nutlin-3a and Inflammation

Uveitis is estimated to account for 10% of all cases of blindness in the United States, including 30,000 new cases of legal blindness each year. Intraocular and oral corticosteroids are the effective mainstay treatment, but they carry the risk of serious long-term ocular and systemic morbidity. New noncorticosteroid therapies with a favorable side effect profile are necessary for the treatment of chronic uveitis, given the paucity of existing treatment choices. We have previously demonstrated that Nutlin-3, a small-molecule inhibitor of murine double minute 2 (MDM2) homolog, suppresses pathologic retinal angiogenesis through a p53-dependent mechanism, but the noncanonical p53-independent functions have not been adequately elucidated. Herein, we demonstrate an unanticipated function of MDM2 inhibition, where Nutlin-3 potently abrogates lipopolysaccharide-induced ocular inflammation. Furthermore, we identified a mechanism by which transcription and translation of NF-κB is mediated by MDM2, independent of p53, in ocular inflammation. Small-molecule MDM2 inhibition is a novel noncorticosteroid strategy for inhibiting ocular inflammation, which may potentially benefit patients with chronic uveitis.

Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Eye Diseases; Gene Expression Regulation; Humans; Imidazoles; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Retinal Pigment Epithelium; Tumor Suppressor Protein p53

Inflammation has been implicated in myocardial infarction (MI). MDM2 associates with nuclear factor-κB (NF-κB)-mediated inflammation. However, the role of MDM2 in MI remains unclear. This study aimed to evaluate the impacts of MDM2 inhibition on cardiac dysfunction and fibrosis after experimental MI and the underlying mechanisms. Three-month-old male C57BL/6 mice were subjected to left anterior descending (LAD) coronary artery ligation for induction of myocardial infarction (MI). Immediately after MI induction, mice were treated with Nutlin-3a (100 mg/kg) or vehicle twice daily for 4 weeks. Survival, heart function and fibrosis were assessed. Signaling molecules were detected by Western blotting. Mouse myofibroblasts under oxygen and glucose deprivation were used for in vitro experiments. MDM2 protein expression was significantly elevated in the mouse heart after MI. Compared with vehicle-treated animals, Nutlin-3a treatment reduced the mouse mortality. Nutlin-3a treatment improved heart function and decreased the infarct scar and fibrosis compared with vehicle. Furthermore, MDM2 inhibition restored IκB and inhibited NF-κB activation, leading to suppressed production of proinflammatory cytokines in the heart after MI. The consistent results were obtained in vitro. MDM2 inhibition reduced cardiac dysfunction and fibrosis after MI. These effects of MDM2 inhibition is mediated through modulating NF-κB activation, resulting in inhibition of inflammatory response.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Fibrosis; Heart Diseases; I-kappa B Proteins; Imidazoles; Inflammation; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myofibroblasts; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Survival Rate

Murine double minute (MDM)-2, an E3 ubiquitin ligase, promotes cancer cell survival and growth, by degrading the cell cycle regulator p53. Antagonism of MDM2 by the small-molecule cis-imidazoline nutlin analogs is under current study for cancer therapy. To test whether MDM2 also promotes regenerative cell growth, we determined the effects of nutlin-3a on tubule cell healing during postischemic acute kidney injury (AKI). Treatment with nutlin-3a impaired tubular cell regeneration during postischemic AKI in wild-type mice in a p53-dependent manner; however, MDM2 blockade also prevented tubular necrosis by suppressing sterile inflammation during the early postischemic phase. This effect also occurred in p53 knockout mice, indicating a second, proinflammatory, p53-independent role for MDM2 in AKI. In vitro experiments confirmed that MDM2 is required to induce mRNA expression and secretion of NFκB-dependent cytokines upon Toll-like receptor stimulation by enhanced binding of NFκB to cytokine promoter-binding sites. Thus, MDM2 links inflammation and epithelial healing during AKI. These additional biological functions need to be regarded when considering MDM2 inhibition therapy.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Gene Expression Regulation; Imidazoles; Inflammation; Inflammation Mediators; Injections, Intraperitoneal; Kidney Tubules; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Regeneration; Reperfusion Injury; RNA, Messenger; Time Factors; Tumor Suppressor Protein p53; Wound Healing

Tumour protein p53 plays an important role in the vascular remodelling process as well as in oncogenesis. p53 is negatively regulated by murine double minute 2 (MDM2). A recently developed MDM2 inhibitor, nutlin-3, is a non-genotoxic activator of the p53 pathway. So far, the effect of MDM2 inhibition on vascular remodelling has not been elucidated. We therefore investigated the effect of nutlin-3 on neointima formation.. Nutlin-3 up-regulated p53 and its downstream target p21 in vascular smooth muscle cells (VSMCs). DNA synthesis assay and flow cytometric analysis revealed that nutlin-3 inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation by cell cycle arrest. This inhibitory effect was abrogated in p53-siRNA-transfected VSMCs. Furthermore, nutlin-3 inhibited PDGF-stimulated VSMC migration. Treatment with nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury in mice, associated with up-regulation of p53 and p21. BrdU incorporation was decreased at 14 days after injury in nutlin-3-treated mice. TUNEL assay showed that nutlin-3 did not exaggerate apoptosis of the injured vessels. Infiltration of macrophages and T-lymphocytes and mRNA expression of chemokine (C-C motif) ligand-5, interleukin-6, and intercellular adhesion molecule-1 were decreased in the injured vessels of nutlin-3-treated mice. Nutlin-3 suppressed NF-κB activation in VSMCs, but not in p53-siRNA-transfected VSMCs.. The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Topics: Animals; Cell Cycle; Cell Movement; Cell Proliferation; Hyperplasia; Imidazoles; Inflammation; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Neointima; NF-kappa B; Piperazines; Proto-Oncogene Proteins c-mdm2; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53