Activation of PERK following the accumulation of unfolded proteins in the endoplasmic reticulum (ER)promotes translation inhibition and cell cycle arrest. PERK function is essential for cell survival following exposure of cells to ER stress, but the mechanisms whereby PERK signaling promotes cell survival are not thoroughly understood. We have identified the Nrf2 transcription factor as a novel PERK substrate. In unstressed cells, Nrf2 is maintained in the cytoplasm via association with Keap1. PERK-dependent phosphorylation triggers dissociation of Nrf2/Keap1 complexes and inhibits reassociation of Nrf2/Keap1 complexes in vitro. Activation of PERK via agents that trigger the unfolded protein response is both necessary and sufficient for dissociation of cytoplasmic Nrf2/Keap1 and subsequent Nrf2 nuclear import. Finally, we demonstrate that cells harboring a targeted deletion of Nrf2 exhibit increased cell death relative to wild-type counterparts following exposure to ER stress. Our data demonstrate that Nrf2 is a critical effector of PERK-mediated cell survival.

In most mammals, prostaglandin F2alpha (PGF2alpha) is believed to be a trigger that induces the regression of the corpus luteum (CL), whereby progesterone synthesis is inhibited, the luteal structure involutes, and the reproductive cycle resumes. Studies have shown that the early growth response 1 (EGR1) protein can induce the expression of proapoptotic proteins, suggesting that EGR1 may play a role in luteal regression. Our hypothesis is that EGR1 mediates the actions of PGF2alpha by inducing the expression of TGF beta1 (TGFB1), a key tissue remodeling protein. The levels of EGR1 mRNA and protein were up-regulated in the bovine CL during PGF2alpha-induced luteolysis in vivo and in PGF2alpha-treated luteal cells in vitro. Using chemical and genetic approaches, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of EGR1 in PGF2alpha-treated cells. Treatment with PGF2alpha increased the expression of TGFB1 mRNA and protein as well as the binding of EGR1 protein to TGFB1 promoter in bovine luteal cells. The effect of PGF2alpha on TGFB1 expression was mimicked by a protein kinase C (PKC)/RAF/MEK1/ERK activator or adenoviral-mediated expression of EGR1. The stimulatory effect of PGF2alpha on TGFB1 mRNA and TGFB1 protein secretion was inhibited by blockade of MEK1/ERK signaling and by adenoviral-mediated expression of NAB2, an EGR1 binding protein that inhibits EGR1 transcriptional activity. Treatment of luteal cells with TGFB1 reduced progesterone secretion, implicating TGFB1 in luteal regression. These studies demonstrate that PGF2alpha stimulates the expression of EGR1 and TGFB1 in the CL. We suggest that EGR1 plays a role in the expression of genes whose cognate proteins coordinate luteal regression.

LH stimulates the production of cAMP in luteal cells, which leads to the production of progesterone, a hormone critical for the maintenance of pregnancy. The mammalian target of rapamycin (MTOR) signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. This study examined the actions of LH on the regulation and possible role of the MTOR signaling pathway in primary cultures of bovine corpus luteum cells. Herein, we demonstrate that activation of the LH receptor stimulates the phosphorylation of the MTOR substrates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1. The actions of LH were mimicked by forskolin and 8-bromo-cAMP. LH did not increase AKT or MAPK1/3 phosphorylation. Studies with pathway-specific inhibitors demonstrated that the MAPK kinase 1 (MAP2K1)/MAPK or phosphatidylinositol 3-kinase/AKT signaling pathways were not required for LH-stimulated MTOR/S6K1 activity. However, LH decreased the activity of glycogen synthase kinase 3Beta (GSK3B) and AMP-activated protein kinase (AMPK). The actions of LH on MTOR/S6K1 were mimicked by agents that modulated GSK3B and AMPK activity. The ability of LH to stimulate progesterone secretion was not prevented by rapamycin, a MTOR inhibitor. In contrast, activation of AMPK inhibited LH-stimulated MTOR/S6K1 signaling and progesterone secretion. In summary, the LH receptor stimulates a unique series of intracellular signals to activate MTOR/S6K1 signaling. Furthermore, LH-directed changes in AMPK and GSK3B phosphorylation appear to exert a greater impact on progesterone synthesis in the corpus luteum than rapamycin-sensitive MTOR-mediated events.

The corpus luteum (CL)2 is a transient endocrine gland derived from an ovulated follicle within the ovary. The steroidogenic cells of the CL produce progesterone, a prerequisite for normal maintenance of pregnancy in mammals. In the event of pregnancy, the CL retains its role in progesterone synthesis in support of early pregnancy. In the absence of pregnancy luteolysis or corpus luteum regression occurs, a physiologic process associated with a reduction in progesterone secretion (functional regression) followed by death of endothelial and steroidogenic cells (structural regression) (1-4). Prostaglandin F2␣ (PGF2␣) was identified as a luteolytic factor over 35 years ago (5, 6). Recent genetic studies in mice lacking the PGF2␣ receptor (FP) further highlight the role for PGF2␣ in CL regression. Mice lacking FP receptors experience defects in CL regression and consequently parturition does not occur because of the maintenance of progesterone secretion at the end of pregnancy (7).The FP receptor is a member of the large class of heterotrimeric G-protein-coupled receptors (GPCRs) (8) and is present on the surface of steroidogenic luteal cells. PGF2␣ binding to its G q -coupled receptor results in activation of phospholipase ␤ (PLC␤) and consequent generation of the second messengers; diacylglycerol and inositol trisphosphate (9). The resultant increase in protein kinase C (PKC) activity in luteal cells contributes to the activation of other downstream protein kinases. PGF2␣ stimulates the activity of the extracellular signal-regulated kinase (Erk) family of mitogen-activated protein kinases (MAPK) through a mechanism that involves the PKC-dependent phosphorylation and activation of Raf (10, 11). These events result in the induction of early response genes that code for transcription factors that in turn, alter the synthesis of proteins that regulate progesterone synthesis (12-14). Little is understood regarding other signaling mechanisms initiated by PGF2␣ that account for the actions of PGF2␣ in the CL.The mammalian target of rapamycin (mTOR) protein is a key regulator of protein translation via mechanisms involving the phosphorylation of the translation regulator eukaryotic initiation factor 4E (eIF4E) -binding protein (4EBP1) and the 70-kDa ribosomal protein S6 protein kinase 1 (S6K1) (15,16 receptor; GPCR, G-protein-coupled receptor; mTOR, mammalian target of rapamycin; m 7 G, 7-methyl-guanosine; PI3K, phosphatidylinositol-3-kinase; PGF2␣, prostaglandin F2␣; S6K1, p70 ribosomal S6 kinase; TSC2, tuberous sclerosis complex 2; FBS, fetal bovine serum; FCS, fetal calf serum; JNK, c-Jun N-terminal kinase; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C.

Little is known about the early intracellular events that contribute to corpus luteum regression. Experiments were designed to determine the effects of prostaglandin F2alpha (PGF2alpha) on phosphatidylinositol-3-kinase (PI3K)/Akt signaling in the corpus luteum in vivo and in vitro. Treatment of midluteal-phase cows with a luteolytic dose of PGF2alpha resulted in a rapid increase in ERK and mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K1) signaling and a rapid suppression of Akt phosphorylation in luteal tissue. In vitro treatment of primary cultures of luteal cells with PGF2alpha also resulted in an increase in ERK and mTOR/p70S6K1 signaling and a diminished capacity of IGF-I to stimulate PI3K, Akt, and protein kinase C zeta activation. Accounting for the reductions in PI3K and Akt activation observed in response to PGF2alpha treatment, we found that PGF2alpha promoted the phosphorylation of serine residues (307, 612, 636) in the insulin receptor substrate 1 (IRS1) peptide sequence in vivo and in vitro. Serine phosphorylation of IRS1 was associated with reduced formation of IGF-I-stimulated IRS1/PI3Kp85 complexes. Furthermore, treatment with inhibitors of the MAPK kinase 1/ERK or mTOR/p70S6K1 signaling pathways prevented PGF2alpha-induced serine phosphorylation of IRS1 and abrogated the inhibitory actions of PGF2alpha on Akt activation. Taken together, these experiments provide compelling evidence that PGF2alpha treatment stimulates IRS1 serine phosphorylation, which may contribute to a diminished capacity to respond to IGF-I. It seems likely that the rapid changes in phosphorylation events are among the early events that mediate PGF2alpha-induced corpus luteum regression.