No abstract

Reactive oxygen species (ROS) play important roles in follicular development and survival. Granulosa cell death is associated with increased ROS, but the mechanism of granulosa cell death induced by ROS is not clear. In order to define the molecular link between ROS and granulosa cell death, COV434, human granulosa tumor cells, were treated with H2O2. Compared to control cells, H2O2 induced granulosa cell death in a dose- and time-dependent manner. H2O2 induced an increase in Bax, Bak and Puma, and a decrease in anti-apoptotic molecules such as Bcl-2, Bcl-xL and Mcl-1. Both knockdown of Puma and overexpression of Bcl-xL could inhibit H2O2-induced granulosa cell death. These results suggest that suppression of Puma and overexpression of anti-apoptotic Bcl-2 family members could improve granulosa cell survival. To explore the mechanisms responsible for these findings, ROS in granulosa cells treatment with H2O2 were measured. The results showed that ROS was increased in a H2O2 dose- and time-dependent manner at the earlier time point. In addition, H2O2 induced an increase in Nrf2 and phosphorylation of JNK and p53. SP600125, an inhibitor of JNK, inhibits H2O2-induced phosphorylation of JNK and p53, and granulosa cell death. Antioxidant N-acetylcysteine (NAC) dose-dependently prevents H2O2-induced granulosa cell death. Furthermore, NAC also prevents phosphorylation of JNK and p53 induced by H2O2. Taken together, these data suggest that H2O2 regulates cell death in granulosa cells via the ROS-JNK-p53 pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be useful for future clinical applications.

The membrane-anchored metalloproteinase tumor necrosis factor-α α α α-converting enzyme (TACE/a disintegrin and metalloproteinase [ADAM] 17) is key in proteolytic ectodomain shedding of several membrane-bound growth factors, cytokines and receptors. The expression and activity of ADAM17 increases under some pathological conditions including stroke, and promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis. Hypoxia initiates cellular invasive processes that occur under both physiological and pathological conditions such as invasion and metastasis of some tumors. In the present study, we sought to elucidate whether ADAM17 contributes to brain tumor invasion. To this end, we examined the role of ADAM17 in the invasiveness of two different brain tumor cell lines, 9L rat gliosarcoma and U87 human glioma, under normoxic and hypoxic conditions. Additionally, we tested the effects of ADAM17 suppression on in vitro tumor cell invasion by means of ADAM17 proteolytic inhibitors and specific small interfering RNA. We found that tumor cells upregulated ADAM17 expression under hypoxia, and that ADAM17 activity correlated with increased tumor cell invasion. Conversely, suppression of ADAM17 proteolysis decreased invasiveness induced by hypoxia in 9L and U87 cells. Furthermore, the contribution of ADAM17 to tumor invasion was independent of matrix metalloproteinase (MMP)-2 and MMP-9 activity. ADAM17 was also found to activate the epidermal growth factor/phosphoinositide-3 kinase/serine/threonine kinase signal transduction pathway. Our data suggest that hypoxiainduced ADAM17 contributes to glioma cell invasiveness through activation of the EGFR signal pathway. (Cancer Sci 2007; 98: 674-684) T NF-α converting enzyme (TACE/ADAM17) is the primary secretase responsible for releasing the soluble form of TNF-α from the plasma membrane.(1) However, the expression of ADAM17 is ubiquitous, (2) and is not limited to TNF-α-producing cells. ADAM17 also plays a pivotal role in development through the processing of numerous growth factors and growth factor receptors.(3-6) A member of the ADAM family of transmembrane, multidomain zinc metalloproteinases, (2,7) ADAM17 has protease activity against multiple substrates including TNF-α, transforming growth factor-α, TNF receptor, interleukin-6, L-selectin and amyloid precusor protein. (3)(4)(5)(6)8) Owing to the physiological importance of ADAM17-mediated shedding of vital regulatory proteins, the mechanism of ADAM17 activation has been investigated. An increase in ADAM17 activity generally occurs when cells are exposed to cell activators such as phorbol esters, ionophores and growth factors.(9-11) The expression and activity of ADAM17 also increases under some pathological conditions including stroke, which promotes neuro-progenitor cell migration and contributes to stroke-induced neurogenesis. (12) ADAM17 is involved in the proteolysis of collagen IV of the ECM and, also in the cell surface release of several integral proteins, (1,13) suggesting that ADAM17 may...