Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients.

BACKGROUND: Cellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet-deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. METHODS: Orthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. RESULTS: We show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet-deficient mice. CONCLUSIONS: Our results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis.

Aurora B kinase is an integral regulator of cytokinesis, as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified that the ubiquitin E3 ligase complex SCF(FBXL2) mediates Aurora B ubiquitination and degradation within the midbody, which is sufficient to induce mitotic arrest and apoptosis. Three molecular acceptor sites (K¹°², K¹°³ and K²°7) within Aurora B protein were identified as important sites for its ubiquitination. A triple Lys mutant of Aurora B (K¹°²/¹°³/(²°7R)) exhibited optimal resistance to SCF(FBXL2)-directed polyubiquitination, and overexpression of this variant resulted in a significant delay in anaphase onset, resulting in apoptosis. A unique small molecule F-box/LRR-repeat protein 2 (FBXL2) activator, BC-1258, stabilized and increased levels of FBXL2 protein that promoted Aurora B degradation, resulting in tetraploidy, mitotic arrest and apoptosis of tumorigenic cells, and profoundly inhibiting tumor formation in athymic nude mice. These findings uncover a new proteolytic mechanism targeting a key regulator of cell replication that may serve as a basis for chemotherapeutic intervention in neoplasia.

F-box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest.

Dysregulated behavior of cell cycle proteins and their control by ubiquitin E3 ligases is an emerging theme in human lung cancer. Here, we identified and characterized the activity of a novel F-box protein, termed FBXL2, belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family. Ectopically expressed FBXL2 triggered G2/M-phase arrest, induced chromosomal anomalies and increased apoptosis of transformed lung epithelia by mediating polyubiquitination and degradation of the mitotic regulator, cyclin D3. Unlike other F-box proteins that target phosphodegrons within substrates, FBXL2 uniquely recognizes a canonical calmodulin (CaM)-binding motif within cyclin D3 to facilitate its polyubiquitination. CaM bound and protected cyclin D3 from FBXL2 by direct intermolecular competition with the F-box protein for access within this motif. The chemotherapeutic agent vinorelbine increased apoptosis of human lung carcinoma cells by inducing FBXL2 expression and cyclin D3 degradation, an effect accentuated by CaM knockdown. Depletion of endogenous FBXL2 stabilized cyclin D3 levels, accelerated cancer cell growth and increased cell viability after vinorelbine treatment. Last, ectopic expression of FBXL2 significantly inhibited the growth and migration of tumorogenic cells and tumor formation in athymic nude mice. These observations implicate SCF(FBXL2) as an indispensible regulator of mitosis that serves as a tumor suppressor.