Inflammation is implicated in the pathogenesis of many diseases. In the lung, some of the examples include asthma, bronchitis, sarcoidosis, and a variety of disorders that ultimately lead to the loss of lung function. Monocytes are important components during the initiation and resolution of the innate immune response. Circulating monocytes normally live 24-48 hrs and in the absence of a survival signal undergo spontaneous apoptosis. In the presence of stimulatory factors, monocytes survive and differentiate into macrophages, which live up to 3 months. Thus, the understanding of the molecular mechanisms that control the apoptotic proteins is essential to manipulate the accumulation of monocyte/macrophages at inflammatory sites. Caspase-3 is a key executioner of the apoptotic pathway in monocytes. We demonstrated that stimulatory stimulus, such as lipopolysaccharide (LPS), induces monocyte survival by blocking the activation of caspase-3. Our preliminary studies demonstrate that caspase-3 is a phosphoprotein that associates and is phosphorylated by a member of the protein kinase C family (PKC). Our results suggest that phosphorylation induces caspase-3 activity and promotes apoptosis. The caspase-3-associated-kinase activity is modulated during monocyte life span and by inflammatory stimuli. The goal of this proposal is to understand the mechanisms that regulate apoptosis in monocytes. Here, we propose in Aim 1 to determine the biological role of caspase-3 phosphorylation. For this purpose, we will determine first the number and location of the phosphorylated sites on caspase-3 in vivo and in vitro. Next, using in vivo labeled caspase-3 and caspase-3 mutants we will elucidate whether phosphorylation affects the activation or the activity of caspase-3. Next, we will determine the role the caspase-3 phosphorylation in apoptosis.
In Aim 2, we propose to identify the kinase/s that are responsible for caspase-3 phosphorylation in vivo using siRNA and animal models. We propose in Aim 3, to investigate whether the phosphorylation changes the localization of caspase-3 and to study whether the localization changes during apoptosis. Finally, in Aim 4 we propose to determine the mechanisms by which caspase-3 phosphorylation and the caspase-3-associated-kinases regulate stimulation,differentiation, and life span of monocytes. Altogether, these studies should advance our understandingof the basic mechanisms that regulate apoptosis and help identifying new therapeutic targets to selectively induced apoptosis of inflammatory cells. Furthermore, it is our goal that the studies proposed here will contribute to improve the treatments for inflammation.
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