Progesterone inhibits apoptosis in part by PGRMC1-regulated gene expression

Abstract

Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)'s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4's anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1's anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.

Figure 1

Detection of PGRMC1 (A) HnRNP A1 (B) and GAPDH (C) in lysates prepared from whole cells (WC), cytoplasmic (C) or nuclear (N) fractions. WC lysate was prepared in RIPA buffer, while the C and N fractions were prepared in hypotonic buffers supplemented with 0.1M DTT. NC indicates a negative control in which a Western blot was conducted in the absence of the primary antibody.

Figure 2

Detection of endogenous PGRMC1 and PGRMC1-GFP fusion protein by Western blots conducted on whole cell lysates prepared in RIPA buffer from cells either transfected with empty GFP vector (A) or a vector encoding PGRMC1-GFP (B,C). Each sample was probed with an antibody to either PGRMC1 or GFP. The Western blot in panel C is restricted to the 50 to 100 kDa range in order to more clearly reveal the 52 and 54 kDa bands. NC indicates a negative control in which a Western blot was conducted in the absence of the primary antibody.

Figure 3

Cellular localization of PGRMC1-GFP fusion protein as assessed by GFP-fluorescence or Western blot analysis. After PGRMC1-GFP transfection, some cells were fixed, counter stained with DAPI and examined under epi-fluorescence using the GFP and DAPI filter sets to reveal the localization of PGRMC1-GFP (A) or the nucleus (B). A merged image is shown in C. The remaining cells were lysed and separated into cytoplasmic (C) or nuclear (N) fractions. The Western blots were probed with an antibody to PGRMC1. The PGRMC1-GFP fusion protein is identified with an arrow. NC indicates a negative control in which a Western blot was conducted in the absence of the primary antibody.

Figure 4

The effect of P4 and PGRMC1-GFP on SIGC apoptosis. In panel A, the effect of increasing concentrations of P4 on the percentage of SIGCs that undergo apoptosis within 5 h in serum-free medium is shown. Panel B shows the effect of either the empty GFP vector or PGRMC1-GFP on the percentage of transfected SIGCs that undergo apoptosis in the presence or absence of 0.1 nM P4. In these graphs, an * indicates a value that is significantly different (p < 0.05) from control (no P4 group).

Figure 5

The effect of treatment with PGRMC1-GFP fusion protein and either scramble or PGRMC1 siRNA on the levels of endogenous PGRMC1 and PGRMC1-GFP fusion protein as assessed by immunocytochemistry. After treatment the cells were fixed, stained for PGRMC1 and counter stained with DAPI (1 μg/ml). In the upper panel, cells were transfected with PGRMC1-GFP and scramble siRNA. The same field of cells was observed for DAPI staining (Blue-total cells), GFP fluorescence (Green-presence of PGRMC1-fusion protein) or PGRMC1 using an Alexa 546-labeled secondary antibody (Red-both endogenous and PGRMC1-fusion protein). The images in the upper panel reveal that a select population of cells expressed PGRMC1-GFP but all cells expressed endogenous PGRMC1. The lower panel shows cells transfected with PGRMC1-GFP and PGRMC1 siRNA. In cells not successfully transfected with the PGRMC1-GFP expression vector (some of these cells are marked by arrows in the DAPI-stained image), endogenous PGRMC1 is not detected in either the cytoplasm or nucleus. PGRMC1 was only detected in PGRMC1-GFP transfected cells with most of the PGRMC1-GFP fusion protein localizing to the cytoplasm. A negative control was conducted for the immunocytochemical localization of PGRMC1 by omitting the primary antibody. The negative control did not show any staining.

Figure 6

The effect of PGRMC1 siRNA and PGRMC1-GFP expression on endogenous and PGRMC1-GFP mRNA levels as assessed by real-time PCR (A) and Western blot analysis (B). In panel A, values are shown as a mean of two and the diamond (◆) indicates the individual value of each replicate. Panel B shows a Western blot that used aliquots of whole cell lysates prepared from PGRMC1-GFP transected cells treated with either scramble or PGRMC1 siRNA. Panel B is divided into four panels. The two panels on the right were probed with the PGRMC1 antibody and represent the same blot exposed for 30 sec (upper panel) and 60 seconds (lower panel). The shorter exposure was required to clearly distinguish the 52 and 54 kDa PGRMC1 bands. The two panels on the left were probed with a GFP antibody. Panel C shows the effect of PGRMC1-GFP and either scramble or PGRMC1 siRNA on the percentage of transfected SIGCs that undergo apoptosis in the presence or absence of 0.1 nM P4. The * indicates a value that is significantly different (p < 0.05) from control (no P4 group).

Figure 7

The effect of actinomycin D (Act D; 4 μM) and P4 (1 μM) on SIGC apoptosis (A). In panel B, the effect of PGRMC1 siRNA and P4 on endogenous levels of PGRMC1 mRNA is shown. In panels A and B the * indicates a value that is significantly different (p < 0.05) from control (no P4 group). The effects of PGRMC1 siRNA treatment of the expression profile of various apoptosis-related genes is shown in panel C. The mRNA levels of both endogenous PGRMC1 and apoptosis-related genes were assessed by real-time PCR and shown as a percentage change from scramble control values. The values are means (± standard error) of three experiments. Genes associated with the induction of apoptosis are shown in red and the anti-apoptotic genes shown in green.

Figure 8

The effects of 1μM P4 plus scramble siRNA (A) or 1μM P4 plus PGRMC1 siRNA treatment (B) on the expression profile of various apoptosis-related genes. The mRNA levels of apoptosis-related genes were assessed by real-time PCR and shown as a percentage change from scramble siRNA control values. The values are means (± standard error) of three experiments. Genes associated with the induction of apoptosis are shown in red and the anti-apoptotic genes shown in green.