Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-κB-mediated survival signaling
Wihuri Research Institute, Kalliolinnantie 4, FI-00140 Helsinki, Finland. Experimental Cell Research
(Impact Factor: 3.25).
06/2006; 312(8):1289-98. DOI: 10.1016/j.yexcr.2005.12.033
Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-kappaB-mediated survival signaling. Following chymase treatment, the translocation of active NF-kappaB/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1beta-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-kappaB-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-kappaB-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.
Available from: James F Collawn
- "In mast cell-deficient mice, there is no detectable chymase mRNA, suggesting that at baseline in the absence of any stress the mast cell is the primary source of chymase . In addition to converting angiotensin I (Ang I) to Ang II , , chymase directly degrades fibronectin , activates matrix metalloproteinases (MMPs) , , , and initiates apoptosis in cardiomyocytes through disruption of the focal adhesion complex in vitro
, . Based on amino acid sequence homology, chymase is divided into two groups, α and β. "
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ABSTRACT: Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.
Available from: ajpcell.physiology.org
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ABSTRACT: Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.
Available from: António Coutinho
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ABSTRACT: The recent progress in the genetic characterization of many primary immunodeficiencies (PIDs) allows for a better understanding of immune molecular and cellular mechanisms. The present chapter discusses associations between PIDs and autoimmune diseases (AIDs) in this new light. PIDs are classified according to the frequency of association with AIDs, defining four groups of conditions: systematic (more than 80% of all patients), strong (10-80%), mild (less than 10%), and absent (no available descriptions). Several general conclusions could be drawn: (1) pathological autoimmune (AI) manifestations are very frequently associated with PIDs, indicating that, contrary to conventional notions, antimicrobial protection and natural tolerance to body tissues share many basic mechanisms; (2) in some gene defects, association is so strong that one could speak of "monogenic" AIDs; (3) basic types of PIDs are selectively associated with AID of a particular set of target tissues; (4) while for some gene defects, current theory satisfactorily explains pathogenesis of the corresponding AID, other situations suggest extensive gaps in the present understanding of natural tolerance; and (5) not exceptionally, observations on the AI phenotype for the same gene defect in mouse and man are not concordant, perhaps owing to the limited genetic diversity of mouse models, often limited to a single mouse strain. Overall, clinical observations on PID support the new paradigm of "dominant" tolerance to self-components, in which AID owes to deficits in immune responses (i.e., in regulatory mechanisms), rather than from excessive reactivity.
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