PKC epsilon and an increase in intracellular calcium concentration are necessary for PGF2 alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

Abstract

The hypotheses that PKC epsilon is necessary for: 1) PGF2 alpha 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 PKC epsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKC epsilon 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 PGF2 alpha 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 PGF2 alpha 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.PKC epsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKC epsilon expression was ablated by 75%, the inhibitory effect of PGF2 alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKC epsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKC epsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2 alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 mumol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2 alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKC epsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2 alpha.

Figure 1

Time-course reduction in PKCε mRNA expression after transfection of luteal steroidogenic cells with PKCε specific siRNA. (A) Representative RT-PCR products obtained from total RNA using the PKCε and GAPDH primers. The amount of total RNA was adjusted to 200 ng per reaction and 40 cycles were used for PKCε ; while 28 cycles were used for GAPDH. The size of the amplified products for the GAPDH and PKCε were 900 and 500 bp, respectively. PKCε and GAPDH mRNA expression after 48, 72, and 96 h of transfection with PKCε specific siRNA are shown. Lanes labeled Media, non-specific (Non-sp) siRNA, and Transfection reagent represent respective treatments without PKCε specific siRNA treatment. GAPDH was used as the control gene to normalize the PKCε mRNA expression. (B) Quantitative analysis of the RT-PCR products obtained in four (n = 4) replicates similar to those shown in panel A. Data are the mean mean ± SEM of the densitometry measurements for PKCε relative to GAPDH mRNA. Statistical comparisons were made between different treatments. Different letters above each SEM represent different values (P < 0.05).

Figure 2

Reduction in PKCε protein. (A) Representative Western blot showing the amount of PKCε and actin expressed in protein samples prepared from luteal steroidogenic cells after 48, 72, and 96 h of transfection with PKCε specific siRNA (lanes 1–3). Lanes labeled 4 – 6, contained protein samples from indicated control treatments (media, Non-sp siRNA, and transfection reagent, respectively). (B) Semi-quantitative analysis of the densitometry derived from four experiments similar to the one shown in panel A. the y-axis shows the ratio of the optical density ratio of PKCε to that of its corresponding β-actin. The data are shown as mean ± SEM, and comparisons were made between different treatments. Values with different letters denote differences by one-way ANOVA followed by Tukey-Kramer honestly significant difference (P < 0.05).

Figure 3

PKCα and PKCβII protein after 96 h transfection of luteal steroidogenic cells with PKCε specific siRNA. (A) Representative Western blot showing the amount of PKCα, PKCβII KCε and actin detected in protein samples prepared from luteal steroidogenic cells after 96 h of transfection with PKCε specific siRNA (lane 1). Lanes labeled 2 – 4, contained protein samples from indicated control treatments (media, Non-sp siRNA, and transfection reagent, respectively. B and C) Semi-quantitative analysis of the densitometry derived from four experiments similar to the one shown in panel A for PKCα (B) and PKCβII (C). The y-axis shows the ratio of the optical density ratio of PKC isozyme to that of its corresponding β-actin. The data are shown as mean ± SEM, and comparisons were made between different treatments by one-way ANOVA followed by Tukey-Kramer honestly significant difference.

Figure 4

Effects of PKCε down-regulation on the ability of PGF₂α to inhibit the LH-stimulated progesterone synthesis/secretion in cultures of steroidogenic luteal cells transfected for 96 h with PKCε specific siRNA (filled bars) or with transfection regents (control, open bars). Progesterone accumulation was determined in culture media after 4 h of incubation in the following treatments: LH (100 ng/ml), PGF₂α (1 μg/ml) and a combination of PGF₂α and LH. Data are presented as mean ± SEM of four individual replicates (n = 4 cows). For each treatment group, statistical comparisons were made between PKCε down-regulated (PKCε siRNA) and control (not PKCε down-regulated); different letters above each SEM denote different values, P < 0.05.

Figure 5

Effect of the Ca²⁺ ionophore, 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. 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 PGF₂α (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 PGF₂α 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).

Figure 6

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. 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 PGF₂α (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 PGF₂α 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.