The Role of Redox-Sensitive Mitogen-Activated Protein Kinases JNK and p38 in the Dysregulation of Apoptosis of Mononuclear Leukocytes under Oxidative Stress

The dissertation is devoted to the study of the role of redox-sensitive mitogen-activated protein kinases JNK and p38 in the mechanisms of dysregulation of apoptosis of mononuclear leukocytes of peripheral blood under oxidative stress. The research was conducted at the Siberian State Medical University (Tomsk) and combines clinical and experimental approaches. The objects of investigation were mononuclear leukocytes of blood from patients with acute appendicitis and nosocomial pneumonia, as well as cultures of these cells under conditions of experimental oxidative stress in vitro. The work includes assessment of kinase activity, level of apoptosis, cytokine production, and expression of the transcription factor p53, which allows the identification of molecular mechanisms of programmed cell death under conditions of imbalance of oxidative metabolism.

Relevance of the Problem. Apoptosis represents an active form of cell death, which is a physiological mechanism for eliminating excessive and/or functionally deficient cells. Disruption of the implementation of the lethal cell program as a result of an imbalance between pro- and antiapoptotic factors leads to pathological changes in structure and functions. Important links in the pathogenesis of various diseases (oncological, cardiovascular, neurodegenerative, acute and chronic inflammatory processes, diabetes mellitus, etc.) may be disorders of apoptosis dysregulation mechanisms leading to its excessive activation or inhibition [Bredesen D.E., 2000; Menytsikova E.B. et al., 2006; Zhukova O.B. et al., 2007]. At the same time, the development of these diseases is associated with cell damage caused by oxidative stress due to prooxidant-antioxidant imbalance [Zenkov N.K. et al., 2001]. Reactive oxygen species (ROS) act as damaging agents, which are an effective tool for local action due to high reactivity. An increase in ROS content leads to oxidative modification of biomolecules, changes in the activity of key enzymatic systems, and disruption of membrane structure [Dubinina E.E., 2001; Menytsikova E.B. et al., 2006]. In addition, excessive generation of ROS in tissues against the background of exhaustion of antioxidant protection reserves affects the functional state of redox-sensitive systems of intracellular apoptosis regulation. Among the latter are mitogen-activated protein kinases JNK and p38, which phosphorylate proteins responsible for the implementation of the cell death program [Gallo K.A., Johnson G.L., 2002]. Activation of JNK plays a leading role in launching the cell death program in response to stress (action of inflammatory cytokines IL-1, TNF-α, and free radicals formed under the influence of ultraviolet radiation, irradiation, ROS; inhibition of protein synthesis) [Gallo K.A., Johnson G.L., 2002]. JNK can induce apoptosis by phosphorylating and activating the transcription factor p53 [Morgunkova A.A., 2005]. It has been established that JNK kinase can penetrate into mitochondria, where it phosphorylates and activates proapoptotic proteins Bax and Bad, and also inactivates antiapoptotic proteins of the Bcl-2 family [Gallo K.A., Johnson G.L., 2002; Vlaopoulos S., Zumpulis B.C., 2004; Das D.K., Molik N., 2004; Teraishi F., Wu S., 2005; Harada S., Nakamura K., 2006]. The p38 kinase activates the factor Nf-kB, MEF2C [Gallo K.A., Johnson G.L., 2002], promotes the expression and mitochondrial translocation of one of the most important proapoptotic proteins — Bax, mediating its influence through p53 phosphorylation [Kim S.J. et al., 2002; Mayr M. et al., 2002]. At the same time, a number of studies indicate the presence of antiapoptotic activity of JNK and p38 [Sabapathy K. et al., 1999; Harada S., Nakamura K., 2006], which depends on the features of inducing signals, combinations of possible pathways of their transmission, and cell types. Discrepancy in the presented views on the designated problem complicates the development of pharmaceutical approaches for targeted correction of programmed cell death and determines the expediency of conducting research aimed at studying the role of MAP kinases under conditions of oxidative stress. In connection with this, there is a need for more detailed study of the role of stress-activated protein kinases JNK and p38 in the implementation of the cell death program under oxidative stress, which is a typical universal mechanism of development of pathological processes of various genesis. Goal of the Research: to establish the molecular mechanisms of the regulatory influence of redox-sensitive MAP kinases on the implementation of programmed death of mononuclear leukocytes under oxidative stress. Tasks of the Research: 1. To assess the level of active forms of stress-activated MAP kinases (JNK and p38) in mononuclear leukocytes of blood under conditions of imbalance of oxidative metabolism in vitro. 2. To establish features of implementation of apoptosis of mononuclear leukocytes of blood under the action of selective inhibitors of JNK and p38 MAPKs under conditions of oxidative stress in vitro. 3. To determine the role of redox-sensitive kinases JNK and p38 in the modulation of the transcription factor p53 under oxidative stress in vitro. 4. To assess the participation of redox-sensitive kinases JNK and p38 in the production of cytokines (IL-8 and IL-10) by mononuclear leukocytes of blood under conditions of oxidative stress in vitro and in acute inflammation. 5. To reveal molecular mechanisms of modulation of programmed death of mononuclear leukocytes of blood mediated through the action of redox-sensitive MAP kinases in the case of acute inflammatory process. Scientific Novelty. Using modern molecular-biological research methods, the assessment of the role of redox-sensitive MAP kinases p38 and JNK in the disruption of the implementation of programmed cell death under conditions of oxidative stress was conducted for the first time. It has been established that under conditions of imbalance of oxidative metabolism, activation of redox-sensitive JNK and p38 MAP kinases occurs, which are an important element of the signaling transduction system of proapoptotic signals. Under conditions of imbalance of oxidative metabolism of mononuclear leukocytes of blood, MAP kinases JNK and p38 act as proapoptotic regulatory molecules. Activation of the transcription factor p53 (due to its phosphorylation by MAP kinases and/or direct action of ROS) leads to a change in the regulation of programmed cell death under conditions of oxidative stress. In the experiment using the selective inhibitor SP600125, it was revealed that the redox-sensitive kinase JNK influences the production of IL-8, while the p38 MAPK inhibitor ML3403 does not have such an effect. For the first time in the experiment using selective MAPK inhibitors, it has been demonstrated that p38 and JNK do not participate in the regulation of IL-10 synthesis under conditions of imbalance of oxidative metabolism. Theoretical and Practical Significance. The results of the conducted research expand existing fundamental representations about the nature of changes in programmed cell death under conditions of imbalance of oxidative metabolism. Priority data have been obtained on the role of redox-sensitive kinases JNK and p38 in the dysregulation of programmed death of mononuclear leukocytes of blood under experimental oxidative stress and acute inflammatory diseases accompanied by intensification of free radical oxidation processes (nosocomial pneumonia and acute appendicitis). The obtained data may serve as a basis for the development of methodology for correction of apoptosis regulation disorders under pathological conditions accompanied by imbalance of oxidative metabolism. Positions to be Defended: 1. Imbalance of oxidative metabolism is accompanied by activation of redox-sensitive systems of signaling transduction of proapoptotic signals, in particular activation of phosphorylation of MAP kinases JNK and p38. 2. Under conditions of oxidative stress, MAP kinases JNK and p38 perform the role of proapoptotic regulatory molecules. 3. Activation of the transcription factor p53 under conditions of oxidative stress in vitro is due to its phosphorylation by MAP kinases and/or the direct effect of ROS. 4. In acute inflammatory diseases (acute appendicitis, nosocomial pneumonia), as well as under experimental oxidative stress in vitro, the balance of IL-8 and IL-10 is disrupted; increased production of IL-8 under disruption of oxidative metabolism is associated with activation of JNK kinase. Presentation and Implementation of the Work. The results of the conducted research were reported and discussed at the VIII All-Russian Scientific and Practical Conference «Actual Questions of Clinical Diagnosis and Treatment of Patients in a Multiprofile Medical Institution» (Saint Petersburg, 2007), the III All-Russian Scientific and Practical Conference «Fundamental Aspects of Compensatory-Adaptive Processes» (Novosibirsk, 2007), the Interregional Scientific and Practical Conference «Actual Problems of Medicine» (Abakan, 2007), the III Congress of the Euro-Asian Respiratory Society (Astana, 2007), the VII Congress of Young Scientists and Specialists «Sciences about Man» (Tomsk, 2007), the Intercity Conference of Young Scientists «Actual Problems of Pathophysiology» (Saint Petersburg, 2007). The research was supported by the Grants Council under the President of the RF for support of leading Russian scientific schools in the framework of the project «Molecular Foundations of Disruption of Cell Homeostasis under Current Diseases of Infectious and Non-Infectious Nature» (NS-4153.2006.7), RFBR — «Molecular Mechanisms of Management of Programmed Cell Death Using Regulatory Molecules» (No. 07-04-12150), as well as carried out within the framework of the Federal Target Program (project «Development of Methods for Correction of Apoptosis Regulation Disorders in Cells under Pathological Processes in Conditions of Oxidative Stress», state contract No. 02.442.11.7276 dated 20.02.2006). The main results of the dissertation research have been included in the lecture course on pathological physiology («Cell Pathophysiology», «Typical Pathological Processes», «The Role of Cell Apoptosis in Pathology») for students of the therapeutic and pediatric faculties of GOU VPO SibGMU of Roszdrav. Publications. Based on the dissertation materials, 8 works have been published, of which 2 are in peer-reviewed journals recommended by VAK. Structure and Volume of the Dissertation. The dissertation is presented on 154 pages of typed text and consists of an introduction, four chapters, conclusions, and a list of references including 279 sources, of which 105 are domestic and 174 are foreign. The work is illustrated with 6 tables and 20 figures.