Despite the wide distribution of Nonylphenol (NP) in the environment and its great hazard to the reproductive health of human and animals, the detailed mechanism of NP toxicity has not been fully elucidated. In the present study, we first demonstrate that NP treatment lead to increased apoptosis of primary cultured rat testicular Sertoli cells. To investigate the detailed mechanism, we find that NP causes disturbance of intracellular Ca2+ homeostasis and ultrastructure changes of endoplasmic reticulum (ER) in Sertoli cells. Furthermore, our results indicate that several gene and protein markers of ER stress are changed after NP exposure. These findings suggest that NP can cause ER stress in rat testicular Sertoli cells, which may underlie the NP-induced apoptosis to Sertoli cells.
NP as an important environment contaminant has been demonstrated to induce apoptosis in several cell types. It was reported by Kim et al. that NP was able to trigger apoptosis in human embryonic stem (hES) cells (Kim et al., 2006). Besides, it was also demonstrated that NP could induce thymocyte apoptosis (Yao et al., 2006). In this study, we identified that NP treatment also led to remarkably increased apoptosis in Sertoli cells. However, there was still no definite explanation about the underlying mechanism. Kim found that Fas/FasL system played vital role in NP induced hES cell apoptosis, while Yao reported that caspase-3 activation and mitochondrial depolarization were involved in NP induced thymocyte apoptosis (Kim et al., 2006; Yao et al., 2006).
Recently, several researchers have focused on ER-stress induced cell apoptosis (Chiang et al., 2005; Yeh et al., 2007). For example, it was suggested in Yeh’s study that genistein induced apoptosis in human hepatocellular carcinomas via ER-stress. Two distinct mechanisms are involved in the process: the accumulation of unfolded or misfolded protein and the Ca2+ signaling, which can interact with each other (Shen et al., 2004). In the present study, can also be a source for Ca2+ release into the cytosol in response to intracellular messengers. According to Hughes, Ruehlmann and Khan’s studies, NP-induced Ca2+ homeostasis might be attributed to the inhibiting effect of NP on endoplasmic reticulum Ca2+ pumps and channels (Khan et al., 2003; Ruehlmann et al., 1998), and our previous data also revealed that NP treatment led to the alterations of Ca2+-ATPase activity (not shown). On the other side, the normal function of the ER requires appropriate concentrations of free Ca2+ within the ER lumen, and disturbance of Ca2+ homeostasis will hamper the activation of Ca2+-dependent chaperones and impair protein folding (High et al., 2000), which will render accumulation of unfolded and misfolded protein in the ER and cause ER stress (Banhegyi et al., 2007). In the present study, swelling ERs accompanied by an enlargement of lumen were observed in NP-treated cells, indicating that NP disturbed the function of ER and caused its morphological changes.
Glucose regulated protein (GRP78) and protein disulfide isomerase (ERp57) are both ER resident proteins and playing pivotal functions in correct protein folding in the ER (Chichiarelli et al., 2007; Lee, 2005; Zapun et al., 1998). Besides, GRP78 is crucial in directing ER-stress signaling. In unstressed cells, GRP78 bind to the lumenal domains of several ER stress sensors such as IRE1, PERK and ATF6 (Bertolotti et al., 2000; Liu et al., 2000; Liu et al., 2002; Morris et al., 1997). As unfolded proteins accumulate in the ER lumen, GRP78 disassociates from these ER stress sensors to help protein folding. Consequently, released IRE1 and PERK undergo activation and result in the opening of ER-stress signaling pathway, which may in turn improve the expression of the target gene GRP78. In our study, we found that GRP78 mRNA expression was severely enhanced by 20 and 30 M NP treatment, suggesting the activation of ER-stress signaling pathway. The protein expression of GRP78 and ERp57 was different from the gene expression. They were both first elevated after short time treatment and then decreased, with the effect of 30 M NP more evident. In the Wang study of ER stress induced by thiamine deficiency, he also found that the GRP78 expression displayed a first elevation and the following decrease (Wang et al., 2007). We speculate that cells under ER stress will first increase GRP78 and ERp57 expression to help protein folding and prevent accumulation of misfolded proteins. However, prolonged ER stress will cause damage to the cell as well as the structure of ER, rendering the decreased GRP78 and ERp57 expression.
gadd153 is another specific marker of ER stress response. It is the first identified pro-apoptotic transcription factor during the activation of ER stress signaling pathway, which can forms stable heterodimers with C/ERP family members and controls expression of a set of stress-induced genes involving in apoptosis (Friedman, 1996; Zinszner et al., 1998). Here, we demonstrated that the gene expression of gadd153 was significantly increased both after 20 and 30 M NP treatments, another evidence of NP-induced ER stress in Sertoli cells. The protein expression of gadd153 exhibited a similar trend, i.e. the continuing increase following NP exposure, which was distinguished from that of GRP78 and ERp57. Furthermore, the result of immunofluorescent staining revealed weak gadd153 staining in control living cells but stronger immunoreactivity in NP-induced apoptotic cells, which suggested that expression of gadd153 was closely related to cell apoptosis.
Taken together, we drew the conclusion that NP could cause disturbance of Ca2+ homeostasis and the normal function of ER, rendering the prolonged activation of ER stress signaling pathway and finally leading to Sertoli cell apoptosis via increased gadd153 expression.
