Plasminogen activator inhibitor 1 RNA-binding protein interacts with progesterone receptor membrane component 1 to regulate progesterone's ability to maintain the viability of spontaneously immortalized granulosa cells and rat granulosa cells

Abstract

Progesterone receptor membrane component 1 (PGRMC1) mediates the antiapoptotic action of progesterone (P4). PGRMC1 interacts with plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), but the functional significance of this interaction is unknown. To examine the function of PGRMC1-PAIRBP1 interaction, PAIRBP1 was depleted from spontaneously immortalized granulosa cells (SIGCs) and the effects on the expression and localization of PGRMC1 as well as P4's ability to bind to SIGCs and prevent apoptosis was assessed. Depleting PAIRBP1 enhanced cellular (3)H-P4 binding and did not alter the expression or cellular localization of PGRMC1 but attenuated P4's antiapoptotic action. Transfection of a PGRMC1-green fluorescent protein (GFP) peptide mimic, which binds PAIRBP1 as demonstrated by in situ proximity assay, doubled the rate at which SIGCs undergo apoptosis compared to cells transfected with either the empty GFP expression vector or Pairbp1 small interfering RNA. Moreover, P4 did not prevent these cells from undergoing apoptosis. Similar studies conducted with granulosa cells isolated from immature rats also showed that PGRMC1 interacts with PAIRBP1 and that transfection of PGRMC1-GFP peptide mimic accelerates the rate of granulosa cell apoptosis by 4-fold even in the presence of serum and P4. These studies support the concept that the interaction between PAIRBP1-PGRMC1 is an essential component of the mechanism through which P4 inhibits apoptosis. Surprisingly, PGRMC1-PAIRBP1 interaction is not required for P4 binding or the cellular localization of PGRMC1 but rather appears to couple PGRMC1 to downstream components of the P4-PGRMC1 signal transduction pathway.

FIG. 1

Expression of PAIRBP1 and PGRMC1 in SIGCs cultured in serum-supplemented medium as assessed by Western blot (upper panel) and immunocytochemistry (lower panel). PAIRBP1 was detected as a single band of ≈55 kDa and localized to the cytoplasm. In contrast, PGRMC1 was detected as multiple bands and distributed throughout the cell. Negative controls for the Western blots were conducted by omitting the primary antibodies and are indicated with a minus sign. Negative controls were also conducted for the immunocytochemistry and did not show any staining (not shown).

FIG. 2

The effect of Pairbp1 siRNA treatment on the expression and localization of PAIRBP1 and PGRMC1 in SIGCs. Compared to scramble (control) siRNA treatment, Pairbp1 siRNA selectively depleted PAIRBP1 protein as assessed by immunocytochemistry (A) and mRNA as measured by real-time PCR (B) without altering Pgrmc1 mRNA or localization in SIGCs maintained in serum-supplemented medium. In C the effect of Pairbp1 siRNA on the ability of progesterone (P4) to suppress SIGC apoptosis induced by serum withdrawal is shown. In B and C, values are expressed as a mean ± one standard error, with * indicating a value that is significantly different from scramble control (P < 0.05). The capacity of SIGCs treated with either scramble or Pairbp1 (D) siRNA and maintained in serum-supplemented medium to specifically bind ³H-P4 is shown. Values represent the mean ± one standard error for each dose of ³H-P4. Data was fitted using a single-binding-site model and represented by either a solid (scramble control) or dashed (PAIRBP1 siRNA) line.

FIG. 3

PAIRBP1-PGRMC1 interaction in SIGCs maintained in serum (A) or cultured for 5 h in serum-free medium (B) or serum-free medium plus P4 (C). Negative control is shown in D. PAIRBP1-PGRMC1 interaction was determined by in situ PLA and revealed by a red dot.

FIG. 4

PAIRBP1-PGRMC1-GFP interaction in SIGCs maintained in serum as assessed by in situ PLA. In order to detect an interaction with PGRMC1-GFP, PGRMC1-GFP must be detectable both by its intrinsic GFP fluorescence and by an antibody to GFP. This is illustrated in A, in which PGRMC1-GFP-transfected SIGCs were fixed and stained for GFP using a GFP antibody and an Alexa Fluor 350-labeled anti-rabbit antibody. The same field of cells was observed under phase or fluorescent optics with filters to detect intrinsic GFP (green fluorescence) or immunodetectable GFP (blue fluorescence). Note that immunodetectable GFP is observed only in cells with intrinsic GFP fluorescence. In B in situ PLA detected PAIRBP1-PGRMC1-GFP interaction (red) only in SIGCs that were transfected with PGRMC1-GFP (green). Note that the transfected cells are marked by * in DAPI-stained preparation.

FIG. 5

The ability of the PGRMC1 peptide mimic (70-130-PGRMC1-GFP) to interact with endogenous PAIRBP1. In A the sequence of the PGRMC1 peptide mimic is shown, indicating that it comprises approximately half of the cytochrome b5 binding domain. B) Western blot indicating that the PGRMC1 peptide mimic (70–130) is expressed in SIGCs and detected as a single band that has a kDa, which is appropriately less than the full-length PGRMC1-GFP fusion protein (1–195). As seen in C the in situ PLA (red) detected the interaction between PAIRBP1 and the PGRMC1-GFP peptide mimic in those cells transfected with PGRMC1-GFP peptide mimic (green). The transfected cells are marked by * in DAPI-stained preparation. The effect of the PGRMC1 peptide mimic (70-130-PGRMC1-GFP) on the ability of P4 to inhibit apoptosis is shown in D. In this experiment cells were transfected with either the empty GFP vector or the PGRMC1-GFP peptide mimic and maintained in serum-supplemented medium for 24 h. After 24 h, the SIGCs were cultured for 5 h in serum-free medium in the presence or absence of P4 and then the transfected cells (green) assessed for apoptotic nuclei by DAPI staining (blue). Values are expressed as means ± one standard error, with * indicating a value that is less (P < 0.05) and ** indicating values greater than (P < 0.05) nontreated (∅) cells transfected with empty vector.

FIG. 6

The localization and interaction of PAIRBP1 and PGRMC1 in granulosa cells maintained in serum-supplemented medium. The localizations of PAIRBP1 shown in red (A) and PGRMC1 shown in green (B) were determined by immunocytochemistry, and the interaction between PAIRBP1 and PGRMC1 was revealed by PLA (red dots, C). In the PLA image, the nuclei were stained with DAPI (blue). The graph in D shows the effect of transfecting the PGRMC1 peptide mimic (70-130-PGRMC1-GFP) on the rate of apoptosis in granulosa cells cultured in the steroid-free serum with or without P4. In this graph, the values are expressed as means ± one standard error, with * indicating a value that is greater than (P < 0.05) granulosa cells transfected with empty vector.