Abstract
Apoptosis is the predominant process controlling cell deletion during post-lactational mammary gland remodeling. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism. Epidermal growth factor (EGF) belongs to a family of proteins that act as survival factors for mammary epithelial cells upon binding to specific membrane tyrosine kinase receptors. Expression of EGF peaks during lactation and dramatically decreases in the involuting mammary gland. Though it was suggested that the protective effect of EGF is mediated through the phosphatidylinositol-3-kinase (PI3K) or MEK/ERK kinases activities, little is known about the downstream mechanisms involved on the anti-apoptotic effect of EGF on mammary epithelial cells; particularly the identity of target genes controlling apoptosis. Here, we focused on the effect of EGF on the survival of mammary epithelial cells. We particularly aimed at the characterization of the signaling pathways that were triggered by this growth factor, impinge upon expression of Bcl-2 family members and therefore have an impact on the regulation of cell survival. We demonstrate that EGF provokes the induction of the anti-apoptotic isoform Bcl-X L and the phosphorylation and down-regulation of the pro-apoptotic protein Bad. The activation of JNK and PI3K/AKT signaling pathways promotes the induction of Bcl-X L while AKT activation also leads to Bad phosphorylation and down-regulation. This protective effect of EGF correlates mainly with the up-regulation of Bcl-X L than with the down-regulation of Bad. In fact, HC11 cells unable to express bcl-X, die even in the presence of EGF. In this context, Bcl-X L emerges as a key anti-apoptotic molecule critical for mediating EGF cell survival.
Highlights
Apoptosis is a tightly regulated and highly efficient process
The Epidermal growth factor (EGF) dependent activation profiles of the protein kinases ERK1/ 2, JNK, p38 MAPK and AKT was analyzed by Western blot of the phosphorylated forms on extracts obtained from HC11 confluent cells treated with EGF for 5 min to 4 h on serum free medium (SFM)
We observed that EGF inhibits the activation of caspase 3 provoked by serum depletion (Fig. 1D, columns 2 and 3). While this EGF effect was blocked by all the kinase inhibitors tested, PD, SP and LY, the caspase 3 activity turned out significantly high in the presence of these two last inhibitors which indicates a per se effect of these drugs. These results suggest that even though three kinase pathways are activated upon EGF addition, only JNK and PI3K/ AKT signaling are relevant on the EGF-dependent cell death protection
Summary
Apoptosis is a tightly regulated and highly efficient process. It occurs throughout the development and growth of the mammary gland, as was described by several authors. According to several lines of evidence it was suggested that members of the EGF family are survival factors for mammary epithelial cells [14,15,16] In this sense, it was demonstrated that mammary tumor cell lines established from mice over-expressing the c-myc oncogene, undergo apoptosis if cultured in the absence of EGF [15,16]. The role of the JNK pathway in controlling cell death or survival in the mammary gland is still controversial In this sense, Faraldo et al demonstrated that a low level or absence of proliferation and an induction of MEC apoptosis in involuting glands correlates with lack of JNK activation [28]. Our results together support the idea that while EGF triggers a variety of responses upon the Bcl-2 family of proteins in HC11 cells, induction of Bcl-XL emerges as a key event in mediating EGF dependent cell survival
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