Cellular mechanisms of luteal regression in the bovine corpus luteum (CL)

The corpus luteum (CL) is a transient endocrine gland that produces progesterone (P4) for the establishment and maintenance of pregnancy. In absence of pregnancy, timely regression of the CL is essential for normal ovarian cyclicity. Several factors are known to participate in luteal regression. In this study, two factors, PGF2 alpha and endothelin (ET-1) are being studied. Protein kinase C (PKC) and calcium are the two main intracellular mediators of PGF2 alpha. The role of ET-1 in the regulation of luteal regression is unclear. The early CL is insensitive to the luteolytic actions of PGF2 alpha and the cellular mechanism(s) involved in this process are poorly understood. This study investigates: (1) the array of PKC isozyme expression as a function of development in the bovine CL and the ability of PGF2 alpha and ET-1 to activate the PKC isozymes in the early (day-4) and late (day-10) luteal phase, (2) the physiological role of the luteal PKC isozymes on PGF2 alpha-induced rise in intracellular calcium concentration and luteinizing hormone (LH) stimulated P4 accumulation at the mid luteal phase and (3) the cellular source of the luteal PKC isozymes. PKC alpha, beta I, beta II, epsilon and meu were observed to be expressed in the bovine CL with beta II and epsilon being differentially expressed as a function of development. In day-10 CL PGF2 alpha and ET-1 were able to activate PKC alpha, beta I and epsilon. More importantly, PKC epsilon was found to be involved in the regulation of PGF2 alpha induced rise in intracellular calcium concentration and antagonized the inhibitory effect of PGF2 alpha and ET-1 on LH-stimulated P4 accumulation in cultures of day-10 luteal steroidogenic cells (SC). PKC epsilon was found exclusively expressed in SC. In contrast, PKC alpha, beta I and beta II were expressed in both SC and endothelial cells (EC), with SC expressing higher amounts than EC. In this study we have proposed that the differential expression and activation of PKC epsilon as a function of development may be one of several factors responsible for the insensitivity of the early CL. Expression of PKC epsilon in the mid luteal phase shifts the PGF2 alpha induced rise in intracellular calcium concentration from a P4 favorable to a P4 inhibitory condition. Based on these observations it is hypothesized that the insensitivity of the early CL towards the luteolytic actions of PGF2 alpha may be due to differences in the intracellular mediators with respect to luteal development

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM

Just when you thought it was safe to go into the membrane: the growing complexities of extra-nuclear progesterone signaling

The diversity of membrane-initiated progesterone actions has made characterization and establishment of its biological importance a complicated endeavor. A new study by Zuo and colleagues shows that progesterone via endogenous membrane progesterone receptor-α acts as a negative regulator of proliferation and epithelial to mesenchymal transition in a breast cancer cell line. These progesterone-mediated actions appear to be regulated through epidermal growth factor receptor and phosphatidylinositol 3-kinase signaling localized in caveolae. Moreover, the study shows expression of membrane progesterone receptor-α in benign and malignant breast cancer tissues. These data bring forth novel concepts with regard to progesterone actions in the breast; however, further work is warranted to fully characterize the physiologic actions of extra-nuclear progesterone signaling in the breast

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour–like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug

Effect of the Caionophore, A23187, on basal and LH-stimulated progesterone synthesis/secretion (ng/ml) in cultured steroidogenic cells collected from Day 4 (panel A) and Day 10 (panel B) bovine CL

<p><b>Copyright information:</b></p><p>Taken from "PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells"</p><p>Reproductive biology and endocrinology : RB&E 2007;5():37-37.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2041951.</p><p></p> Progesterone accumulated in culture media was determined after 4 h of incubation in the following treatments: media alone (Media), LH (100 ng/ml), LH and PGFα (1000 ng/ml), or LH and A23187 (0.1, 1, 10, and 100 μmol). As explained in Materials and Methods, these treatments also contained 0.1% of the solvent used for PGFα and A23187, DMSO. Data are presented as the mean ± SEM of four Day 4 and 10 Day 10 individual replicates (n = 4 and 10 cows respectively). Statistical comparisons were made across treatments, and means with different letters, differ within each panel (P < 0.05)

Effect of the cell-permeable calcium chelator, Bapta-AM, on basal and LH-stimulated progesterone synthesis/secretion (ng/ml) in cultured steroidogenic cells collected Day 10 bovine CL

<p><b>Copyright information:</b></p><p>Taken from "PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells"</p><p>Reproductive biology and endocrinology : RB&E 2007;5():37-37.</p><p>Published online 30 Aug 2007</p><p>PMCID:PMC2041951.</p><p></p> Progesterone accumulated in culture media was determined after 4 h of incubation in the following treatments: media alone (Media), LH (100 ng/ml), LH and PGFα (1000 ng/ml), or LH and Bapta-AM (0.1, 1, 10, and 100 μmol). As explained in Materials and Methods, these treatments also contained 0.1% of the solvent used for PGFα and Bapta-AM, DMSO. Data are presented as the mean ± SEM of four Day 10 individual replicates (n = 4 CL obtained from 4 cows). Statistical comparisons were made across treatments, and means with different letters denote different values, P < 0.05

Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation

In prostate cancer, the signals that drive cell proliferation change as tumors progress from castration-sensitive (androgen-dominant) to castration-resistant states. While the mechanisms underlying this change remain uncertain, characterization of common signaling components that regulate both stages of prostate cancer proliferation is important for developing effective treatment strategies. Here, we demonstrate that paxillin, a known cytoplasmic adaptor protein, regulates both androgen- and EGF-induced nuclear signaling. We show that androgen and EGF promoted MAPK-dependent phosphorylation of paxillin, resulting in nuclear translocation of paxillin. We found nuclear paxillin could then associate with androgen-stimulated androgen receptor (AR). This complex bound AR-sensitive promoters, retaining AR within the nucleus and regulating AR-mediated transcription. Nuclear paxillin also complexed with ERK and ELK1, mediating c-FOS and cyclin D1 expression; this was followed by proliferation. Thus, paxillin is a liaison between extranuclear MAPK signaling and nuclear transcription in response to androgens and growth factors, making it a potential regulator of both castration-sensitive and castration-resistant prostate cancer. Accordingly, paxillin was required for normal growth of human prostate cancer cell xenografts, and its expression was elevated in human prostate cancer tissue microarrays. Paxillin is therefore a potential biomarker for prostate cancer proliferation and a possible therapeutic target for prostate cancer treatment