Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.

Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD(-/-) thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II(-/-)CAD(-/-) embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-beta gene was strongly up-regulated in the thymus of DNase II(-/-)CAD(-/-) embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.

Apoptotic cells are rapidly phagocytosed by professional phagocytes, such as macrophages and dendritic cells. This process prevents the release of potentially noxious or immunogenic intracellular materials from dying cells, and is thought to play a critical role for the maintenance of normal functions in surrounding tissues. Milk fat globule-EGF-factor 8 (MFG-E8), secreted by activated macrophages and immature dendritic cells, links apoptotic cells and phagocytes, and promotes phagocytosis of apoptotic cells. Here, we report that an MFG-E8 mutant, designated as D89E, carrying a point mutation in an RGD motif, inhibited not only the phagocytosis of apoptotic cells by a wide variety of phagocytes, but also inhibited the enhanced production of IL-10 by thioglycollate-elicited peritoneal macrophages phagocytosing apoptotic cells. When intravenously injected into mice, the D89E protein induced the production of autoantibodies including antiphospholipids antibodies and antinuclear antibodies. The production of autoantibodies was enhanced by the coinjection of syngeneic apoptotic thymocytes. After the induction of autoantibody production by D89E, the treated mice showed a long-term elevation of the titer for autoantibodies, and developed IgG deposition in the glomeruli. These results indicated that the impairment of apoptotic cell phagocytosis led to autoantibody production.

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf 2 activity through the interaction with Nrf 2 Neh2 domain. However, the mechanism(s) of Nrf 2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule.

The major cause of death among pulmonary hypertension patients is right heart failure, but the biology of right heart is not well understood. Previous studies showed that mechanisms of the activation of GATA4, a major regulator of cardiac hypertrophy, in response to pressure overload are different between left and right ventricles. In the left ventricle, aortic constriction triggers GATA4 activation via post-translational modifications without influencing GATA4 expression, while pulmonary artery banding enhances GATA4 expression in the right ventricle. We found that GATA4 expression can also be increased in the right ventricle of rats treated with chronic hypoxia to induce pulmonary hypertension, and investigated the mechanism of increased GATA4 expression. Examination of Gata4 promoter revealed that CCAAT box plays an important role in gene activation; and hypoxic pulmonary hypertension promoted the binding of CBF/NF-Y to CCAAT box in the right ventricle. We found that CBF/NF-Y forms a complex with annexin A1, which inhibits DNA binding activity. In response to hypoxic pulmonary hypertension, annexin A1 gets degraded, resulting in CBF/NF-Y-dependent activation of Gata4 gene transcription. The right ventricle contains a higher level of CBF/NF-Y compared to the left ventricle, and this may allow for efficient activation in response to annexin A1 degradation. Signaling via iron-catalyzed protein oxidation, mediates hypoxic pulmonary hypertension-induced annexin A1 degradation, Gata4 gene transcription, and right ventricular hypertrophy. These results establish a right heart-specific signaling mechanism in response to pressure overload, which involves metal-catalyzed carbonylation and degradation of annexin A1 that liberates CBF/NF-Y to activate Gata4 gene transcription.

Keywords: apoptosis; genes; pulmonary hypertension; smooth muscle Pulmonary hypertension is characterized by the elevation of pulmonary vascular resistance, which interferes with the ejection of blood by the right ventricle and ultimately causes heart failure. It is often developed secondary to various cardiovascular and pulmonary diseases such as left ventricular failure, congenital heart defects, chronic obstructive pulmonary disease, sleep apnea syndrome, and post-thrombotic diseases. Pulmonary arterial hypertension can also occur as a genetic disorder. Pulmonary hypertension is associated with increased vasoconstriction in the pulmonary circulation and vascular remodeling in part due to thickening of pulmonary vascular wall because of increased number of smooth muscle cells (SMC). Although therapeutic agents are available that target the vasoconstrictive aspect of this condi-

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Obstructive sleep apnea (OSA) is associated with cardiovascular diseases such as hypertension through mechanisms involving intermittent hypoxia (IH). However, it is not yet clear whether IH directly affects the heart. In a mouse model of OSA, we found that IH causes time-dependent alterations of the susceptibility of the heart to oxidative stress. Acute IH can exert preconditioning-like cardioprotection, in part, through the transcriptional activation of genes such as bcl-x(L) and gata4. We cloned the mouse gata4 promoter and identified an IH-responsive region. The exposure of mice to prolonged IH results in the increased susceptibility of the heart to ischemia-reperfusion injury by increasing the oxidative stress status. This might resemble conditions of OSA patients. In our mouse model, further exposure to prolonged IH allowed reversal of the enhancement of myocardial damage. Understanding the complex effects of IH on the heart should help ultimately to develop therapeutic strategies against OSA-induced complications.