Akt2 Is The Major Akt Kinase Against Oxidative Stress And Regulates Multiple Signaling Pathways To Resist H₂O₂-Induced Apoptosis

Akt kinase (also called protein kinase B) plays a central role in regulating myriad physical and pathological activities including apoptosis, cell proliferation, cell differentiation, metabolism, ageing, disease and cancer. Although Akt kinase family members Aktl and Akt2 share high amino acid homogeneity and similar protein primary structure, accumulating evidence suggests that Aktl and Akt2 have differential functions in regulating physiological and pathological activities.It is generally acknowledged that oxidative stress is a common environmental stress that the lens epithelium encounters, and it has been shown that oxidative-stress-induced apoptosis of lens epithelial cells is one of common pathological mechanisms for the initiation and progression of non-congenital cataract. Akt kinase being one of the most significant kinases promoting cell survival, its specific function in regulating cellular responses against oxidative stress in lens epithelial cells has not been well established. Thus the major focus of the present study is to define the molecular mechanism by which Akt kinase family members modulate oxidative-stress-induced lens epithelial cell apoptosis.In this study, we first identified the expression pattern of three Akt kinase family members Aktl, Akt2 and Akt3 in mouse ocular tissues (retina, lens epithelium, lens fiber and cornea) during different embryonic developmental stages. Then we identified the expression pattern of Aktl, Akt2 and Akt3 as well as the phosphorylation level of Akt on Thr450, Thr308 and Ser473 in the four ocular tissues of adult mouse. Our data revealed that Aktl is the predominantly expressed Akt kinase family member in adult mouse lens tissue. To investigate the mechanism by which Akt1 regulates cellular activities against oxidative-stress-induced lens epithelial cell apoptosis, taking Human Lens Epithelial (HLE) cells as the function-study ing-model, we specifically knockdown Akt1 with specific Akt1 shRNA in HLE cells and established the stable Akt1 knockdown HLE cell line. Correspondingly, we also established the control Mock knockdown HLE cell line. Then we treated both Akt1 knockdown HLE cells and Mock knockdown HLE cells with a method producing constant 50μM hydrogen peroxide (H2O2) for 2 hours, and the experimental result indicated that, compared with Mock knockdown HLE cells, the Akt1 knockdown HLE cells were endowed with much more resistance against oxidative-stress-induced apoptosis. Analysis of the underlying mechanism of differential apopotosis between two different cell lines revealed that 1) Akt1 knockdown promoted Akt2 upregulation and enhanced Akt kinase activity; and that 2) the enhanced Akt kinase activity significantly modulated three main downstream signaling pathways including MDM2-p53-Bak, GSK3β-MCL-1 and FOXO3A-Bim. As a result, such modulation confers HLE cells enhanced antiapoptotic ability in the Akt1 knockdown cells under oxidative stress insult.These experimental data suggested that in HLE cells, Akt2, but not Akt1, is the major Akt kinase regulating cellular responses and antagonizing apoptosis under oxidative stress. To confirm this proposition, we specifically knockdown Akt2 with Akt2 shRNA in the HLE cells and Akt1 knockdown HLE cells, and established the stable Akt2 knockdown HLE cell line and Akt 1/2 knockdown HLE cell line, respectively. Under the same H2O2 treatment (as being conducted in previous experiments), the knockdown of Akt2 significantly increased the sensitivity of these two cell lines to oxidative-stress-induced apoptosis. As a result, both Akt2 knockdown HLE cells and Akt1/2 knockdown HLE cells displayed increased apoptosis rate compared with corresponding Mock knockdown cells. Besides that, we identified the similar changes in MDM2-p53-Bak, GSK3β-MCL-1 and FOXO3A-Bim cascades which were modulated by Akt kinase in H2O2 treated-Akt2 knockdown HLE cells and Aktl/2 knockdown HLE cells. To further testify the critical function of Akt2 in resisting against oxidative-stress-induced apoptosis, we transfected the Akt2 expression plasmid in HLE cells, and we found that the overexpression of Akt2 significantly attenuated oxidative-stress-induced apoptosis.In summary, this study presented the first evidence that Aktl knockdown induces Akt2 compensatory upregulation and significantly enhanced Akt kinase activity, which confers the HLE cells resistance against oxidative-stress-induced apoptosis. More importantly, this study demonstrated that Akt2 is the major Akt kinase in HLE cells counteracting oxidative-stress-induced apoptosis, and three signaling pathways:MDM2-p53-Bak, GSK3β-MCL-1 and FOXO3A-Bim are involved in the molecular mechanism. These conclusions have shed new light on the precaution and therapy of non-congenital cataract induced by oxidative stress.