Lactational exposure to CrVI during the postnatal days 1–21 decreased development of antral follicles and arrested follicular development at the secondary follicular stage in rat (Banu et al., 2008b
; Samuel et al., 2010
). The underlying molecular and cellular mechanisms that regulate CrVI-induced follicular atresia/apoptosis are not known. Results of the present study for the first time showed that CrVI induces apoptosis of granulosa cells through multiple mechanisms.
Bcl-2 family members Bcl-2, Bcl-XL, Bax and Bad proteins are the key mediators of intrinsic apoptotic pathway. In addition, HSP70 protects the cells against apoptosis by inhibiting translocation of BAX protein from the cytosol to the mitochondria, release of cytochrome c
from the mitochondria into the cytosol, and activation of caspase-3 and PARP proteins (Stankiewicz et al., 2005
; Bivik et al., 2007
; Joly et al., 2010
). HSP90 protein located in the mitochondria regulates mitochondrial membrane permeabilization and release of cytochrome c
(Kang et al., 2007
; Neckers et al., 2007
). Results of the present study indicated that CrVI decreased expression of antiapoptotic and cell survival proteins Bcl-2, Bcl-XL, HSP70 and HSP90 proteins, translocated BAX and BAD proteins from cytosol to the mitochondria, increased mitochondrial membrane permeability, facilitated the release of cytochrome c
, and activated caspase-3 and PARP proteins, and thus induced apoptosis of granulosa cells. These results suggest that CrVI attenuates antiapoptotic pathways in order to stabilize pro-apoptotic members to execute apoptosis of granulosa cells.
The fate of cells to die or survive depends on balance between survival and apoptosis signaling (Matsuzawa et al., 2002
). Further, expression of Bcl-2 and Bcl-XL proteins are regulated by MAPK, JNK and AKT pathways (Matsuzawa et al., 2002
). Therefore, we determined effects of CrVI on ERK1/2, AKT, p38MAPK, and JNK pathways in granulosa cells. Interestingly, CrVI inhibited phosphorylation of AKT proteins, and in contrast, increased phosphorylation of ERK1/2 and JNK proteins, and did not alter activation of p38MAPK protein. ERK1/2 pathways are mainly associated with mitogenesis and cell survival (Meloche and Pouyssegur, 2007
). Inactive ERKs are bound to anchoring proteins in resting cells, mostly confined to the cytosol. Upon phosphorylation, ERK becomes active, translocates to the nucleus, and activates transcription of several proteins (Lidke et al., 2010
). Interestingly, recent findings have documented a role for delayed and sustained ERK activation in apoptosis (Gladys et al., 1999
; Stanciu and DeFranco, 2002
). ERK can be activated often in the same cell type by pro-survival factors and toxic/apoptotic stimuli (Hetman et al., 1999
) and thus ERK activation alone may not be predictive of subsequent cellular survival responses (Stanciu et al., 2000
; Stanciu and DeFranco, 2002
). It has been shown that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax (Tsuruta et al., 2004
). It is evident from the present data that CrVI activates ERK1/2 temporally in a delayed and sustained manner and activates JNK in granulosa cells.
DNA damage promotes phosphorylation and subsequent stabilization of p53 and leads to apoptosis (Meek, 2009
). Phosphorylation of p53 protein at one serine residue is not sufficient to induce apoptosis, whereas phosphorylation at multiple serine residues is required (Kurihara et al., 2007
). Site specific phosphorylation of p53 is induced by activation of diverse cell signaling pathways and DNA damage (Kurihara et al., 2007
). Phosphorylation of p53 at ser-392 is required for p53-mediated growth arrest (Cox and Meek, 2010
). Phosphorylation of p53 at ser-15 can be induced by oxidative stress (Long et al., 2007
(Verschoor et al., 2010
), and ionization (Sluss et al., 2010
) and UV irradiation (Milne et al., 1995
). In addition, function of p53 is regulated by its negative regulator MDM2 (Shieh et al., 1997
). In order to understand the role of p53 in CrVI-induced apoptosis of granulosa cells we determined phosphorylation of p53 protein at multiple serine sites and expression of MDM2 protein. Our results indicate that CrVI increased phosphorylation of p53 protein at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392 and decreased expression of MDM-2 protein in granulosa cells in a time-dependent manner. These results suggest that CrVI increases p53 phosphorylation at multiple serine sites, decreases its interaction with its negative regulator MDM2 and thereby stabilizes p53 and promotes apoptosis of granulosa cells.
One of the interesting findings of the present study is that CrVI selectively translocated active p53 protein into mitochondria in granulosa cells. p53-mediated cell death is primarily routed through the mitochondrial pathways (Schuler and Green, 2001
) which require translocation of p53 protein into mitochondria (Zhao et al., 2005
). Recent studies showed translocation of p53 from the cytosol to mitochondria and its association with antioxidants and apoptotic proteins (Pani et al., 2004
; Siu et al., 2009
; Galluzzi et al., 2010
; Holley et al., 2010a
). Mitochondrial translocation of p53 triggers a rapid pro-apoptotic response (Erster and Moll, 2004
). After translocation into mitochondria, p53 protein could interact with endogenous antiapoptotic Bcl-XL and/or Bcl-2 protein, induce oligomerization of Bak protein, increase permeabilization of the outer mitochondrial membrane in order to facilitate cytochrome c
release (Mihara et al., 2003
), or interact with MnSOD and inhibit its ability to scavenge free radicals (Holley et al., 2010a
). The results of the present study along with available information suggest that p53 could play a central role in CrVI-induced apoptosis by inhibiting association or balance between pro-and anti-apoptotic proteins.
p53 regulates transcription of several genes (Meek, 1998a
) that regulate cell cycle, growth arrest, and apoptosis (Agarwal et al., 1998
; Giaccia and Kastan, 1998
). However, cell signaling associated with phosphorylation of p53 is complex and largely unknown. Our data indicated that CrVI activated ERK1/2 and JNK pathways. Therefore, we tested whether the inhibition of ERK1/2 or JNK decreases CrVI-induced p53 transcriptional activity and apoptosis. Our data showed that inhibition of ERK1/2 decreased CrVI-induced apoptosis of granulosa cells through suppression of transcriptional activity of p53. By contrast, inhibition of JNK did not decrease transcriptional activity of p53 although it decreased apoptosis of granulosa cells. These results indicate that ERK1/2 might be a potential upstream kinase that activates p53 and mediates CrVI-induced apoptosis of granulosa cells through p53.
ERK1/2 proteins are localized in several microenvironments of mitochondria and regulate survival or apoptosis of cells or modulate steroid synthesis (Dagda et al., 2008
; Poderoso et al., 2008
). Phosphorylation of p53 by ERK1/2 is important for doxorubicin-induced p53 activation and cell death (Yeh et al., 2004
). Therefore, we hypothesized that CrVI translocates active ERK1/2 proteins into mitochondria in addition to nucleus in granulosa cells. Our results indicated that CrVI translocated active ERK1/2 proteins not only into the nucleus but also to the mitochondria. The present study indicates that CrVI translocates active p53 protein into mitochondria. Based on these data, we propose that sustained activation of ERK1/2 by CrVI could phosphorylate p53, which in turn, interacts with other mitochondrial proteins of cell survival pathways and or antioxidants, and thus promotes apoptosis. In addition, CrVI translocates active ERK1/2 to the nucleus in granulosa cells and induces apoptosis. This finding is consistent with other evidence that prolonged nuclear retention of activated ERK promotes cell death (Stanciu et al., 2000
; Stanciu and DeFranco, 2002
). Moreover, association of ERK1/2 activation with granulosa cell apoptosis in the present study supports the recent finding that ERK1/2 is not essential for the active proliferation of granulosa cells from preovulatory follicles; rather ERK1/2 plays an essential role to cease granulosa cell proliferation and to initiate the terminal differentiation response to LH in preovulatory follicles (Fan et al., 2009
In the present study, vitamin C exhibited a selective and time-dependent molecular intervention of CrVI effects in several signaling pathways that lead to granulosa cell apoptosis. Vitamin C was more effective in mitigating CrVI effects at 12 h of treatment compared to 24 h in most of the end-points studied. This suggests that with short-time (12 h) CrVI exposure, the cells may still retain DNA repair machinery and operational survival signals. However, after 24 h of CrVI treatment, the DNA damage may have exceeded native DNA repair mechanisms so that vitamin C can not rescue granulosa cells from apoptosis. In conclusion, the novel findings of the present study are that CrVI: (i) decreased expression or activity of Bcl-2, Bcl-XL, and AKT proteins; (ii) increased activation and mitochondrial translocation of pro-apoptotic BAD, BAX, (iii) increased sustained activation of ERK1/2 and its sub-cellular translocation into nucleus and mitochondria; (iv) increased phosphorylation of p53 at multiple serine sites and thereby induced apoptosis of granulosa cells. Vitamin C partially mitigated the adverse effects of CrVI on granulosa cells; therefore, vitamin C could be a potential intervention to prevent or reduce the toxic effects of CrVI on the ovary to preserve the fertility.