TOPK inhibits TNF-α-induced granulosa cell apoptosis via regulation of SIRT1/p53.

T-LAK cell originated protein kinase (TOPK) has been shown to regulate proliferation, invasion or migration of various cancer cells. However, the role of TOPK in follicle environments remains unknown. Here we reveal that TOPK inhibits TNF-α-induced human granulosa COV434 cell apoptosis. The expression of TOPK were increased in COV434 cells in response to TNF-α. TOPK inhibition also decreased TNF-α-induced SIRT1 expression but promoted TNF-α-induced p53 acetylation and expression of PUMA or NOXA. Accordingly, TOPK inhibition attenuated TNF-α-mediated SIRT1 transcriptional activity. In addition, SIRT1 inhibition augmented acetylation of p53 or expression of PUMA and NOXA in response to TNF-α, leading to COV434 cell apoptosis. We conclude that TOPK suppresses TNF-α-induced COV434 granulosa cell apoptosis via regulation of p53/SIRT1 axis, suggesting a potential role of TOPK in regulation of ovarian follicular development.

Role of the SIRT1/p53 regulatory axis in oxidative stress­mediated granulosa cell apoptosis.

Oxidative stress has been suggested to induce granulosa cell apoptosis, which contributes to follicular atresia. However, the mechanism via which oxidative stress mediates granulosa cell apoptosis remains elusive. Therefore, the aim of this study was to elucidate the molecular mechanisms regulating oxidative stress­induced granulosa cell apoptosis. The present study demonstrated that reactive oxygen species induced by H2O2 resulted in human granulosa COV434 cell apoptosis via the regulation of sirtuin 1 (SIRT1)­mediated p53 activity. Endogenous SIRT1 expression was alleviated by H2O2 treatment of COV434 cells in a time­dependent manner. In addition, knockdown or inhibition of SIRT1 promoted H2O2­induced poly(ADP­ribose) polymerase (PARP) cleavage and p53 acetylation, which led to an increase in COV434 cell apoptosis. Treatment with H2O2 enhanced the expression levels of the p53­dependent proteins, p53­upregulated modulator of apoptosis (PUMA) and phorbol­12­myristate­13­acetate­induced protein 1 (PMAIP1), as well as those of p53; however, knockdown of p53 decreased cleaved PARP, PUMA and PMAIP1 expression levels induced by H2O2 treatment. Moreover, knockdown of PUMA or PMAIP1 attenuated the H2O2 induction of PARP cleavage and COV434 cell apoptosis. In conclusion, the present findings suggested that H2O2­induced oxidative stress causes granulosa COV434 cell apoptosis via the upregulation of p53 activity by SIRT1 suppression, indicating a mechanistic role of the SIRT1/p53 axis in H2O2­induced granulosa cell apoptosis.

TOPK inhibition accelerates oxidative stress­induced granulosa cell apoptosis via the p53/SIRT1 axis.

It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that T­lymphokine­activated killer cell­originated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stress­induced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa COV434 cell apoptosis induced by hydrogen peroxide (H2O2). Co­treatment with the TOPK inhibitor, OTS514, in combination with H2O2 increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H2O2­induced COV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrin­µ, diminished the augmentation in poly(ADP­ribose) polymerase (PARP) cleavage induced by OTS514 plus H2O2, while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H2O2, but promoted the H2O2­induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53­mediated granulosa cell apoptosis through SIRT1 downregulation in response to H2O2. Therefore, it can be concluded that TOPK suppresses H2O2­induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.

KLOTHO gene polymorphism is associated with coronary artery stenosis but not with coronary calcification in a Korean population.

Experimental studies have demonstrated KLOTHO gene polymorphism might be associated with vascular atherosclerosis and calcification. However, the impact of this genetic variant on human coronary arteries still remains to be elucidated. We investigated the effect of a KLOTHO gene variant on coronary artery stenosis and calcification. Four hundred and thirty-four patients referred for chest pain were enrolled. All the patients underwent coronary angiography and were investigated for polymorphism of the KLOTHO G395A gene. Coronary artery disease (CAD) was defined as > or = 50% diameter stenosis in at least one coronary artery. The other patients were considered to be controls. Homozygotes or heterozygotes for G395A were significantly more common in the CAD patients than in the controls (30.2% versus 21.5%, P = 0.039). In the subgroup aged < 60 years, the G395A mutant was more frequent in CAD than in control (35.3% versus 18.8%, P = 0.016), but in patients > or = 60 years, there was no difference (28.0% versus 24.1%, P = 0.473). Using multivariate analysis, we identified the KLOTHO gene G395A mutant as an independent risk factor of CAD (OR 1.712, 95% CI [1.066-2.749], P = 0.026). The frequency of the KLOTHO gene G395A mutant was not different between the calcified and noncalcified coronary artery groups (25.7%, 26.4%, respectively, P = 0.861) and an A allele carrier state was not an independent risk factor of coronary artery calcification. In conclusion, the KLOTHO gene G395A allele carrier state may be associated with CAD but not with coronary artery calcification in this Korean population.