Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases.

Department of Medicine, Indiana University, Indianapolis, Indiana 46202, USA.
Molecular Medicine (Impact Factor: 4.51). 01/2012; 18(1):445-54. DOI: 10.2119/molmed.2011.00207
Source: PubMed


α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

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    • "The current studies provide substantial evidence for a novel, intracellular role for A1PI-M in limiting caspase-1 activity and IL-1β release in monocytes. These results are similar to previous studies which invoke A1PI-M interactions with cytosolic executioner caspases 3 and 6 [25]–[26], and recently caspase-1 [27], as critical for staving off cell death in endothelial cells, epithelial cells, and cardiomyocytes. As in previous studies, we observed that an excess of A1PI was required to limit caspase activity in a cell free assay, which is highly reliant on the purity and potency of the enzyme preparation. "
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    ABSTRACT: Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli. IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro. IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner. Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.
    PLoS ONE 11/2012; 7(11):e51078. DOI:10.1371/journal.pone.0051078 · 3.23 Impact Factor
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    ABSTRACT: Serine elastase inhibitors have been proposed as a treatment for cigarette smoke-induced emphysema, but little is known about whether such agents actually are effective. We recently reported that a synthetic serine elastase inhibitor, ZD0892, provided some protection against emphysema in a guinea pig model. For these experiments, we used transgenic mice that expressed extremely low levels of human alpha-1-antitrypsin (A1AT) but were tolerant of exogenous human A1AT. Mice were exposed to daily cigarette smoke for up to 6 months; some animals received 20 mg of human A1AT (Prolastin) every 48 hours. Treatment with A1AT produced an approximate twofold increase in serum A1AT levels and elastase inhibitory capacity and abolished smoke-induced elevations in lavage neutrophils and matrix breakdown products (desmosine and hydroxyproline) measured from 2 to 30 days of smoke exposure. A1AT oxidized to remove antiproteolytic activity did not increase serum elastase inhibitory capacity but did prevent neutrophil influx. Treatment with A1AT for 6 months provided 63% protection against increased airspace size (emphysema) and abolished smoke-mediated increases in plasma tumor necrosis factor-alpha. We conclude that A1AT therapy ameliorates smoke-induced inflammation and matrix breakdown, possibly via an antiinflammatory mechanism related to tumor necrosis factor-alpha suppression, and provides partial protection against emphysema.
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    ABSTRACT: Alpha-1 antitrypsin (A1AT) is an acute phase reactant, but also a major protective factor against the development of chronic obstructive pulmonary disease (COPD), a complex disease with sustained chronic inflammation. The lung-protective effects of A1AT have been attributed to inhibition of proteases involved in lung matrix fragmentation, macrophage activation, and endothelial cell apoptosis. More recently, A1AT has been shown to directly interact with or modulate the action of cytokines such as TNFα or IL-1 on inflammatory cells, but its effect on the lung endothelium, an active participant in the amplification and resolution of inflammation, has received little attention. An important role of A1AT in modulating lung endothelial inflammatory responses is expected, given the high levels of circulating A1AT during inflammation and its active uptake by endothelial cells. We investigated the role of A1AT on primary lung microvascular endothelial cell activation by relevant cytokines such as TNFα or IL-1β. Despite initial marked augmentation of TNFα self-induced transcription, A1AT inhibited TNFα receptor 1 upregulation and significantly reduced TNFα secretion, effects that were associated with inhibition of the TNFα converting enzyme (TACE) activity. Furthermore, A1AT inhibited calpain activity, whose activation by TNFα contributed to decreased intracellular A1AT levels. These data indicate that A1AT initially facilitates acute responses of endothelium to TNFα, followed by selective inhibition of TNFα-induced-self amplification, which may assist the vasculature in the resolution of chronic inflammation.
    American Journal of Respiratory Cell and Molecular Biology 03/2013; 49(1). DOI:10.1165/rcmb.2012-0515OC · 3.99 Impact Factor
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