A genetic variant of p53 restricts the mucous secretory phenotype by regulating SPDEF and Bcl-2 expression.

Despite implications for carcinogenesis and other chronic diseases, basic mechanisms of p53 and its variants in suppressing Bcl-2 levels are poorly understood. Bcl-2 sustains mucous cell metaplasia, whereas p53(-/-) mice display chronically increased mucous cells. Here we show that p53 decreases bcl-2 mRNA half-life by interacting with the 5' untranslated region (UTR). The p53-bcl-2 mRNA interaction is modified by the substitution of proline by arginine within the p53 proline-rich domain (PRD). Accordingly, more mucous cells are present in primary human airway cultures with p53(Arg) compared with p53(Pro). Also, the p53(Arg) compared with p53(Pro) displays higher affinity to and activates the promoter region of SAM-pointed domain-containing Ets-like factor (SPDEF), a driver of mucous differentiation. On two genetic backgrounds, mice with targeted replacement of prolines in p53 PRD show enhanced expression of SPDEF and Bcl-2 and mucous cell metaplasia. Together, these studies define the PRD of p53 as a determinant for chronic mucous hypersecretion.

Deacetylation of p53 induces autophagy by suppressing Bmf expression.

Interferon γ (IFN-γ)-induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-γ on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-γ down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death-stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-γ did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-γ-induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53's proline-rich domain. Suppression of Bmf facilitated IFN-γ-induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf(-/-) but not in bmf(+/+) cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy.

Adam8 limits the development of allergic airway inflammation in mice.

To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8(-/-) mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite-treated Adam8(-/-) mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8's anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8(-/-) mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8(-/-) macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.

Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes.

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.

Mutual dependence of Mob1 and the chromosomal passenger complex for localization during mitosis.

The spatial and temporal coordination of chromosome segregation with cytokinesis is essential to ensure that each daughter cell receives the correct complement of chromosomal and cytoplasmic material. In yeast, mitotic exit and cytokinesis are coordinated by signaling cascades whose terminal components include a nuclear Dbf2-related family kinase and a noncatalytic subunit, Mps one binding (Mob) 1. There are five human Mob1 isoforms, all of which display redundant localization patterns at the spindle poles and kinetochores in early mitosis, and the spindle midzone during cytokinesis. Mob1 shares similar localization patterns to Polo-like kinase (Plk1) and the chromosomal passenger complex (CPC), and although depletion of Plk1 resulted in a loss of Mob1 from the spindle poles, Mob1 recruitment to kinetochores was unaffected. Conversely, disruption of CPC signaling resulted in a loss of Mob1 from kinetochores without disrupting recruitment to the spindle poles. In Mob1-depleted cells, the relocalization of the CPC and mitotic kinesin-like protein (MKLP) 2 to the spindle midzone was delayed during early anaphase, and as a consequence, the midzone recruitment of MKLP1 also was affected. Together, these results suggest that Mob1 and the other mammalian orthologues of the mitotic exit network regulate mitotic progression by facilitating the timely mobilization of the CPC to the spindle midzone.