analysed using nanoflow ultrahigh performance liquid chromatography tandem mass spectrometry (nUPLC-MS/MS). Overall, lipids from the soleus tissues were more affected by p53 KO than those from the gastrocnemius in most lipid profiles. In p53 KO, lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), phosphatidic acid (PA), sphingomyelin (SM), and triacylglycerol (TAG), including 6 TAG (44:2, 46:0, 58:5, 58:8, 58:9, and 50:0), were significantly increased (p < 0.05) by 1.4–2-fold only in the soleus tissue. Overall monohexosylceramide (MHC) levels, including those of 3 MHC species (d18:0/24:0, d18:1/22:0, and d18:1/24:0), were significantly increased (p < 0.05) by 2–4 fold, only in the gastrocnemius tissue. The results suggest that lipid profiles are significantly altered by the lack of p53 in muscle tissues.