Prostaglandin F2a activates stress response signaling and induces expression of activating transcription factor 3 (ATF3) in bovine large luteal cells

Authors: MAO, DAGAN - Nebraska Medical Center; ZHANG, PAN - Nebraska Medical Center; CORDES, CRYSTAL - Nebraska Medical Center; STEPHANY, MATHEW - Nebraska Medical Center; TALBOTT, HEATHER - Nebraska Medical Center; CUPP, ANDREA - University Of Nebraska; Cushman, Robert - Bob; HOU, XIAOYING - Nebraska Medical Center; DAVIS, JOHN - Nebraska Medical Center

Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/19/2012
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The pulsatile uterine secretion of prostaglandin F2 alpha (PGF) triggers the regression of the corpus luteum (CL). Recent studies have explored global changes in gene expression in response to PGF that may contribute to structural and functional regression of the CL. Activating transcription factor 3 (ATF3) is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. It is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family that represses transcription from promoters with ATF sites. It is believed that ATF3 may repress transcription by stabilizing the binding of inhibitory cofactors at the promoter. The present study investigated induction and possible role of ATF3 during CL regression. Postpubertal cattle of composite breeding were treated at midcycle with saline (n=3) or PGF (Lutalyse, n=12). Ovariectomies were performed after 0, 0.5, 1, 2, 4h and corpora luteal were processed for immunohistochemistry, protein or RNA isolation. Ovaries were also collected at a local slaughterhouse from first trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to isolate small luteal cells (SLC) and large luteal cells (LLC). Western blot analysis showed that PGF treatment in vivo increased activity of several members of the MAPK family including phosphorylated ERK, and the stress kinases JNK and p38 MAPK and their downstream targets JUN and HSP27. Real-time PCR analysis and in situ hybridization showed that ATF3 mRNA increased within 1h of PGF treatment in vivo. Western blot and immunohistochemistry confirmed elevation of ATF3 protein within 1h of PGF treatment in vivo; ATF3 was prominently expressed in the nuclei of LLC within 1h and was maintained for at least 4h. In vitro treatment with PGF stimulated concentration- and time-dependent increases in ERK, JNK and p38 MAPK phosphorylation. Western blot demonstrated that PGF treatment increased ATF3 expression in LLC. SLC were much less responsive to PGF compared to LLC. The cytokine tumor necrosis factor alpha (TNF) robustly increased JNK and p38 MAPK phosphorylation and ATF3 expression in luteal cells. In order to identify a role for ATF3 in the regressing CL, we transduced SLC and LLC with an adenovirus to express ATF3 and a control adenovirus expressing galactosidase GLB1. Expression of GLB1 had little effect of progesterone synthesis. Expression of ATF3 reduced basal and LH-stimulated progesterone production in LLC (28.1±8.2% and 42.6±12.0% inhibition, respectively; mean±sem), and in SLC (34.7±4.9% and 67.0±16.7% inhibition, respectively; mean±sem), but it did not change the expression of STAR, CYP11A1 and HSD3B proteins in 24h incubations. In conclusion, the action of PGF in LLC is associated with the rapid activation of stress activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during CL regression. Because ATF3 did not affect expression of key regulators of progesterone synthesis, it may increase cholesterol metabolism or export, thus diverting cholesterol from the steroidogenic pathway. Supported by the Department of Veterans Affairs, USDA NIFA-AFRI 2011-67015-20067 and the Olson Center for Women’s Health, UNMC.