Parturition is an inflammatory process mediated to a significant extent by macrophages. Progesterone (P4) maintains uterine quiescence in pregnancy, and a proposed functional withdrawal of P4 classically regulated by nuclear progesterone receptors (nPRs) leads to labor. P4 can affect the functions of macrophages despite the reported lack of expression of nPRs in these immune cells. Therefore, in this study we investigated the effects of the activation of the putative membrane-associated PR on the function of macrophages (a key cell for parturition) and discuss the implications of these findings for pregnancy and parturition. In murine macrophage cells (RAW 264.7), activation of mPRs by P4 modified to be active only extracellularly by conjugation to BSA (P4BSA, 1.0×10−7 mol/l) caused a pro-inflammatory shift in the mRNA expression profile, with significant upregulation of the expression of cyclooxygenase 2 (COX2 (Ptgs2)), Il1B, and Tnf and downregulation of membrane progesterone receptor alpha (Paqr7) and oxytocin receptor (Oxtr). Pretreatment with PD98059, a MEK1/2 inhibitor, significantly reduced P4BSA-induced expression of mRNA of Il1B, Tnf, and Ptgs2. Inhibition of protein kinase A (PKA) by H89 blocked P4BSA-induced expression of Il1B and Tnf mRNA. P4BSA induced rapid phosphorylation of MEK1/2 and CREB (a downstream target of PKA). This phosphorylation was inhibited by pretreatment with PD98059 and H89, respectively, revealing that MEK1/2 and PKA are two of the components involved in mPR signaling. Taken together, these results indicate that changes in membrane progesterone receptor alpha expression and signaling in macrophages are associated with the inflammatory responses; and that these changes might contribute to the functional withdrawal of P4 related to labor.
Parturition is an inflammatory process observed at term and preterm. Evidence from human and animal studies has demonstrated that leukocytes infiltrate myometrium, cervix, and decidua during and before the process of labor, playing a critical role in parturition (Mackler et al. 1999, Thomson et al. 1999, Hamilton et al. 2012, Care et al. 2013, Shynlova et al. 2013). In mice, numbers of uterine macrophages increase during the period of early pregnancy and then decline near term (Mackler et al. 1999). In contrast, numbers of macrophages in the cervix increase at term and peak on the day before delivery. Therefore, it has been suggested that macrophage trafficking between uterus and cervix, and associated cytokine production, contribute to the termination of pregnancy (Mackler et al. 1999). Decidual macrophage infiltration has also been shown in both term labor and idiopathic preterm labor in humans and rats before parturition, indicating an initiating role of inflammatory events in labor (Hamilton et al. 2012). Taken together, the results of these studies highlight the importance of inflammatory cell infiltration into reproductive tissues as a physiological mechanism regulating pregnancy maintenance and parturition.
17-Hydroxyprogesterone caproate injections have been shown to prevent preterm delivery in high-risk women (Meis et al. 2003). Progesterone (P4) maintains pregnancy by promoting uterine quiescence until parturition is initiated by certain forms of withdrawal of this ‘P4 block’ (Csapo 1956). In humans, maternal levels of circulating P4 do not change during spontaneous labor or in the weeks preceding labor (Pieber et al. 2001). Therefore, alternative mechanisms of functional P4 withdrawal have been proposed (Zakar & Hertelendy 2007, Mesiano et al. 2011). P4 exerts its actions through the classical intracellular nuclear progesterone receptors (nPRs; Mulac-Jericevic et al. 2000, Conneely et al. 2003, Merlino et al. 2007), leading to the translocation of hormone-receptor complexes into the nucleus, where they bind to hormone-responsive elements of DNA to regulate gene transcription (Webster et al. 2002). However, some of the effects of P4 are not related to its transcriptional activity (Gellersen et al. 2009). In 2003, putative mPR receptors (mPRα, β, and γ) were cloned, shedding new light on PR (Zhu et al. 2003).
P4 elicits a variety of functional effects on immune cell types, including dendritic cells (DCs), monocytes, lymphocytes, and macrophages. P4 shifts the proinflammatory activity of DCs toward a more tolerogenic state (Kammerer et al. 2000, Liang et al. 2006) and promotes a T helper 2 (Th2)-biased profile that is a prerequisite for fetal survival and the maintenance of pregnancy (Piccinni et al. 1995, Raghupathy 1997, Szekeres-Bartho et al. 2009, Sykes et al. 2012). Functional P4 withdrawal may contribute to the switch from a Th2- to a Th1-dominant phenotype via the actions of P4-induced blocking factor in lymphocytes toward the end of pregnancy (Szekeres-Bartho & Chaouat 1990, Szekeres-Bartho et al. 1990, Druckmann & Druckmann 2005, Raghupathy et al. 2009). Interestingly, classical nPRs are undetectable or expressed at very low levels in these immune cells. For instance, results from several studies have demonstrated the absence of nPRs in peripheral blood leukocytes, T lymphocytes, immortalized T cells (Jurkat cells), and the murine RAW 264.7 macrophage cell line, as well as in mouse bone marrow-derived macrophages (Mansour et al. 1994, Mulac-Jericevic et al. 2000, Merlino et al. 2007, Dosiou et al. 2008, Ndiaye et al. 2012). Since then, results from several studies have provided evidence for the activation of mPRs in reproductive tissues and immune cells, and indicated that these mPRs act as G-protein-coupled receptors (GPCRs) in fish oocytes (Zhu et al. 2003), human myocytes (Karteris et al. 2006), human T lymphocytes, and Jurkat T cells (Dosiou et al. 2008, Ndiaye et al. 2012). As P4 elicits a variety of functional effects on immune cell types, even in those lacking nPRs (Dressing et al. 2011), the functions of P4-mediated mPR activation and signaling are of great interest. As mPRs are putative GPCRs (Dosiou & Giudice 2005, Karteris et al. 2006, Thomas et al. 2007, Dosiou et al. 2008, Dressing et al. 2011) and activation of GPCRs leads to downstream activation of cAMP-dependent protein kinase A (PKA), MAPK kinase (MEK), and phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways, we investigated the potential involvement of mPR, PKA, MEK1/2, and PI3K/AKT in murine macrophage responses to P4.
Materials and methods
Reagents and antibodies
P4 3-(O-carboxymethyl)oxime: BSA–FITC conjugate (P4BSA; a cell-impermeable form of P4; Gaetjens & Pertschuk 1980), lipopolysaccride (LPS, cat #L2262, a MEK1/2 activation positive control), forskolin (cat #F3917, a PKA activation positive control), and dihydrochloride hydrate (H89; cat #B1427, a PKA inhibitor) were purchased from Sigma Chemical Co. PD98059 (cat #9900, a MEK1/2 inhibitor) and LY294002 (cat #99901, a PI3K inhibitor) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti-IL1B (cat #ab9722) was from Abcam (Cambridge, MA, USA). Antibodies to GAPDH (cat #5174), CREB (cat #9197), phospho-CREB (cat #9198), MEK (cat #9126), phospho-MEK (cat #9154), p38 (cat #9212), and phospho-p38 (cat #9211) were from Cell Signaling Technology. Antibodies to ERK (cat #sc-135900), phospho-ERK (cat #sc-7383), and HRP-conjugated anti-rabbit (cat #sc-2030) and anti-mouse (cat #sc-2031) secondary antibodies were from Santa Cruz Biotechnology.
Mouse RAW 267.4 macrophage cells from the American Type Culture Collection (ATCC, Rockville, MD, USA) were maintained from passages 5 to 25 in DMEM (Life Technologies) supplemented with 10% FBS, 1% l-glutamine, 1% penicillin, and streptomycin in a humidified incubator with 5% CO2 at 37 °C to 80–90% confluence. For treatment, RAW 264.7 cells were cultured in duplicate in 12-well plates (for RNA and signal transduction experiments) or 100 mm Petri-dishes for protein extraction for 24 h before being subjected to the serum-free medium with 1% l-glutamine and 1% penicillin and streptomycin overnight. The cells were then pre-treated with either a PKA inhibitor H89 (3.0×10−5 mol/l), a MEK1/2 inhibitor PD98059 (2.0×10−5 mol/l), or a PI3K/AKT inhibitor LY294002 (1.0×10−5 mol/l) for 1 h in a fresh serum-free medium followed by incubation with control (medium or medium plus vehicle) or P4BSA (1×10−7 mol/l) for 2, 15, or 30 min for signal transduction experiments and 4 h for RNA and protein isolation.
Total RNA was extracted using TRIzol Reagent (Life Technologies). RNA concentrations were measured using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) and 500 ng of RNA from each sample was used to generate cDNA using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA). mRNA expression was determined by semiquantitative real-time PCR (RT-PCR) using an ABI StepOnePlus RT-PCR instrument (Applied Biosystems). The reactions were performed in duplicate in 10 μl volumes using 1 μl diluted cDNA (5×) and 9 μl of a mixture of TaqMan Universal Master Mix Reagents (Roche) and TaqMan Gene Expression Assay for mPRα (Paqr7, Mm00510958_m1), tumor necrosis factor (TNF (Tnf), Mm00443260_g1), interleukin 1B (IL1B (Il1B), Mm00434228_m1), cyclooxygenase 2 (COX2 (Ptgs2), Mm00478374_m1), iNOS (Nos2, Mm00440502_m1), oxytocin receptor (Oxtr, Mm01182684_m1), Creb3 (Mm00457268_m1), and steroid receptor coactivator 2 (SRC2 (Ncoa2), Mm00500749_m1) (Life Technologies). The cycling conditions were 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The mRNA level of each gene of interest was normalized against GAPDH (Gapdh, Mm99999915_g1), as the levels of the reference gene (Gapdh) did not differ among treatment groups in our study (data not shown). Gene expression is presented as fold change relative to respective controls and is plotted on a logarithmic scale with base 2.
To extract total cell lysates for detection of protein expression, at the indicated time points cells were washed with 1× ice-cold PBS twice in dishes and scraped into conical tubes and again washed with PBS. The cells were then solubilized in cold lysis buffer, containing 0.02 mol/l HEPES at pH 7.4, 0.15 mol/l NaCl, 1.0×10−9 mol/l EDTA, 1% Nonident P-40 (IGEPAL-CA-630) supplemented with cOmplete ULTRA Protease Inhibitors (Roche Applied Science) on ice for 20 min. The supernatants were collected after centrifugation at 12 000 g for 10 min at 4 °C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) and a total of 30 μg of protein lysate was subjected to electrophoresis. For signal transduction studies, cells were washed with 1× PBS twice and then immediately lysed by adding 200 μl of 2× SDS sample buffer with 10% 2-mercaptoethanoland kept on ice for 10 min. The suspension was sonicated for 20 s to shear DNA and to reduce the sample viscosity. The samples were heated at 95–100 °C for 5 min. After being cooled on ice for 2 min, the samples were centrifuged for 2 min at 12 000 g before 10 μl of supernatant was loaded into each well of 4–12% precast SDS–PAGE gels (Life Technologies) and transferred to PVDF membranes using a semi-dry transfer system (Bio-Rad). The membranes were blocked with 5% nonfat milk (NFM, Bio-Rad) in Tris-buffered saline (TBS, 0.02 mol/l Tris–HCl, 0.137 mol/l NaCl, pH=7.5) with 0.1% Tween-20 (TBST) for an hour on a shaker at room temperature and then probed with appropriate primary antibodies in 5% NFM in TBST overnight at 4 °C. The membranes were washed four times for 10 min each time with TBST and then incubated with secondary antibodies for 1 h at room temperature. The chemiluminescent signal was detected using ECL Plus from Amersham (GE Healthcare Life Science, Piscataway, NJ, USA) and captured using a STORM phosphor imager (Molecular Dynamics, Piscataway, NJ, USA). The density of each band was quantified with ImageJ (NIH, Bethesda, MD, USA) and normalized to GAPDH or the respective total protein and presented as fold change relative to the control.
Kruskal–Wallis ANOVA was used to test overall heterogeneity and differences among groups. If significant differences were identified, post hoc tests were performed by multiple comparisons of means, allowing for nonnormality in the data. Adjusted P values were computed using a bootstrap re-sampling method with step-down tests. Statistical analysis was performed on the SAS 9.3 (Cary, NC, USA) platform, and P values <0.05 were considered statistically significant.
RAW 264.7 cells express mPRα but do not express nPRs
To verify reports that RAW 264.7 cells lack classical nPRs, protein expression of PR-A and PR-B, the two isoforms of PR, was evaluated by western blotting. As shown in Fig. 1, total cell extracts were used to detect protein levels of PRs in several cell lines. While MCF7 (a human breast adenocarcinoma cell line) and T47D (a human ductal breast epithelial tumor cell line) cells are known to highly express nPRs (Horwitz et al. 1982, Cho et al. 1994), MDA-MB-231 (a human breast adenocarcinoma cell line) cells are known not to express nPRs (Dressing et al. 2011). We detected PR-A and PR-B protein in both MCF7 and T47D cells, but neither was present in MDA-MB-231 or RAW 264.7 cells, confirming the previous reports that RAW 264.7 cells lack nPRs.
Although we were able to detect the expression of Paqr7 mRNA in RAW 264.7 cells (see data below), attempts to detect mPRα protein by western blotting were not fully confirmed, probably due to the nonspecificity of commercially available antibodies (see Supplementary Fig. 1, see section on supplementary data given at the end of this article). Other groups, using a custom-made mPRα antibody generated by Dr Peter Thomas at University of Texas (Thomas 2008), have demonstrated that RAW 264.7 cells express mPRα protein (Dressing et al. 2011).
P4BSA-induced mPR activation induces MEK-dependent increases in Il1B and Ptgs2 mRNA expression
To study the functions of membrane-bound PRs specifically, we used P4BSA, which is cell-impermeable. Cellular responses to P4BSA were assessed, along with whether these effects are dependent on MEK1/2, signaling components of GPCR pathways. RAW 264.7 murine macrophages were pretreated without or with 2.0×10−5 mol/l PD98059 (a specific inhibitor of MEK1/2) for 1 h and then incubated with P4BSA (1.0×10−7 mol/l) for 4 h. We found that stimulation of mPRs with P4BSA resulted in significant increases of Il1B, Tnf, Ptgs2, and Nos2 mRNA transcripts (expressed as fold change relative to untreated controls; Fig. 2a, b, c and d), indicating a pro-inflammatory role for P4 in RAW 264.7 macrophage cells. PD98059 alone did not change the expression of Il1B, Ptgs2, and Nos2 mRNA (Fig. 2a, c and d), but significantly reduced the basal expression of Tnf (Fig. 2b). Pre-incubation with PD98059 followed by stimulation with P4BSA significantly reduced P4BSA-induced Il1B, Tnf, and Ptgs2 mRNA expression (Fig. 2a, b and c), but not Nos2 mRNA expression (Fig. 2d). These results indicate that MEK1/2 activity contributes to P4BSA-induced expression of Il1B and Ptgs2 and is partially responsible for P4BSA-induced Tnf expression.
P4BSA-induced upregulation of Il1B and Tnf, but not Ptgs2 or Nos2, is PKA-dependent
As PKA is one of the main kinases in the cAMP-related signal transduction pathway upon GPCR activation, we evaluated the potential involvement of PKA in P4BSA-induced mPR activation. RAW 264.7 cells were pretreated without or with 3.0×10−5 mol/l H89 (a PKA inhibitor) for 1 h and then stimulated with P4BSA (1.0×10−7 mol/l) for 4 h. Inhibition of the activity of PKA by H89 completely eliminated the P4BSA-induced increase in Il1B mRNA expression (Fig. 2e). P4BSA-induced Tnf mRNA was significantly reduced, but not completely blocked, by H89 treatment (Fig. 2f). In contrast, inhibition of PKA had no effect on P4BSA-induced Ptgs2 or Nos2 expression (Fig. 2g and h), although it increased baseline expression of these mRNAs. These results indicate that PKA is an important regulator of mPR-mediated Il1B and Tnf and baseline Ptgs2 and Nos2 expression.
P4BSA-induced upregulation of Il1B, Tnf, and Ptgs2 mRNA is not dependent on PI3K/AKT signaling
RAW 264.7 cells were pretreated without or with 1.0×10−5 mol/l LY294002, a specific inhibitor of PI3K/AKT, for 1 h followed by stimulation with P4BSA (1.0×10−7 mol/l) for 4 h. Pharmacological blockade of the PI3K/AKT pathway did not normalize P4BSA-induced Il1B, Tnf, and Ptgs2 mRNA expression (Fig. 2i, j and k), indicating that PI3K/AKT signaling does not mediate the mPR-induced upregulation of these pro-inflammatory mediators.
P4BSA-induced IL1B protein expression is dependent on PKA and MEK1/2
To further characterize the involvement of PKA and MEK1/2 in the P4BSA-induced Il1B upregulation seen in RAW 264.7 cells, IL1B protein expression was measured by western blotting analysis using whole-cell extracts after treatments. P4BSA administration at 1.0×10−7 mol/l for 4 h caused a significant increase in IL1β protein expression (Fig. 3a, b and c); Fig. 3a is a representative western blot, and Fig. 3b and c contain the relevant densitometric analyses (n=4). Pretreatment with either 3.0×10−5 mol/l H89 or 2.0×10−5 mol/l PD98059 for 1 h before P4BSA treatment significantly diminished P4BSA-induced IL1B protein expression, confirming the involvement of PKA and MEK1/2 in this pathway.
Potential downstream targets of MEK1/2 involved in mPR signaling
As MEK1/2 are activated by serine phosphorylation and, in turn, phosphorylate downstream kinases to initiate the signal cascade (Wortzel & Seger 2011), we examined the effects of MEK1/2 inhibition on P4BSA-induced phosphorylation of MEK1/2, ERK1/2, and p38. Figures 4a, 5a and 6a are representative blots demonstrating that stimulation with 1.0×10−7 mol/l P4BSA for 2 min significantly increases phosphorylation of MEK1/2 at Ser217/221, but not ERK1/2 at Tyr202/204 or p38 at Tyr180/182. Two minutes of stimulation with 10 ng/ml LPS induced phosphorylation of MEK, but not ERK1/2 or p38 phosphorylation. Stimulation with P4BSA for 15 min did not increase the phosphorylation levels of either MEK1/2 at Ser217/221 (Fig. 4c), ERK1/2 (Fig. 5c) at Tyr202/204 or p38 at Tyr180/182 (Fig. 6c). The observations after 15 min treatment were similar to those after 30 min (data not shown). At all three time points P4BSA treatment also did not alter MEK1/2, ERK1/2, or p38 total protein levels. These experiments were repeated five to ten times and the results of optical densitometry analyses are summarized in Figs 4c, d, 5c, d, and 6c, d. Pretreatment with the MEK1/2 inhibitor 2.0×10−5 mol/l PD98059 significantly suppressed basal phosphorylation of MEK (Fig. 4a, b, c and d) and ERK1/2 (Fig. 5a, b, c and d), but it had no effect on p38 (Fig. 6a, b, c and d) phosphorylation in the absence of a stimulus. The increase in phosphorylation of MEK1/2 at Ser217/221 after treatment with P4BSA for 2 min was prevented by pretreatment with PD98059 (Fig. 4a and b). These data together with results displayed in Figs 2 and 3 indicated that P4BSA-induced phosphorylation of MEK1/2 is involved in P4BSA-induced inflammatory responses.
Potential targets of PKA in the mPR-activated pathway
As PKA catalytic subunit alpha (Cα) phosphorylates CREB on Ser133 (Gonzalez & Montminy 1989) and CREB is an essential component of the cAMP signaling pathway in the regulation of Il1B and Ptgs2 transcription (Chandra et al. 1995, Ghosh et al. 2007), we tested whether the inhibition of PKA affects phosphorylation of CREB in macrophages stimulated with P4BSA. RAW 264.7 cells were pretreated with 3.0×10−5 mol/l H89 for 1 h before treatment with 1.0×10−7 mol/l P4BSA for 2, 15, or 30 min. The cell lysates were collected and subjected to western blotting to determine the phosphorylation levels of CREB at Ser133 using total CREB as a loading control. P4BSA significantly increased phosphorylation of CREB at Ser133 after stimulation for 2 min (Fig. 7a and b), but not after stimulation for 15 min (Fig. 7c and d). Inhibition of PKA by H89 did not affect phosphorylation of CREB at any time point. The rapid and transient P4BSA-induced CREB phosphorylation was blocked by pretreatment with H89 (Fig. 7a and b). The observations after 15 min of treatment were similar to those after 30 min (data not shown). These results indicate that the signaling of PKA through CREB might be responsible for the effects of P4BSA on the gene expression of the targets studied.
P4BSA did not affect the mRNA levels of the transcription factors Creb3 and Ncoa2
As studies have demonstrated the involvement of CREB in the regulation of transcription of Il1B and Ptgs2 (Chandra et al. 1995, Ghosh et al. 2007) and downregulation of SRC2 (Ncoa2) has been implicated in the effects of mPRs activation in human myometrial cells (Karteris et al. 2006), we determined the effects of activation of mPR via P4BSA treatment on expression of Creb3 and Ncoa2 in RAW 264.7 cells. As shown in Fig. 8a and c, pretreatment with 2.0×10−5 mol/l PD98059 did not affect the basal levels of Creb3 and Ncoa2 mRNA expression. Subsequent stimulation with 1.0×10−7 mol/l P4BSA also did not affect the transcriptional levels of both genes. Pretreatment with 3.0×10−5 mol/l H89 did not change the expression levels of Creb3 (Fig. 8b), but did significantly decrease the mRNA level of Ncoa2 (Fig. 8d). There was no additional downregulation of Ncoa2 transcription when PKA inhibition was followed by P4BSA stimulation (Fig. 8d). These results indicate that H89 regulates basal mRNA level of Ncoa2.
P4BSA downregulates mPRa (Paqr7) and Oxtr mRNA expression in RAW 264.7 cells
As ligand-induced downregulation of receptor mRNA levels is a mechanism for diminishing receptor signaling (Hadcock & Malbon 1988), and OXTR is reportedly downregulated by P4 through miR-200 (Renthal et al. 2010), we explored the mechanisms through which P4 might regulate cellular responses in murine macrophages. Expression of Paqr7 and Oxtr was evaluated by RT-PCR after the administration of the treatments described earlier. We observed that P4BSA treatment significantly reduced the mRNA expression levels of Paqr7 (Fig. 8e and f) and Oxtr (Fig. 8g and h). Interestingly, MEK1/2 inhibition (Fig. 8e) did not affect either baseline Paqr7 or P4BSA-induced downregulation of Paqr7, while PKA blockade (Fig. 8f) resulted in significant downregulation of Paqr7 expression at baseline. Pretreatment with H89 did not affect P4BSA's ability to downregulate Paqr7. In comparison, inhibition of either MEK or PKA alone reduced the baseline mRNA levels of Oxtr (Fig. 8g and h). Oxtr downregulation by P4BSA stimulation was not affected by pretreatment with the MEK inhibitor or PKA inhibitor. These results indicate that P4BSA induced downregulation of expression of both Paqr7 and Oxtr. PKA regulates steady-state expression of Paqr7 whereas both PKA and MEK regulate steady-state Oxtr expression in murine macrophages.
During pregnancy, decidual macrophages exhibit an immunosuppressive phenotype that is required for maintaining immunological homeostasis and supporting immune tolerance of the fetus. As the placenta develops, macrophages are recruited around spiral arteries to support vascular remodeling by producing pro-angiogenic factors. At the end of the pregnancy, classically activated macrophages engage in the cervical remodeling process toward the onset of labor (Lee et al. 2012). Therefore, macrophages, among other immune cells, are pivotal for the maintenance of pregnancy and initiation of labor. P4 has both immunosuppressive and immunostimulatory effects on macrophages (Miller & Hunt 1996), demonstrating the plasticity and versatility of these cells, depending on the biological environment. Despite the reported various effects of P4 on macrophages, most studies have failed to detect the expression of the so-called classical nPRs in macrophages (Miller & Hunt 1996, Dressing et al. 2011). On the other hand, recent advances in this area have revealed the existence of mPRs in macrophages (Dressing et al. 2011). This indicates that they might be responsible for P4's action in these immune cells.
In this study, we demonstrate that activation of a putative family of mPRs by a cell-impermeable form of P4 resulted in a pro-inflammatory profile in murine macrophages. This cell-impermeable form of P4 induced robust increases in the expression of mRNA for pro-inflammatory markers such as Il1B, Tnf, Ptgs2, and Nos2. Both PKA and MEK1/2 are involved in the regulation of the mRNA and protein expression of Il1B. MEK1/2 also regulates transcription of Tnf and Ptgs2. P4BSA-stimulated production of Tnf mRNA is also mediated by PKA. Furthermore, P4 regulates mRNA expression of one of its own receptors, Paqr7, and of Oxtr, thus providing potential mechanisms by which P4 affects cellular events in macrophages.
The roles of membrane PRs in pregnancy and labor are diverse and complex. These roles are almost certainly tissue-, cell type-, and gestational age-specific. Pro-inflammatory actions of P4 have been shown in other cell types (i.e. other than the macrophages we studied). For example, exposure to P4 activates a wide array of genes involved in several biological processes, including cell adhesion, cell survival, and inflammation, in the mammary gland (Santos et al. 2009). Feng et al. (2014) demonstrated diminished progesterone receptor membrane component 1 (PGRMC1) at the rupture site among preterm prelabor rupture of membranes (PPROM) subjects compared with preterm nonlabor or term-nonlabor subjects. They deduced from the data that PGRMC1 functions to maintain fetal membrane integrity on the basis of results from the previous studies, which indicated that nPR expression is negative in the chorion and amnion. Our results were in agreement with the results from previous studies (Mackler et al. 1999, Thomson et al. 1999, Shynlova et al. 2013), which demonstrate that infiltration of macrophages to decidua and cervix and the inflammatory responses associated with the infiltration might be the keys for the initiation of labor. The uterus also undergoes a switch from quiescence to contraction near term due to a proposed functional withdrawal of P4 (Tan et al. 2012). In this model, classical anti-inflammatory nPR-B is downregulated and PR-A (pro-inflammatory) is increased at the onset of labor. The net results of this altered expression of PR-A/PR-B in the uterus are thought to increase the expression of pro-inflammatory factors and contraction-associated proteins to promote labor. Therefore our results on macrophages stimulated with P4BSA are in agreement with the overall concept that labor is an inflammatory event in which macrophages play an active role. Furthermore, we provide evidence that not the classical nPRs, but mPRs are responsible for the action of P4 on macrophages.
The actions of mPRs activated specifically by P4BSA have been described in recent years. Blackmore and colleagues demonstrated that P4 covalently linked to BSA is capable of eliciting Ca2+ influx in human sperm. The conclusion drawn from the study is that there is a receptor that is most probably present in the plasma membrane of the spermatozoa and that the P4-binding site resides in the extracellular portion of the receptor (Blackmore et al. 1991). In a cell model system of human parafollicular cells, P4BSA increases the release of calcitonin through activation of adenylyl cyclase and PKA (Lu & Tsai 2007). Flock et al. (2013) reported that activation of mPRs by P4BSA is responsible for the increased secretion of glucagon-like peptide 1 in enteroendocrine cells in vitro and improvement in glucose tolerance in vivo. The results from our study, using a murine macrophage cell line expressing at least one of the mPRs, mPRα, but not expressing classical nPRs, are consistent with the results of the above-mentioned studies and indicate that the site of P4's action in macrophages is the plasma membrane.
Several lines of evidence indicate that mPRs have the functional characteristics of GPCRs in fish oocytes (Zhu et al. 2003), MDA-MB-231 (human breast adenocarcinoma) cells transfected with mPRα (Zhu et al. 2003, Kelder et al. 2010), human myocytes (Sulke et al. 1985), human T lymphocytes, and Jurkat T cells (Dosiou et al. 2008, Ndiaye et al. 2012). However, others question the subcellular localization and signal coupling of this family of receptors (Fernandes et al. 2008, Smith et al. 2008). To date, the seven-transmembrane-domain topology, a hallmark of the GPCR, has never been conclusively demonstrated in mPRs, but has been deduced from the amino acid sequence of mPRs. Furthermore, there is a large deviation from canonical G-protein-induced signaling pathways downstream of activation of mPRs. Although early reports indicated that P4 can promote phosphorylation of ERK1/2 in fish oocytes and MDA-MB-231 cells transfected with mPRα (Zhu et al. 2003), in human myometrial cells both P4 and P4BSA did not increase the levels of phosphorylation of ERK1/2 (Karteris et al. 2006). P4BSA was also reported to induce phosphorylation of p38 MAPK in human myometrial cells (Karteris et al. 2006), but when Krietsch et al. (2006) stably expressed human, sea trout, and Fugu mPRα in HEK293 and MDA-MB-231 cells, administration of P4 did not decrease cAMP levels or increase levels of phosphorylation of ERK and p38. In this study, P4BSA induces a rapid and transient phosphorylation of MEK1/2 and CREB, but not ERK and p38, demonstrating that MEK1/2 and CREB/PKA are two of the signaling components involved in the pathways triggered by the activation of mPRs in macrophages.
Downregulation of GPCRs through ligand binding (a form of negative feedback) is one of the distinctive characteristics of this large family of receptors (Hadcock & Malbon 1988). The diminished steady-state level of GPCR expression following prolonged treatment with an agonist is the combined result of increased degradation and decreased synthesis as a consequence of a decrease in the level of its mRNA (Drake et al. 2006). Our results indicate that reduced expression of Paqr7 at 4 h may be one of the adaptive mechanisms that function to desensitize macrophages to the inflammatory environment, representing a potential negative feedback mechanism for immune cells to withstand further inflammatory insults. Further study using prolonged stimulation with P4BSA is warranted to test whether the downregulation of Paqr7 parallels diminishing pro-inflammatory responses in these macrophages and whether this downregulation is in concert with a global functional withdrawal of P4 in myometrium before labor.
Oxytocin induces uterine contraction in labor (Fuchs et al. 1984), and increased expression of Oxtr mRNA in the uterus occurs before delivery in many species (Murata et al. 2000). P4 blocks both preterm labor and the associated increase in myometrial expression of Oxtr in ovariectomized rats (Ou et al. 1998), indicating a role for P4 in the regulation of the expression of Oxtr mRNA. In fact, Renthal et al. (2010) demonstrated that P4, through binding to its nPRs, directly upregulated zinc finger E-box-binding homebox protein 1 and suppression of miR-200b/429, which in turn resulted in the downregulation of contraction-associated genes such as connexin 43 and Oxtr in immortalized human myocytes. Other studies also revealed that P4 suppressed expression of Oxtr mRNA in endometrial epithelial cells (Kombe et al. 2003) and bovine lymphocytes (Ndiaye et al. 2008). RAW 264.7 macrophages express OXTR (Szeto et al. 2008) and in our study we demonstrated that extracellular P4 downregulates the expression of Oxtr in these murine macrophages. Further studies are needed to examine the physiological significance of hormonal regulation of expression of Oxtr in macrophages within the context of reproductive systems.
In summary, a non-cell-permeable form of P4 elicits pro-inflammatory responses and downregulates gene transcripts of Paqr7 and Oxtr in macrophages, most probably through binding to mPRs in a process dependent on PKA and MEK1/2. Changes in expression of mPR or of its activation by P4 may represent a novel pathway that contributes to the regulation of inflammatory responses in macrophages and overall regulation of parturition by P4.
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-14-0470.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This study is funded by the National Institutes of Health NIH R01HD056118 March of Dimes #21-FY10-202 and the Satter Foundation.
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